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Genome-wide analysis of endocytic recycling in S. cerevisiae Burston, Helen Elizabeth 2011

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GENOME-WIDE ANALYSIS OF ENDOCYTIC RECYCLING IN S. CEREVISIAE by Helen Elizabeth Burston B.Sc. Simon Fraser University, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2011  © Helen Elizabeth Burston, 2011 i  ABSTRACT The process of endocytic recycling, in which cell surface proteins are internalized and re-delivered to the plasma membrane, is essential in all eukaryotes for maintaining plasma membrane composition and regulating the surface levels of signaling receptors. The applicability of Saccharomyces cerevisiae as a model to study endocytic recycling is a subject of debate, as there appears to be critical differences between yeast and mammalian cells. For example, while clathrin and its adaptors are critical for uptake in mammals, they do not seem to be essential in yeast. Endocytic recycling has not been comprehensively studied on a genetic level in yeast, and only limited cargo have been considered, making it difficult to accurately assess the similarity between the two systems. Furthermore, the transport of SNARE proteins is poorly understood, but appears to involve specialized mechanisms. This study uses a genome-wide screening approach to systematically and quantitatively identify genes required for the endocytic recycling of the yeast SNARE protein Snc1, homologous to the mammalian VAMP2/synaptobrevin. Endocytic defects for mutants of many yeast homologs of mammalian endocytosis genes were identified, for the first time. Significantly, a cargo-selective and partiallyredundant role for clathrin and its adaptors yAP1801 and yAP1802 was identified. The lipid phosphatase Inp52 was found to mediate AP180 release from endocytic vesicles. Additionally, the previously uncharacterized protein Ldb17, homologous to the mammalian endocytic protein SPIN90, was identified as a new component of the endocytic machinery, and regulates both coat and actin dynamics at endocytic sites. Factors regulating Snc1 recycling were also identified, including the variant clathrin adaptor AP-1R. This is the first reported function for this complex. The previously uncharacterized protein Ima1 was found to be a putative enzyme that specifically binds to AP-1R, and may have activity related to AP-1R function. Overall, this study demonstrates that endocytic recycling in yeast and mammals is more similar than previously appreciated, and identifies new factors in this process. Furthermore, it raises awareness of the degree of cargo-selectivity underlying this pathway, and demonstrates quantitative methods that can be further applied to future studies in both systems.  ii  PREFACE List of publications generated from work presented in this dissertation:  1. CHAPTER 2:  Burston HE, Maldonado-Báez L, Davey M, Montpetit B, Schluter C, Wendland B, and Conibear E. (2009) Regulators of Yeast Endocytosis Identified by Systematic Quantitative Analysis. Journal of Cell Biology. 185(6):1097-110. A re-formatted version of this publication is included as chapter 2.  Contributions I was responsible for the writing of the manuscript, all experiments and data analysis, with the exception of the following:  The primary, E-Mapping, and cargo-specificity screens for Invertase activity were carried out by Michael Davey (Conibear lab). Data resulting from these screens were analyzed by Elizabeth Conibear, in collaboration with Jochen Brumm and Jenny Bryan (University of British Columbia).  Figure 2.7 (A-F, H) and Figure A2 (A-D): Live cell microscopy and TIRF analysis were carried out by Lymarie Maldonado-Báez and Beverly Wendland (Johns Hopkins University)  Figure A2 (E, G, and H): Co-immunoprecipitations and Yeast-two-hybrid analyses were carried out by Benjamin Montpetit (University of Berkley)  2. CHAPTER 3: Burston HE, Tam C, Davey M, Raghuram N, Maldonado-Báez L, Wendland B, and Conibear E. (2010). The Alternate AP-1R Clathrin Adaptor Mediates Snc1 Endocytic Recycling. A reformatted version of this manuscript (in preparation) is included as chapter 3.  iii  Contributions  I was responsible for the writing of this manuscript, and for the design, implementation, and analyses of all experiments, with the following exceptions:  Figure 3.1 (B): live cell imaging and TIRF analysis was carried out by Lymarie MaldonadoBáez and Beverly Wendland (Johns Hopkins University)  Figure 3.3 (B, C): Co-immunoprecipitations were carried out by Chris Tam (University of British Columbia)  Figure 3.3 (D) and 3.5 (D): Yeast-two-hybrid experiments and Invertase assays were carried out by Nandini Raghuram (University of British Columbia)  iv  TABLE OF CONTENTS ABSTRACT ........................................................................................................................................................................... ii PREFACE ..............................................................................................................................................................................iii TABLE OF CONTENTS .................................................................................................................................................... v LIST OF TABLES ............................................................................................................................................................ viii LIST OF FIGURES ............................................................................................................................................................. ix LIST OF ILLUSTRATIONS ..............................................................................................................................................x LIST OF SYMBOLS AND ABBREVIATIONS .......................................................................................................... xi ACKNOWLEDGEMENTS ............................................................................................................................................ xiii DEDICATION ....................................................................................................................................................................xiv CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW ........................................................................ 1 1.1. Foreward: Membrane protein transport in the endosomal system ........................................ 2 1.1.1. Endocytic trafficking in cellular function and human disease ........................................... 2 1.1.2. Overview of intracellular transport and the regulation of vesicle formation............ 3 1.1.2. Markers of organelle identity and the specificity of vesicular transport ..................... 4 1.1.4. Coat proteins mediate vesicle formation ...................................................................................... 6 1.2. Clathrin adaptors are key regulators of cellular transport........................................................... 8 1.2.1. Heterotetrameric clathrin adaptors: structure and function ............................................. 8 1.2.2. Physiological importance of clathrin adaptors .......................................................................... 9 1.2.3. Recruitment and un-coating of AP complexes: membrane phospholipids ................. 9 1.2.4. AP appendage domains interact with multiple regulators ............................................... 10 1.2.5. Mechanisms of AP cargo recognition ........................................................................................... 11 1.2.6. Non-canonical cargo sorting signals are recognized by CLASPS ................................... 12 1.3. Transport of SNARE proteins.................................................................................................................... 13 1.3.1. Alternative sorting mechanisms regulate SNARE transport ........................................... 13 1.3.2. What is the role of palmitoylation in SNARE transport? ................................................... 14 1.4. Endocytosis and endocytic recycling pathways .............................................................................. 15 1.4.1. Yeast as a model for endocytic recycling ................................................................................... 15 1.4.2. Do yeast and mammals share common mechanisms of endocytosis?........................ 16 1.4.3. A comparison of the endosomal system in yeast and mammalian cells .................... 17 1.5. Yeast functional genomics: a tool to dissect trafficking pathways ........................................ 19 1.5.1. Systems biology approaches to study membrane transport ........................................... 19 1.5.2. Phenotypic screening in yeast to identify transport components ................................ 21 1.6. Research objectives and hypotheses..................................................................................................... 22 1.6.1. A new approach for identifying endocytic recycling components in yeast.............. 22 1.7. Illustrations ........................................................................................................................................................ 24  v  CHAPTER 2. REGULATORS OF YEAST ENDOCYTOSIS IDENTIFIED BY SYSTEMATIC QUANTITATIVE ANALYSIS. ..................................................................................................................................... 27 2.2. Introduction ....................................................................................................................................................... 28 2.3. Results ................................................................................................................................................................... 30 2.3.1. A Snc1-based quantitative assay for endocytic recycling.................................................. 30 2.3.2. Genetic interaction mapping clusters functionally related genes................................. 31 2.3.3. Integration of genetic and physical interaction data identifies complexes required for Snc1 transport ............................................................................................................................................... 32 2.3.4. Clathrin and clathrin assembly proteins are required for Snc1 endocytosis.......... 33 2.3.5. Systematic analysis of cargo specificity ...................................................................................... 34 2.3.6. Ldb17 is a new regulator of yeast endocytosis that is transiently recruited to cortical patches .................................................................................................................................................... 35 2.3.8. Aberrant actin distribution in LDB17 mutants....................................................................... 37 2.4. Discussion............................................................................................................................................................ 37 2.4.1. Role for clathrin adaptors in yeast endocytosis ..................................................................... 38 2.4.2. Identification of novel components of the yeast endocytic machinery...................... 39 2.5. Materials and methods ................................................................................................................................. 40 2.6. Figures and Tables .......................................................................................................................................... 44 CHAPTER 3: THE YEAST VARIANT AP-1R CLATHRIN ADAPTOR AND IMA1 ARE REQUIRED FOR SNC1 ENDOCYTIC RECYCLING1................................................................................................................... 53 3.1. Synopsis................................................................................................................................................................ 54 3.2. Introduction ....................................................................................................................................................... 54 3.3. Results ................................................................................................................................................................... 56 3.3.1. A role for the variant AP-1R complex in endocytic recycling.......................................... 56 3.3.2. AP-1R mediates intracellular recycling ...................................................................................... 57 3.3.3. AP-1R and Ima1 localize to Golgi/endosomal compartments ........................................ 57 3.3.4. Ima1 and AP-1R are not required for the transport of AP-1-dependent cargo ..... 58 3.3.5. Ima1 specifically binds the AP-1R C-terminal domain ....................................................... 59 3.3.6. Ima1 is not required for AP-1R recruitment or interaction with cargo..................... 60 3.3.7. Ima1 is a conserved protein with a consensus α/β hydrolase catalytic motif ....... 61 3.3.8. The Ima1 GXSXG catalytic motif is required for normal Snc1 surface levels .......... 62 3.4. Discussion............................................................................................................................................................ 63 3.4.1. AP-1R mediates Snc1 intracellular recycling........................................................................... 63 3.4.2. Apm2 binds to Snc1 as cargo............................................................................................................ 63 3.4.3. Distinct sorting functions for AP-1/AP-1R ............................................................................... 64 3.4.4. AP-1/AP-1R localize to similar intracellular compartments........................................... 65 3.4.5. Role of Ima1 in AP-1R regulation................................................................................................... 65 3.4.6. Ima1 has putative enzymatic activity .......................................................................................... 67  vi  3.5. Materials and methods ................................................................................................................................. 68 3.6. Figures................................................................................................................................................................... 73 CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS .............................................................................. 81 4.1. Overview .............................................................................................................................................................. 82 4.2.1. Role of clathrin and yeast AP180 homologs in Snc1 endocytosis................................. 83 4.2.2. How is yAP180 recruited to endocytic sites? .......................................................................... 83 4.2.3. Role of synaptojanin in yAP180 vesicle un-coating.............................................................. 85 4.2.4. How does yAP180 recognize Snc1 for internalization? ..................................................... 87 4.2.5. Does AP-2 mediate endocytosis in yeast?.................................................................................. 88 4.2.6. Role of actin and its regulators in Snc1 internalization ..................................................... 89 4.3. Regulators of Snc1 recycling: AP-1R and Ima1 ................................................................................ 91 4.3.1. What is the role of AP-1R in Golgi/endosomal recycling? ................................................ 91 4.3.2. What is the Snc1 recycling signal recognized by AP-1R? .................................................. 94 4.3.3. How do AP-1 and AP-1R regulate distinct sorting pathways?........................................ 95 4.3.4. Do AP-1 and AP-1R localize to different lipid domains? .................................................... 96 4.3.5. Ima1 is a putative enzyme and binds the Apm2 C-terminal domain .......................... 97 4.3.6. Is Ima1 important for the generation of lipids required for AP-1R function? ......100 4.3.7. Determination of Ima1 substrates...............................................................................................100 4.3.8. Investigating the role of TMC04 in higher cells ....................................................................101 4.4. Genetic interaction profiling to identify complexes and pathways required for Snc1 transport .....................................................................................................................................................................102 4.4.1. Phenotypic profiling and the construction of genetic interaction networks ........102 4.4.2. Endocytic processes are part of a broader cellular context ...........................................104 4.4.3. Assessing the role of essential genes in Snc1 transport...................................................105 4.4.4. Relationships between endocytic recycling factors; implications in disease .......106 4.4.5. Future perspectives.............................................................................................................................107 4.4. Illustrations ......................................................................................................................................................109 BIBLIOGRAPHY.............................................................................................................................................................111 APPENDIX A: Supplemental material for chapter 2 ..................................................................................130 APPENDIX B: Supplemental material (chapter 3) ......................................................................................221  vii  LIST OF TABLES Table 2.1. Predicted yeast endocytic genes identified in the screen................................................. 45 Table A1. Plasmids and yeast strains used in this study .......................................................................130 Table A2. Full list of mutants with cell surface GSS levels greater to or equal to the median value for all strains. ....................................................................................................................................................162 Table B.1. Plasmids and yeast strains used in this study ......................................................................221  viii  LIST OF FIGURES Figure 2.1. Genome-wide screen for endocytic recycling mutants.................................................... 44 Figure 2.2. Endocytosis mutants have related genetic interaction profiles. ................................. 46 Figure 2.3. Integration of genome-wide genetic and physical interaction data identifies pathways and complexes. ......................................................................................................................................... 47 Figure 2.4. Yeast AP180 homologs have a conserved role in Snc1 internalization................... 48 Figure 2.5. Cargo sorting defects of yap1801∆ yap1802∆ mutants. ................................................. 49 Figure 2.6. Cargo specificity of genes required for Snc1 endocytosis. ............................................. 50 Figure 2.7. Ldb17 is required for proper coat and actin dynamics ................................................... 51 Figure 3.1. AP-1R is required for correct sorting of Snc1 ....................................................................... 73 Figure 3.2. AP-1R and Ima1 localize to Golgi and endosomal compartments ............................. 74 Figure 3.3. Ima1 associates functionally and physically with AP-1R................................................ 75 Figure 3.4. Ima1 is not required for Apm2 membrane recruitment or cargo binding ............ 77 Figure 3.5. Ima1 contains putative catalytic residues, required for Snc1 sorting ..................... 78 Figure 3.6. Overexpression of Ima1 does not affect Snc1 palmitoylation ...................................... 80 Figure A1. Full hierarchical clustering of the genetic interaction data..........................................218 Figure A2. Characterization of LDB17 mutants and identification of Ldb17-interacting proteins.............................................................................................................................................................................219  ix  LIST OF ILLUSTRATIONS Illustration 1.1. Intracellular compartments and transport pathways. ......................................... 24 Illustration 1.2. Steps in clathrin-coated vesicle formation and transport. ................................. 25 Illustration 1.3. Clathrin Adaptor (AP) structure....................................................................................... 26 Illustration 1.4. Comparison of endocytic recycling compartments and pathways in yeast and mammalian cells. .................................................................................................................................................. 26 Illustration 4.1. New factors required for Snc1 endocytic recycling. ............................................109 Illustration 4.2. AP-1R transport function: Model 1 ...............................................................................110 Illustration 4.3. AP-1R transport function: Model 2 ...............................................................................110  x  LIST OF SYMBOLS AND ABBREVIATIONS AAK1 ABE ABPP AD ALP ANTH AP AP-1R APP ARF ARH BAR BFA CCV CLASP CPY DAB2 DEP DUF Dvl E-Map EGF EH ENTH ER ERC ESCRT FP GAE GAP GAT GDP GEF GGA GLUT4 GPCR GSS GTP HD Hip1/Hip1R HPS Ima1 LacZ LatA LBPA MVB NGSS  Adaptor-associated kinase 1 Acyl-biotin exchange Activity-based protein profiling Alzheimer’s disease Alkaline phosphatase AP180 Amino-terminal homology domain Adaptor Protein AP-1 related Amyloid precursor protein ADP Ribosylation Factor Autosomal recessive hypercholesterolemia Bin/Amphiphysin/Rvs domain Brefeldin A Clathrin-coated vesicle Clathrin-associated sorting protein Carboxypeptidase Y Disabled2 Dishevelled, egl-10, and pleckstrin domain Domain of unknown function Dishevelled Epistatic miniarray profiling Epidermal growth factor Eps15 homology domain Epsin amino terminal homology domain Endoplasmic reticulum Endocytic recycling compartment Endosomal sorting required for transport Fluorophosphonate Gamma-adaptin ear domain GTPase-activating protein GGA and tom1 domain Guanosine di-phosphate Guanine nucleotide exchange factor Golgi-localized, gamma-ear containing, ARF-binding Insulin-sensitive glucose transporter 4 G Protein-coupled receptor GFP-Snc1-Suc2 Guanosine triphosphate Huntington's disease Huntington interacting/Huntington interacting-related protein Hermansky Pudlak syndrome Interacts with µ adaptin 1 β-galactosidase Latrunculin A Lysobisphosphatidic acid Multivesicular body 3x NPFxD-GFP-Sso1-Suc2  xi  NPF PA PC PE PH PIPK PLD PRD PS Ptb PX RNAi SH3 SNARE SNC1 SORL1 TfR TGN TMCO4 TS VAMP VHS VPS WASP Y2H  Nucleation promoting factor Phosphatidic acid Phosphatidylcholine Phosphatidylethanolamine Pleckstrin homology domain Phosphatidylinositol phosphate kinase phospholipase D Proline-rich domain Phosphatidylserine Phosphotyrosine-binding domain Phox homology domain RNA interference SRC homology domain Soluble NSF attachment protein receptor Suppressor of the Null allele of CAP 1 Sortilin-related receptor 1 Transferrin receptor Trans-Golgi network Transmembrane and coiled-coil domain 4 Temperature-sensitive Vesicle-associated membrane protein Vps-27, Hrs and STAM domain Vacuolar protein sorting Wiskott-Aldrich syndrome protein Yeast two-hybrid  xii  ACKNOWLEDGEMENTS First, and foremost, I extend my greatest gratitude to Dr. Elizabeth Conibear. Throughout my degree, Liz has been a constant source of support, guidance, and friendship. Her wisdom and unrelenting enthusiasm is truly inspirational, and she is, and will always be my ultimate mentor. I am grateful to my colleagues in the Conibear lab, past and present. They have offered support, guidance, and expertise during the course of this work, and it would not have been possible without them. I would like to thank Mike Davey and Cayetana Schluter for their assistance in the design and implementation of many experiments, and for countless helpful discussions. I would also like to thank Benjamin Montpetit, Chris Tam, and Nicole Quenneville for their ongoing advice, encouragement, and career mentorship, and Karen Lam for her support, and for the critical reading of this dissertation. I am also amazed by the talent of the two undergraduate students, whom I had the opportunity to supervise during my degree. Thank you to Nandini Raghuram and Sarah Konefal for your incredible work ethic and enthusiasm. I would also like to thank our collaborators, Dr. Beverly Wendland and Dr. Lymarie Maldonado-Báez, who have been instrumental in the work presented here. Dr. Nick Davis and Dr. Amy Roth have also contributed many insights and have aided in the development of experimental approaches. I extend my gratitude to my graduate committee: Dr. Michael Kobor, Dr. Phil Hieter, and Dr. Pamela Hoodless, for their ongoing support and advice, and for the critical reading of this dissertation. Finally, I would like to thank NSERC and the Michael Smith Foundation for providing generous financial contributions.  xiii  DEDICATION  To my family and the many people whom have helped me along the way. Thank you for your support and encouragement.  xiv  CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW  1  1.1. Foreward: Membrane protein transport in the endosomal system 1.1.1. Endocytic trafficking in cellular function and human disease In every eukaryotic cell, proteins are directed to the appropriate location by membrane transport pathways. These pathways facilitate communication within the cell, and between the cell and its environment, maintaining cell homeostasis and function (Schu, 2001; Van Vliet et al., 2003). Endocytosis allows cells to internalize extracellular material, ligands, and plasma membrane proteins. This removal of membrane from the cell surface by endocytosis is balanced by recycling pathways that return many of these proteins and lipids back to the plasma membrane. These endocytic recycling pathways control the composition of the plasma membrane and contribute to diverse cellular processes, including nutrient uptake, cell adhesion and junction formation, cell migration, cytokinesis, cell polarity, and signal transduction (Jovic et al., 2010; Grant and Donaldson, 2009). Many human diseases caused by changes in cellular homeostasis arise through compromised membrane trafficking within the endosomal system (Howell et al., 2006; Aridor and Hannan, 2000 and 2002). Endocytosis has a major role in neuronal function, as it is required for synaptic vesicle (SV) recycling within the pre-synaptic compartment (Jung and Haucke, 2007; Girard et al., 2005). It also regulates the activity-dependent exoendocytic trafficking of postsynaptic receptors, allowing for the fine-tuning of signal strength during neurotransmission and plasticity-associated changes, uptake of growth factors in the pre-synapse, and sculpting of synaptic structure (Sheng and Kim, 2002; Dickman et al., 2006). Disturbance of trafficking is implicated in severe neurodegenerative disorders like Alzheimer’s disease (AD), which is characterized by deposition of amyloid β (Aβ) plaques in the brain (reviewed in Mayeux and Hyslop, 2008; Selkoe, 2001). Endosomes appear to be one of the major sites of Aβ generation and several reports indicate that altered endocytic trafficking may play role in development of AD. For instance, the neuronal sortilin-related receptor SORL1 directs trafficking of amyloid precursor protein into recycling pathways and prevents it from accumulating in the compartments generating Aβ (Andersen et al., 2005; Shah and Yu, 2006). Mutations in SORL1 are associated with both inherited and sporadic cases of Alzheimer’s disease (Mayeux and Hyslop, 2008). Impaired trafficking has also been shown to contribute to the pathogenesis of Huntington's disease (HD). HD is characterized by the mutant protein huntingtin, which  2  aggregates and affects membrane trafficking in neurons, leading to neurodegenerative changes (Berman and Greenamyre, 2006; Caviston and Holzbaur, 2009). Abnormal trafficking of the insulin-sensitive glucose transporter GLUT4 has been identified as a possible mechanism of disease in patients with type 2 diabetes. In some of these patients, GLUT4 accumulates in dense membrane compartments, suggesting that defects in GLUT4 membrane trafficking may be involved in the pathogenesis of insulin resistance (Bouché et al., 2004; Karylowski et al., 2004). Membrane traffic is also intimately connected to infection and immunity. Viruses and bacteria often use the endocytic pathway of host cells for invasion (Mercer et al., 2010; Lin and Guttman, 2010). In addition, large DNA viruses such as adenovirus, herpes simplex virus, Epstein-Barr virus, and cytomegalovirus have evolved sophisticated mechanisms to evade host immune surveillance by perturbing the membrane traffic of host cells, thereby preventing antigen presentation of their own products (Ohno, 2006; Ohmura-Hoshino et al., 2006). Despite the importance of these pathways, the molecular mechanisms governing their function remain poorly understood. A better understanding membrane transport is therefore crucial for our understanding of human disease, and the development of targeted treatments. 1.1.2. Overview of intracellular transport and the regulation of vesicle formation The secretory and endocytic pathways of eukaryotic cells connect membrane-bound organelles, whose primary function is to modify newly-synthesized proteins and deliver them to the appropriate location, and to regulate the uptake and turnover of cell surface proteins (Munro, 2004; van Vliet et al., 2003). Each of these organelles is functionally specialized, and contains a unique composition of proteins and lipids pertinent to its function (Munro, 2004). Vesicular transport pathways are essential for maintaining organelle composition and identity. The transport of cellular material between organelles is highly organized and regulated. A large number of factors ensure that each protein is accurately delivered to its appropriate location, thus preventing the inappropriate mixing of cellular compartments. The main compartments of the secretory and endocytic pathway are the endoplasmic reticulum (ER), Golgi apparatus, plasma membrane, and endosomal compartments (Illustration 1.1). Following delivery to the cell surface, proteins are internalized by endocytosis, and reach early endosomes, where they are then either recycled back to the  3  cell surface directly or by retrograde transport through the TGN, or targeted for degradation in the vacuole (Munro, 2004). Recycling of cell surface proteins is essential in all organisms to maintain the composition of the plasma membrane, by balancing the removal of proteins by endocytosis. This process, called endocytic recycling is the focus of this dissertation. Proteins are transported as ‘cargo’ within membrane-bound vesicles, which shuttle them between organelles. The selective incorporation of cargo into forming vesicles is mediated by protein coats, which are assemblies of proteins that are recruited from the cytosol. Vesicle coats recognize various components of the target membrane, including sorting signals present in the cytosolic domain of cargo proteins. Transport between each compartment is mediated by distinct sets of coats and sorting signals, which confer specificity to these pathways (Bonifacino and Lippincott-Schwartz, 2003). The steps of cargo recognition and vesicle formation will be described in more detail below. 1.1.2. Markers of organelle identity and the specificity of vesicular transport Vesicle transport is a precise process that requires the specific recognition of cellular membranes by components of the trafficking machinery. How do these components ‘‘know’’ how to reach a particular cellular destination? Most internal organelles have ‘signposts’ of identity to distinguish them from other organelles. The main components specifying organelle identity include specific GTPases of the Rab/Arf family, SNARE proteins, and membrane lipids (Reviewed in Behnia and Munro, 2005).  GTPases The small GTPases of the Rab/Arf family are molecular switches that can alternate between a membrane-associated GTP-bound active form and a cytosolic GDP-bound inactive form. The activity of these GTPases is controlled by proteins that regulate their GDP/GTP cycle. These include guanine nucleotide exchange factors (GEFs), which replace GDP by GTP, and GTPase-activating proteins (GAPs), which stimulate GTP hydrolysis. There are more than 60 different Rabs in mammalian cells, and fewer in lower eukaryotes. Each organelle is enriched for a particular subset of these Rabs (Segev, 2010; Pereira-Leal and Seabra, 2001). Regulatory GEFs/GAPs also show a restricted distribution, which increases the functional specificity of their cognate GTPases by allowing specific and rapid control of their activity at these compartments (Kawasaki et al., 2005; Itzen and Goody, 2010).  4  Membrane phospholipids Phosphoinositide phospholipids (PIs) are also enriched on one or a small subset of organelles. PI(4,5)P2 is enriched at the plasma membrane, PI3P at endosomal compartments, PI(3,5)P2 at the vacuole, and PI4P at the Golgi. Much like GTPases, localized synthesis and turnover of these lipids is regulated by specific factors, namely PI kinases and phosphatases, which are also spatially restricted to a subset of organelles. This ensures that the steady state concentration of each PI species varies between organelles. Many components of the vesicle coat have domains that recognize these PI species. These domains include pleckstrin homology (PH), phox homology (PX), epsin amino terminal homology (ENTH) and AP180 amino-terminal holomology (ANTH) domains (Reviewed in De Matteis and Godi, 2004).  SNARE proteins The selectivity of membrane transport is also provided in part by SNARE proteins, which catalyze the fusion of the opposing membranes of the vesicle and target membrane. Functionally, SNAREs are classified into v-SNAREs, which associate with the vesicle, and tSNAREs, which are present on the target compartment. Specific interaction between vSNAREs on an incoming transport vesicle with t-SNAREs on the target membrane is the central event of the docking and fusion process (Pelham, 2001). As each SNARE is restricted to a subset of specific compartments, SNAREs contribute to the specificity of the fusion process. There are 25 members of this family in S. cerevisiae and 36 in Homo sapiens, most of which are small membrane proteins (Pelham, 2001; Hong, 2005)). The major, N-terminal portion of the SNARE protein is exposed in the cytoplasm, followed by a single membrane-spanning region and a few amino acids that face either the lumen of an intracellular compartment or the extracellular space. The core feature of SNAREs is the evolutionarily conserved SNARE motif, which is present in all family members. During membrane fusion, SNAREs assemble into a four helical bundle via interactions between specific residues within their SNARE domains. SNAREs can also be classified based on the residue contributed to this interaction. While Q SNAREs contribute a glutamine residue to the core layer of the assembled complex, R SNAREs contribute an arginine residue. The assembly of SNARE motifs of cognate v- and t-SNAREs generates a trans-SNARE complex bridging two membranes, which brings the lipid bilayers in close proximity and drives membrane fusion. Following fusion, the v- and t-SNAREs are present  5  on the same membrane, and the complex is thus called a cis-SNARE complex. The ciscomplex is dissembled by ATP-hydrolysis generated by the coordinated activity of two proteins: SNAP and NSF. The SNAREs are then returned to the donor membranes (Ungar and Hughson, 2003; Brunger, 2005) In addition to SNAREs, tethering complexes have a major role in the specificity of vesicle fusion with the target membrane. Tethering factors are the initial connection, or tether, between an intracellular trafficking vesicle and its target membrane (Barlowe 1997, Cao et al., 1998). Tethering complexes recognize the features of both the originating upstream compartment and the downstream target compartment, including SNARE proteins of both compartments, as well as activated Rab proteins and phospholipids.  Coincidence detection Individually, Rab/Arf GTPases, SNAREs and phosphoinositides are not sufficient to explain the degree of specificity achieved in vesicle transport. The precision of this process requires the simultaneous recognition of multiple determinants. Coat recruitment at the donor membrane is achieved through combinatorial recognition of an activated Arf GTPase, specific phospholipids, and cargo. Similarly, Rab, SNAREs and membrane lipids all contribute to tether recruitment and SNARE assembly at the target membrane. Hence, organelle identity is more precisely defined by the combination of these restricted components. The process by which the vesicular transport machinery recognizes multiple determinants at a given membrane is often referred to in the literature as ‘coincidence detection’ (Carlton and Cullen, 2005). 1.1.4. Coat proteins mediate vesicle formation The biogenesis of transport vesicles is initiated through the recruitment of large multi-subunit protein complexes termed coats. These coats control both cargo selection and deformation of the lipid bilayer into a budding vesicle. There are three well-defined vesicle coats: COPI, COPII, and clathrin, each of which are involved in distinct transport pathways (Bonifacino and Lippincott-Schwartz, 2003; Kirchhausen, 2000). COPI mediates both intraGolgi transport and retrograde transport from the Golgi to the ER, while COPII functions in anterograde ER to Golgi traffic. Clathrin was the first coat to be identified, and is the best characterized, both structurally and biochemically. Clathrin coats are considerably more complex than the COPI/COPII coats, as they participate in multiple post-Golgi transport pathways, including receptor-mediated endocytosis, and several sorting pathways between  6  the TGN and endosomes (Kirchhausen, 2000). To fulfill its role in each of these pathways, clathrin associates with an additional subset of regulatory proteins. The clathrin-coated vesicle itself is a three-layered structure, consisting of an outer clathrin layer, an inner membrane layer and an adaptor protein layer sandwiched between the two (Brodsky et al., 2001; Edeling et al., 2006). Clathrin is a large hetero-hexameric protein complex composed of three heavy chains and three light chains. Clathrin molecules self-assemble to make a spherical ‘‘clathrin lattice’’ structure, a polyhedron made of regular pentagons and hexagons. The clathrin lattice serves as a mechanical scaffold but is unable to bind directly to the membrane. Adaptor protein (AP) complexes are required for recruitment of clathrin. These complexes interact directly with components of the lipid bilayer, cargo, and clathrin, and thus link clathrin to the membrane and to the specific cargo to be transported (Robinson, 2004). Both clathrin and its adaptors interact with many additional regulatory components. The formation and delivery of clathrin-coated vesicles is divided into 5 major steps: initiation, coated pit formation, vesicle budding, uncoating, and vesicle fusion (Illustration 1.2, Ritter and Wendland, 2009; Ungewickell and Hinrichsen, 2007). Initiation is thought to begin by the recruitment of APs to specific membrane domains where they recruit cargo proteins. Clathrin then binds to these docked AP/cargo complexes, and polymerizes to form the coat. Further stabilization of the budding site is achieved through enhanced affinity of APs with membrane and cargo, and the recruitment of accessory proteins. As clathrin polymerizes, accessory proteins that include BAR (Bin/amphiphysin/Rvs) domaincontaining proteins, such as amphiphysin and endophilin, promote the membrane curvature required for vesicle budding. The crescent-shaped BAR domain has a highly positively charged concave surface, contributing to electrostatic interactions that aid membrane deformation. Further deformation of the membrane and polymerization of clathrin leads to a coated vesicle attached to the plasma membrane by a narrow neck, which pinches off to form a free coated vesicle. Once the vesicle is formed, the clathrin coat is removed. This allows the vesicle to fuse with its target membrane and deliver its cargo, and ensures that coat proteins are returned to the soluble pool to initiate new rounds of coat assembly (Ritter and Wendland, 2009).  7  1.2. Clathrin adaptors are key regulators of cellular transport 1.2.1. Heterotetrameric clathrin adaptors: structure and function There are three main AP complexes (AP-1, AP-2, and AP-3) in most organisms, including yeast, while higher organisms also have AP-4. Each of these complexes regulates distinct vesicle transport pathways, localizes to different compartments, and binds distinct sets of cargo (Boehm and Bonifacino, 2002). AP-2 mediates the formation of endocytic CCVs from the plasma membrane. AP-1 regulates transport between the TGN and endosomes, although it is not clear in which direction it operates. AP-3 traffics cargo from the TGN to the yeast vacuole, or the mammalian lysosome, and is involved in the biogenesis of lysosome-like organelles. The function of AP-4 is not well understood, but it appears to be involved in TGN to endosome trafficking (Robinson, 2004). All APs share a similar hetero-tetrameric structure, consisting of two large subunits of 100–130 kDa (β and one of either α, γ, δ, or ε), a medium-sized subunit of ~50 kDa (μ1–4 in AP1–4, respectively) and a small subunit of ~20 kDa (σ1–4 in AP1–4, respectively), (Illustration 1.3). The large subunits are divided into an N-terminal core domain, which assembles with the medium and small subunits, and a C-terminal appendage domain, which is connected to the core by an unstructured hinge. Each subunit and domain mediates distinct sets of regulated interactions pertinent to AP function. The core domain of the large subunits (α, γ, δ, and ε) is required for binding to the target membrane. The other large subunit (β) contains a clathrin box within its hinge, and is required for the recruitment of clathrin. The medium (μ) subunits have a direct role in the recognition of cargo sorting signals, while the small (σ) subunit is mainly involved in complex stability. Together with regions of the large subunits, the small subunits also bind to cargo sorting signals that are distinct from those recognized by the medium subunit. Interactions between APs and their binding partners are dynamic, weak, and under strict spatiotemporal control, ensuring that vesicles form at the correct time and place, and can be rapidly disassembled once the vesicle has formed (Owen et al., 2004). Some AP complexes can exist as different isoforms that have different sorting functions. Exchange of the large, medium, and small subunits are all observed in mammals (Owen et al., 2004; Nakatsu and Ohno, 2003). Mammals have two isoforms of both AP-1 and AP-3, which differ by the inclusion of an alternate medium subunit. These are expressed in a cell-type specific manner. AP-3B is expressed exclusively in neurons, and is involved in synaptic vesicle biogenesis (Newell-Litwa et al., 2009). AP-1B is expressed only in polarized 8  epithelial cells, and mediates sorting to the basolateral membrane (Traub and Apodaca, 2003). From these studies, it is clear that exchange of the medium subunit can dramatically affect the sorting function of each complex. Two isoforms of the AP-1 complex also exist in yeast, containing the alternate medium subunits Apm1 or Apm2. While the Apm1containing complex has a well-established role in the clathrin-mediated sorting of the Chitin synthase Chs3 and the -factor modification enzyme Ste13 between the endosomes and Golgi (Valdivia et al, 2002; Foote and Nothwehr, 2006), no role has been established for the alternate Apm2-containing complex (Stepp et al., 1995). 1.2.2. Physiological importance of clathrin adaptors The physiological importance of AP complexes is illustrated by studies in multiple organisms. Knockout of AP-1A or AP-2 results in embryonic lethality in C. elegans and mice, indicating that they are essential for normal development (Ohno, 2006). Mutations in AP3A underlie the autosomal recessive disorder Hermansky-Pudlak syndrome 2 (Dell'Angelica et al., 1999). This disease is characterized by defective transport to lysosome-related organelles, including melanosomes and platelet dense granules, and results in clinical features including albinism, platelet storage pool deficiency, defective immune function, and bleeding defects (Huizing and Gahl, 2002 and Huizing et al., 2002). In mice, knockout of the neuronal-specific AP-3B causes epileptic seizures and neurotransmission defects, as a result of mis-regulation of synaptic vesicle formation (Nakatsu et al., 2004; Seong et al., 2005). In addition to demonstrating the critical role of APs, these studies illustrate that different phenotypic consequences can result from deficiency of closely related AP isoforms. The physiological importance of AP complexes has driven considerable research into their function. The mechanisms regulating AP membrane recruitment, cargo recognition, and uncoating are described below. 1.2.3. Recruitment and un-coating of AP complexes: membrane phospholipids Interaction with membrane phospholipids appears to be an early event in the recruitment and activation of APs at their target membranes. Accordingly, the control of phosphoinositide levels by kinases and phosphatases is important in regulating AP recruitment and uncoating (Edeling et al., 2006). For example, higher rates of endocytic internalization by AP2 are associated with increased levels of PI(4,5)P2, caused by overexpression of the type I phosphatidylinositol 4-phosphate 5-kinase h (PIP5KIh) (Krauss et al., 2006) . There is a good correlation between PI(4,5)P2 levels, association of AP-2 with  9  the cell surface, and the number of clathrin coated structures (Gaidarov and Keen, 1999; Padrón et al., 2003; ) Conversely, synaptojanin 1, which hydrolyses PtdIns(4,5)P2 to PtdIns(4)P, promotes the dissociation of AP-2 from the membrane of CCVs following budding. In mice knockout studies, the absence of synaptojanin 1 leads to 100% mortality within 2 weeks of birth, elevated steady-state levels of PI(4,5)P2, an increased number of clathrin-coated vesicles in presynaptic terminals, and increased synaptic depression in hippocampal slices (Cremona et al., 1999). The Golgi contains the main cellular pool of PI(4)P, generated by the phosphorylation of PtdIns by the type II alpha phosphatidylinsositol 4-kinase PI4KII (D'Angelo et al., 2008). In both yeast and mammalian cells, PI4KII homologs are involved in sorting at the late Golgi (De Matteis and D'Angelo, 2007). AP-1 has been shown to bind directly to PI(4)P in mammalian cells, and reduction of PI(4)P levels results in the redistribution of AP-1 from Golgi compartments to the cytosol (Wang et al., 2003). 1.2.4. AP appendage domains interact with multiple regulators The appendage domains of the large AP subunits serve as a major interaction hub for various regulatory proteins that help drive rapid assembly of AP-containing vesicles and aid in cargo recognition. These proteins, called “accessory” factors, fall into three categories: regulatory proteins, mechanical/assembly proteins, and alternate adaptors (Traub, 2005). Examples of regulatory proteins include the kinases AAK1, and GAK, which phosphorylate the  subunit of AP-2 and AP-1, respectively. These phosphorylation events are required for productive association of AP-1 and AP-2 with cargo. Mechanical/assembly proteins act during and after cargo recognition to synchronize interactions between adaptors and other components of the vesicle formation machinery, and may also contribute directly to the vesicle budding event. Although there seem to be multiple mechanisms involved, interaction of clathrin with these proteins appears to increase the intrinsic curvature and rigidity of coated vesicles at the membrane. Many of these proteins are lipid-binding proteins that contribute to membrane deformation (Lundmark and Carlsson, 2010). A well-studied example is epsin, an ENTH domain protein, which inserts a hydrophobic helix into the membrane, leading to membrane curvature (Horvath et al., 2007). Proteins containing BAR domains facilitate the membrane bending required at late stages of vesicle formation. These include amphiphysin1 and Sorting nexin 9 (SNX9) (Itoh and De Camilli, 2006). Alternate sorting adaptors or CLASPs link a subset of cargo to AP complexes. Most  10  CLASPs identified to date interact with the AP appendage domain. Mechanisms of AP cargo recognition and the role of CLASPs are discussed below. 1.2.5. Mechanisms of AP cargo recognition Most signals recognized by AP complexes consist of short, linear sequences of amino acid residues present in the cytosolic domain of cargo proteins. Two of the most widelyused and best understood signals conform to YXXΦ ‘Tyrosine’ motif (in which X represents any amino acid and Φ represents a large hydrophobic residue) and [D/E]XXXL[L/I] ‘acidic dileucine’ motif. All APs recognize YXXΦ motifs, and some additionally bind dileucine motifs (Heilker et al., 1999; Bonifacino and Traub, 2003). Examples of YXXΦ-containing cargo include mammalian LAMPs, the Transferrin receptor and CD63, and yeast Yck3 (Bonifacino and Traub, 2003; Sun et al., 2004). Dileucine-based cargo includes mammalian tyrosinase and GLUT4, and yeast ALP and Gap1 (Bonifacino and Traub, 2003). The medium (μ) subunit of each AP interacts with a distinct but overlapping set of YXXΦ signals, as defined by deviation in the residues other than the critical tyrosine. This partially explains how each is able to recognize a different subset of YXXΦ-based cargo. The μ subunit is organized into two main domains, separated by a flexible linker. The Nterminal domain assembles with the core complex, while the C-terminal domain contains the YXXΦ binding site (Owen et al., 2004). The C-terminal domain is further divided into two β-sandwich subdomains (A and B). Subdomain A is folded to create a cargo-binding pocket. The critical Y and Φ residues fit into hydrophobic pockets on the surface of this domain, and binding is mediated through both hydrogen bonding and backbone interactions (Edeling et al., 2006, Brodsky et al., 2001). Structural studies have demonstrated that in order to efficiently bind cargo, the μ subunit undergoes a large-scale rearrangement relative to the rest of the complex. In the inactive form, the YXXΦ binding site on the μ subunit is occluded by interaction with the large subunit (closed form), and is freed from this occlusion by the movement of the C-terminal cargo binding domain (open form). This rearrangement occurs following binding of the complex to membrane lipids, and is stabilized by phosphorylation of a specific threonine residue within the unstructured linker of μ2 by the kinase AAK1, and by association with cargo. This complex rearrangement is thought to couple cargo binding to membrane attachment, therefore preventing inappropriate interactions the complex and cytosolic proteins (Jackson et al., 2010).  11  The dileucine signal is structurally distinct from the tyrosine-based signal, and binds to the AP complex in a unique manner. The interaction surface is primarily positioned on the small (σ) subunit. Similarly to the recognition of tyrosine-based signals by the μ subunit, interaction of dileucine-based signals requires a structural rearrangement of the complex. In this case, the binding site on the σ subunit is blocked by the large β subunit, which must shift to expose the binding site. This shift may be mediated through phosphorylation of a tyrosine residue on the β subunit, and is less dramatic than the largescale movement of the μ subunit relative to the core. Dileucine-based peptides can engage in binding while the tyrosine binding site remains in a closed conformation (Huang et al., 2003). It seems that cargo recognition is not a mandatory step in AP recruitment, as APs with mutated cargo binding sites still assemble into CCVS. Cargo binding has, however, been shown to enhance the membrane affinity of APs. It is likely that cargo capture occurs concomitantly with coat polymerization. The numerous simultaneous contacts that APs can establish with other factors and accessory proteins serve to explain how APs can associate with the membrane before binding to cargo. It also appears that cargo load is dynamically monitored in the forming vesicle by APs and their regulators, in order to prevent unproductive budding (Traub, 2009). For example, overexpression of the transferrin receptor (TfR), a well-studied AP-2 cargo, decreases the number buds that abort prior to formation or scission (Loerke et al., 2009). 1.2.6. Non-canonical cargo sorting signals are recognized by CLASPS Not all cargo sorting motifs conform to the YXXΦ or [D/E]XXXL[L/I] based motif. In fact, there a broad range of alternative sorting signals. These include variants of the tyrosine and dileucine motifs, but can also involve various post-translational modifications, including phosphorylation or ubiquitination. These signals are not recognized directly by AP complexes, but by a family of AP-associated monomeric adaptors called clathrinassociated sorting proteins (CLASPs), which link APs to cargo containing these alternative signals. Although they share little overall sequence homology with APs, all have a clathrinbinding box, and/or AP interaction sequences that mediate interaction with the clathrin heavy chain and the AP appendage domain, and contain binding sites for membrane phospholipids (reviewed in Traub, 2009 and Maldonado-Baez and Wendland, 2006). The phosphotyrosine-binding (Ptb) CLASPs, including β-arrestins, recognize [FY]XNPX[YF] signals. These mediate internalization of phosphorylated GPCR, by recruiting  12  activated receptors to pre-existing AP-2 containing endocytic pits. ARH and disabled2 (DAB2) recognize the tyrosine-based FXNPXY signal within the LDL receptor, and mediate its internalization. The epsin superfamily is another subset of CLASPs, which includes AP180/CALM, epsin1, and Hip1/Hip1R. Each of these has structurally related ENTH/ANTH domains, and primarily recognizes ubiquitylated cargo. These include the yeast epsin homologs Ent1 and Ent2, which mediate internalization of monoubiquitinated cargo, which is the primary internalization signal in yeast (Maldonado-Baez and Wendland, 2006). Another family of Golgi regulatory adaptors is the Golgi-localized, gamma-ear containing, ARF-binding (GGA) proteins. There are two forms of Gga in yeast (Gga1 and Gga2), which are thought to be largely redundant, and three forms of Gga in mammalian cells, which may have unique sorting roles. These monomeric ubiquitously-expressed proteins have 4 modular domains (VHS, GAT, a hinge and GAE) that essentially recapitulate all of the functional attributes of AP-1 and AP-2. The VHS domain binds to DXXLL-type acidic cluster sorting signals in mammalian cells, and recognizes the receptor Vps10 in yeast. The GAT domain is required for membrane recruitment by activated Arf1 and PI4P at the Golgi, and interacts with ubiquitylated cargo. Clathrin binding is mediated by a variant of the clathrin box motif that is present in the hinge region of Gga. Finally, the GAE domain interacts with accessory proteins that are involved in cargo selection and membrane binding (reviewed in Ghosh and Kornfeld, 2004 and Bonifacino, 2004). Gga proteins are required for sorting of proteins, including the SNARE Pep12, from the Golgi to the late endosome (Black and Pelham, 2000). The use of multiple adaptors provides plasticity to allow precise temporal control in the face of high traffic volumes, ensuring that cargo do not compete with each other for inclusion into coated vesicles (Sorkin, 2004; Traub, 2003). Alternate adaptors can be regulated to alter the sorting of individual cargo, without affecting the sorting of other cargo by the main AP complexes.  1.3. Transport of SNARE proteins 1.3.1. Alternative sorting mechanisms regulate SNARE transport SNARE proteins are critical regulators of vesicle fusion. An important concept is that SNAREs themselves must be accurately transported in the cell. To ensure that a vesicle can fuse with its target membrane, sufficient amounts of the correct v-SNARE must be incorporated into a vesicle during formation, and its cognate t-SNARE must be correctly  13  sorted to its target membrane. Following fusion, SNAREs must also be recycled back to the donor compartment in order for fusion processes to continue (Miller et al., 2007). Some SNAREs interact with directly with AP complexes through canonical motifs, including the TGN-localized VAMP4, which binds to AP-1 through a dileucine-based signal (Peden et al., 2001). Most SNAREs, however, lack the canonical motifs recognized by adaptor proteins. Recent studies have demonstrated that SNARE sorting often involves recognition by unconventional adaptors, and novel methods of cargo interaction. In both yeast and mammals, epsin family proteins interact with various SNAREs involved in the late endocytic pathway. In mammals, enthoprotin, also called epsinR (Wasiak et al., 2002) interacts with the Q- SNARES vti1b, syntaxin 7 and syntaxin 8, involved in late endosome/lysosome transport (Chidambaram et al., 2004 and 2008). The yeast epsinR homolog Ent3 binds the related SNAREs Vti1, Pep12 and Syn8, which participate in fusion with the late endosome (Chidambaram et al., 2004). The localization of Vti1 and Pep12 is dependent on Ent3 together with the partially redundant Ent5, indicating that the cargo-sorting function of Ent3 is conserved in yeast and extends to more than one SNARE (Chidambaram et al., 2008). The clathrin-mediated endocytic retrieval of VAMP7 from the plasma membrane requires the protein Hrb1 (HIV Rev-binding protein), (Pryor et al., 2008). Hrb1 is an ArfGAP, which binds both the AP-2 appendage domain and clathrin, demonstrating the properties of an alternate adaptor. For SNAREs, the transport signal recognized by adaptors can consist of a folded epitope instead of a short cytosolic motif. The ENTH domain of EpsinR interacts with the Nterminus of vti1b via a conformational motif, which includes at least two turns and one helix of epsinR and amino acid residues from three helices of vti1b (Miller et al., 2007). VAMP7 binds Hrb1 through its N-terminal longin domain, a highly structured region of 120 amino acids (Pryor et al., 2008). These studies demonstrate that mechanistically novel interactions occur between given SNARE/adaptor pairs. These specific mechanisms may ensure that there is no competition between SNAREs and standard motif-containing cargo for inclusion in a nascent vesicle. This is important, as a vesicle must include both cargo and SNAREs to be productively delivered to the target membrane (Miller et al, 2007). As the proper sorting of SNAREs is critical to the integrity of essential transport processes, there is considerable interest in uncovering how they are recognized and transported. 1.3.2. What is the role of palmitoylation in SNARE transport? Several SNARE proteins are post-translationally modified by palmitoylation, the  14  reversible addition of palmitate to a cysteine via thioesterification (Roth et al., 2006). SNAREs lacking transmembrane regions require palmitoylation for membrane anchoring (Dietrich et al., 2005; Veit et al., 1996). Many integral membrane SNAREs are also palmitoylated, but the functional consequences of these modifications are not clear (Roth et al., 2006). There is growing evidence that this could regulate the trafficking of these transmembrane SNAREs. For example, palmitoylation of the yeast SNARE Tlg1, which mediates endosome to Golgi transport, protects it from ubiquitination and subsequent degradation in the vacuole (Valdez-Taubas and Pelham, 2005). The mammalian endosomal SNARE syntaxin7 is also palmitoylated. While it normally localizes to early and late endosomes, preventing syntaxin7 palmitoylation leads to its accumulation on the cell surface (He and Linder, 2009). Several studies have highlighted regulatory effects of palmitoylation on either retention or anterograde trafficking of proteins at the ER–Golgi or protein cycling within the endosomal/lysosomal system (Linder and Deschenes, 2007; Greaves et al., 2009). Palmitoylation can also aid in the partitioning of proteins into cholesterol-rich membrane subdomains, and in changing protein orientation and thus affecting protein-protein interactions that could regulate trafficking (Linder and Deschenes, 2007). The mechanisms by which palmitoylation regulates the dynamics of SNARE transport are an important unresolved issue in the field. It remains unclear if palmitoylation directly affects the recognition of SNAREs by components of the transport machinery, and whether the consequences of this modification are generally applicable to all SNARES.  1.4. Endocytosis and endocytic recycling pathways 1.4.1. Yeast as a model for endocytic recycling The protein families that mediate vesicle trafficking are conserved from yeast to mammals, as is the compartmentalization throughout the cell from the endoplasmic reticulum to the plasma membrane. Yeast has therefore been used as a model for both genetic and biochemical analyses to study transport processes for decades (Bock et al., 2001). These studies have revealed that identifying the transport machinery and their pathway relationships in yeast can inform relationships between gene products in mammalian cells. Despite this, there do appear to be some important differences between transport in yeast and mammalian cells, especially in mechanisms regulating endocytosis  15  and recycling. The similarities and differences in these pathways, and possible explanations for these discrepancies are discussed below. 1.4.2. Do yeast and mammals share common mechanisms of endocytosis? Elegant studies in both yeast and mammalian systems have revealed an intricate network of endocytic regulatory proteins, and have used novel methods to investigate their dynamics at endocytic sites. This has revealed that most components of the endocytic machinery are highly conserved from yeast to humans. These studies also show that actin polymerization has an important role in the endocytic process (Engqvist-Goldstein and Drubin, 2003). Most progress in defining the order and timing of endocytic internalization events has resulted from real-time, live cell fluorescence microscopy. A striking feature revealed by these studies is that proteins fall into discrete groups based on their dynamic relationship to clathrin-coated structures, which is also conserved in both mammalian cells and yeast (Kaksonen et al., 2005; Perrais and Merrifield, 2005). In yeast, four protein modules cooperate to drive the distinct stages of endocytic vesicle formation, based on their dynamic localization. The early coat complex is the first to arrive which recruit cargo and establish the endocytic site. The late coat module is then recruited, followed by components of the actin polymerization machinery including the Arp2/3 complex and its activators. An actin network is formed by the Arp2/3 complex, which then drives the rapid internalization of the vesicle. Finally, the vesicle scission machinery pinches the vesicle off the membrane, and coat components are released (Kaksonen et al., 2005; Toret et al., 2008). Despite the similar timeline of recruitment of endocytic homologs in yeast and mammals, there appear to be some critical differences in their relative importance. The endocytic adaptor AP-2 is required for endocytosis in mammals, but appears to be dispensable for uptake in yeast. Targeted disruption of AP-2 is lethal in mammalian cells (Mitsunari et al., 2005), and has been shown to block the uptake of multiple cargo, including the transferrin receptor (Conner and Schmid, 2003), as well as a population of lysosome-associated membrane proteins that traffic via the plasma membrane (Janvier and Bonifacino, 2005). Yeast AP-2 deletion mutants, however, are viable, and have no defects in the internalization of commonly studied endocytic cargo, including Ste6 (Burston et al., 2009; Conibear, 2010). Actin is an essential component of the cytoskeleton, and localizes to endocytic sites in both yeast and mammalian cells (Engqvist-Goldstein and Drubin, 2003). The importance of dynamic actin regulation in endocytic vesicle formation seems to differ between yeast and  16  mammals. In yeast, actin and actin-regulatory proteins are essential for this process. Treatment with inhibitors of actin polymerization, such as latrunculin, or deletion of actinregulatory proteins blocks endocytosis completely in yeast. Actin is required to drive invagination and post-scission movement of the newly formed vesicle (Kaksonen et al., 2006). In mammalian cells, actin appears to be less important, as blocking polymerization results in only partial endocytic defects (Engqvist-Goldstein and Drubin, 2003). Finally, the GTPase dynamin has a key role in the scission of endocytic vesicles in mammalian cells. Upon GTP-binding, it self-assembles into a ring-like collar around the neck of deeply invaginated, mature coated pits. Subsequent GTP hydrolysis causes constriction of this collar, which facilitates vesicle fission. Dynamin was not previously thought to play a role in yeast endocytosis, although a recent study has suggested that it may be involved (Smaczynska-de Rooij et al., 2010). 1.4.3. A comparison of the endosomal system in yeast and mammalian cells The basic organization of the endosomal system in mammalian cells is now fairly well defined. It has functionally and physically distinct compartments, which includes early/sorting endosomes, recycling endosomes, lysosomes, and the TGN (Illustration 1.4.). The sorting endosome is the first destination for endocytosed cargo. Sorting events initiated at this compartment determine the subsequent fate of internalized proteins, directing them for either recycling to the plasma membrane, delivery to the trans-Golgi network, or for degradation. The sorting endosomes have a characteristic morphology, consisting of thin tubular extensions, and vesicular structures. These endosomes contain distinct membrane subdomains, in which cargo are concentrated for different downstream transport steps. Proteins targeted for recycling have been shown to cluster within tubules, while those destined for degradation are concentrated within the vesicular structures. There are multiple routes back to the cell surface from sorting endosomes. Molecules are delivered directly back to the plasma membrane either through an intermediate compartment called the endocytic recycling compartment, or can alternatively pass through the Golgi from sorting endosomes prior to surface redelivery. Each of these pathways has different kinetics of transport. Proteins targeted for degradation are transported from sorting endosomes, which mature into compartments called the multivesicular body (MVB), and subsequently reach lysosomes. The first step in targeting these proteins for lysosomal degradation is often the ubiquitination of their cytoplasmic domain. These ubiquitinated receptors are recognized by the ESCRT (endosomal sorting required for transport) complex  17  at the MVB, which sorts them for delivery to later compartments. The ESCRT complex invaginates the endosomal membrane to form the internal vesicles of the MVB. Fusion of the limiting membrane of the MVB with the lysosome ultimately delivers the internalized vesicles to the lumen for degradation. Most mammalian endocytic compartments can be distinguished based on specific lipid composition. Sorting and recycling endosomes contain high amounts of PI(3)P, while later compartments are enriched in PI(3,5)P2 and the lipid lysobisphosphatidic acid (LBPA). As different endocytic compartments are quite distinct in mammalian cells, most approaches used to study them are based on morphology, and there are a variety of markers available for proteins and lipids at each of these compartments, also facilitating their study (Gruenberg, 2003) Yeast cells share many conserved recycling components and mechanisms with mammalian cells (Illustration 1.4.; left). In yeast, however, the morphological distinction between different endocytic compartments is more elusive than in mammals, and LBPA has not been detected in yeast. It is not clear whether yeast has distinct populations of endosomes, or if subdomains with different lipid compositions exist on these organelles, like those in mammals (Russell et al., 2006). Due to the difficulty in discerning endosomal components in yeast, most compartments have traditionally been defined by genetic analysis, through identifying mutants that lead to the mistargeting of cargo known to transit through the endosomal system. Despite the apparent differences, some key aspects of these sorting pathways appear to be remarkably conserved. In fact, the molecular machinery involved in sorting at the MVB was first identified in yeast. Loss of the ESCRT machinery results in a readily identifiable phenotype: the accumulation of endosomal membranes resulting from the failure in the formation of MVB luminal vesicles (Raymond et al., 1992). Despite progress in identifying components of the late endocytic (MVB) pathway, the early endocytic pathway has not been as well characterized in yeast (Pelham, 2002). Several proteins have been used as markers to study these recycling pathways. For example, the pheromone-induced endocytosis of Ste3 results in its recycling back to the plasma membrane (Chen and Davis 2000, 2002), while the chitin synthase Chs3 translocates between sites of chitin deposition on the cell surface and endosomes (Ziman et al. 1998; Valdivia et al., 2002). Another marker that has been used to study endocytic recycling in yeast is the exocytic v-SNARE Snc1, which is involved in fusion of Golgi-derived secretory vesicles with  18  the plasma membrane, and recycles through early endosomes to the Golgi before redelivery to the cell surface. The localization of GFP-Snc1 has typically been used to identify genes required for its early endosomal recycling. In wild-type cells, Snc1 is localized at the plasma membrane with some punctate staining of internal structures, and has been found to either accumulate in intracellular structures or is enhanced at the plasma membrane in various recycling mutants (Lewis et al. 2000; Galan et al. 2001). Multiple proteins have been implicated in Snc1 recycling, based on the effects of deleting these proteins on the localization of GFP-Snc1. These include the sorting nexin, Snx4, which interacts specifically with the endosome-enriched PI3P (Hettema et al., 2003), and the Arf-GAP Gcs1, which binds directly to Snx4 (Robinson et al., 2006). This process also requires Rcy1, the Ypt31/32 GTPases that facilitate Golgi export and act upstream of Rcy1 (Chen et al., 2005), and components of the coatomer coat complex (Robinson et al., 2006). Importantly, the recycling of Snc1 is not blocked by loss of the MVB sorting machinery, including the ESCRT and retromer complexes, suggesting that Snc1 transits through a distinct endosomal population (Lewis et al., 2000; Schluter et al., 2008; Hettema et al., 2003). The Snc1 recycling pathway has not previously been genetically well characterized, and in most cases, the relationships between genes required for this pathway are not well understood. To understand the similarities and differences between these sorting pathways in yeast and other organisms, it will be important to develop new methods to better define these early sorting steps. It is likely that the development and application of novel technologies to study these pathways in yeast will help resolve these differences, and lead to a better understanding of endosomal sorting in eukaryotic cells.  1.5. Yeast functional genomics: a tool to dissect trafficking pathways 1.5.1. Systems biology approaches to study membrane transport The work described in this dissertation uses yeast functional genomics to study mechanisms regulating endocytic recycling. This approach identifies protein complexes and pathways that are coordinately required in a broad cellular context. By considering the requirements for endocytic recycling on a genome-wide level, we aim to elucidate basic principles that may not be apparent at the level of individual components, and to understand how individual genes and pathways are connected to carry out a common process on the level of a whole organism.  19  S. cerevisiae is an important model system for the development of large-scale genomic and proteomic technologies, due to its compact genome and relatively simple genetics. The yeast genome was the first eukaryotic genome to be successfully sequenced, and over the last decade, numerous genomic methodologies have been developed to identify functions for the roughly 5800 genes in yeast. A collection of ~4800 yeast mutant strains, comprised of systematic knock-out disruptions of all non-essential genes, is available in haploid (MATa and MATα), homozygous and heterozygous diploid strains (Giaever et al., 2002). The deletion collection has allowed for a variety of “reverse genetics” investigations, leading to the discovery of novel components in genetic pathways and protein complexes (Scherens and Goffeau, 2004). In recent years, additional genome wide collections have been developed, allowing for studies of over-expression, localization, protein-protein interactions, and essential genes (Boone et al., 2007). Finally, yeast is easy to manipulate for cell biological and biochemical studies, providing a robust system for functional genomics. Genetic interactions, which represent the modulation of the phenotype of one mutation by the presence of a second mutation, have long been used as a tool to dissect the functional relationships among sets of genes (Guarente, 1993; Kaiser and Schekman, 1990). Classically, researchers have looked for strong qualitative differences between observed phenotypes of double mutants and the phenotypes of the two related single mutants. For example, a relationship referred to as synthetic lethality is observed when two mutations are not lethal when present individually but, when combined, result in an inviable organism. Synthetic sick/ lethal, or negative, interactions have been used as evidence that two genes act in independent but complementary pathways. Strong negative genetic interactions, in which the double mutant phenotype is stronger than the phenotype of either single mutant, often suggests that these genes work in parallel/redundant pathways. On the other hand, a positive interaction, in which a normally deleterious mutation has no additive effect in the context of a second mutation, often identifies genes that act in the same pathway or complex. Investigating these interactions has provided a wealth of data about functional relationships between genes in yeast (Collins et al., 2010). In recent years, technology has developed allowing genetic interactions to be evaluated on a large scale. Genes that are closely related in function often share highly similar patterns of genetic interactions. Thus, the pattern of genetic interactions of each mutant can be considered as a phenotypic signature. Genes that share similar signatures can be identified and grouped, a useful method for identifying functionally related genes (Schuldiner et al., 2005; Tong et al.,  20  2004). One problem with this approach is that with 6000 genes in the yeast genome, a complete genetic interaction map requires accurately evaluating 18 million double mutant phenotypes, which is beyond the capacity of most studies. The E-MAP (Epistatic miniarray profiling) approach provides a shortcut, in which pairwise interactions are measured among a rationally chosen subset of genes, predicted to to have a shared function in a given process (Schuldiner et al., 2005; Collins et al., 2010). The subset of genes can be selected based on multiple criteria, including shared protein localization (Schuldiner et al., 2005), or protein-protein interaction data (Collins et al., 2007). 1.5.2. Phenotypic screening in yeast to identify transport components Genetic approaches are a well-established, commonly used approach to evaluate gene function for a wide variety of cellular pathways. In yeast, genetic screens have been immensely successful for discovering genes involved in the secretory, Golgi, endosomal and vacuolar transport pathways (Raymond et al., 1996; Novick et al., 1980; Bankaitis et al., 1996). Characterization of mutants discovered in these screens has revealed roles in cargo sorting, vesicle budding, and tethering/fusion, emphasizing the power of this approach to discover genes with direct roles in trafficking. In recent years, genetic screens have been developed which focus on phenotypes directly related to trafficking. This involves detection of missorting of specific cargo proteins within different transport pathways in the genome-wide deletion collection, allowing for systematic identification of the pathway requirements. Many genes, for example, have been identified that are required for the sorting of the CPY receptor Vps10, between the Golgi and late endosome through these phenotypic screening methods. While in the Golgi, Vps10 binds newly synthesized pro-CPY, and together, Vps10 and pro-CPY are transported to the late endosome. At the late endosome, CPY dissociates from Vps10, and is delivered to the vacuole, where it matures and functions. In the absence of Vps10, or other factors required for transport of pro-CPY, this pathway is blocked, resulting in the missorting of pro-CPY to the extra-cellular space. CPY secretion can be detected based on a colony overlay assay, in which yeast colonies are grown on a nitrocellulose membrane, and secreted pro-CPY is detected by western blotting. Identifying mutants in the genome-wide collection has that lead to pro-CPY secretion have been a well-used screening approach to identify genes involved in Golgi/vacuolar transport (Bonangelino et al., 2002; Schluter et al., 2008). Despite the progress in yeast functional genomics, over 20% of all yeast genes remain functionally uncharacterized (Peña-Castillo and Hughes, 2007). Furthermore, many  21  yeast homologs of mammalian genes known to be involved in characterized transport steps have not yet been shown to be functionally important. A prime example is the discrepancy between the requirement for many yeast and mammalian endocytosis genes, as previously described (Conibear, 2010). One explanation for this discrepancy is that deletion of many of these genes may lead to a weak phenotype in yeast that is undetectable by standard assays of transport defects. This issue necessitates the development of novel quantitative strategies, which are sensitive enough to detect these mild defects.  1.6. Research objectives and hypotheses 1.6.1. A new approach for identifying endocytic recycling components in yeast The endocytic recycling pathway of Snc1 appears to involve many genes not required for the transport of other recycling cargo (Kama et al., 2007; Robinson et al., 2006). It is therefore expected that developing a functional genomics approach to dissect the Snc1 recycling pathway in yeast could identify previously unrecognized endocytic recycling factors, and generate insight into their functional relationships. Snc1 was selected as a reporter for endocytic recycling due to the similarity of its sorting pathway to that of mammalian VAMP2/synaptobrevin. In mammalian neuronal cells, VAMP2 mediates the fusion of synaptic vesicles with the presynaptic membrane, and is required for neurotransmitter release. Its recycling allows continued rounds of synaptic vesicle fusion, and sustained neurotransmission (Grote et al., 1995). As defects in pathways of synaptic vesicle formation and recycling are prominent in a variety of neurological disorders, systematically characterizing the genes required for its sorting pathway will aid in the identification of causal variants in human disease, and ultimately will contribute to the development of drugs that are targeted to correct the resulting defects. The work described in this dissertation uses a genome-wide phenotypic screen to identify components involved in Snc1 endocytic recycling based on quantitative genomewide techniques to measure the localization of the Snc1 in the genome-wide deletion collection. In this study, a reporter for Snc1 recycling was created by fusing the sucrose converting enzyme invertase, encoded by the gene SUC2, to the C-terminus of GFP-Snc1. The invertase portion is exposed to the extracellular space, allowing detection of plasma membrane localization by whole colony assays of invertase activity. The identification of transport defects in the genome-wide collection relies on densitometry measurements of cell surface invertase activity in each mutant. This provides a sensitive and quantitative  22  assay, which is compatible with high-throughput approach. The following chapters are based on our genome-wide phenotypic assay for increased Snc1 surface levels. The goal of this screen was to identify genes regulating Snc1 endocytosis and recycling in a systematic and non-biased manner. Due to its sensitive and quantitative nature, we anticipated that it would reveal functions for genes that were previously uncharacterized due to weak mutant phenotypes or cargo-specific functions. By evaluating the transport pathway of Snc1, we also aimed to address the mechanisms regulating the sorting of SNARE proteins, one of the least understood issues in the field. We anticipated that we would identify novel factors required for Snc1 sorting, including cargo-specific adaptor proteins and regulators that are not required for the uptake or recycling of other proteins. Chapter 2 is focused on genes required for the endocytosis of Snc1 from the plasma membrane, and the downstream analysis of novel components regulating this process. Chapter 3 is an extension of the results of the primary screen, and investigates the function of the previously uncharacterized clathrin adaptor AP1R and its interacting protein Ima1 in Snc1 endosomal recycling.  23  1.7. Illustrations  Illustration 1.1. Intracellular compartments and transport pathways. Following synthesis in the endoplasmic reticulum (ER), proteins are transported to the Golgi (1). The last compartment of the Golgi (the trans-Golgi network) sorts cargo for delivery to multiple possible destinations. From the TGN, proteins move by anterograde transport to the plasma membrane (2), or are diverted to endosomes (3), or to the vacuole for targeted degradation (4). Following cell surface delivery, some proteins are retrieved through endocytosis (5), and are recycled back to the surface via the endosomes and Golgi (Endocytic recycling, (6)).  24  Illustration 1.2. Steps in clathrin-coated vesicle formation and transport. Vesicle formation is initiated when adaptor proteins are recruited to the donor membrane and bind transmembrane cargo (1). Adaptor/cargo complexes then recruit components of the clathrin coat, leading to its polymerization. These include BAR domain proteins, which promote the membrane curvature required for budding (2). The vesicle is internalized, and pinches off of the target membrane (3). Adaptors and other components are subsequently removed from the vesicle (4). Finally, the uncoated vesicle fuses with the target membrane, delivering its cargo (5).  25  Accessory proteins   Appendage  b Appendage  Accessory proteins Clathrin  Cargo D/ExxLL Membrane phospholipids   b    Core   Cargo (Yxx) Membrane phospholipids  Illustration 1.3. Clathrin Adaptor (AP) structure Structure of the heterotetrameric AP-1 complex. The complex consists of two large subunits (γ and β1), one medium subunit (μ1), and one small subunit (σ1). The large subunits are divided into a core domain, which assembles with the medium and small subunits, and appendage domains, connected to the core by flexible hinges. Each subunit interacts with a different subset of transport components, as indicated.  Illustration 1.4. Comparison of endocytic recycling compartments and pathways in yeast and mammalian cells. Following endocytosis, yeast cell surface proteins are transported the the early endosome (EE), from which they can recycle back to the plasma membrane directly, or by passing throught the TGN. Alternatively, they can be targeted to the vacuole for degradation through transport to the late endosome (LE). In mammalian cells, internalized proteins first reach the sorting endosome (SE), from which they can take a variety of routes back to the surface. They are either recycled directly back from early endosomes (rapid recycling), or can pass through the intermediate endocytic recycling compartment (ERC, slow recycling). They can also recycle by passing through the TGN from the endocytic recycling compartment. Proteins targeted for degradation are transported through late endosomes (LE), to the lysosome, the mammalian equivalent of the yeast vacuole.  26  CHAPTER 2. REGULATORS OF YEAST ENDOCYTOSIS IDENTIFIED BY SYSTEMATIC QUANTITATIVE ANALYSIS. 1 I II  A version of this chapter has been published as: Burston HE, Maldonado-Baez L, Davey M, Montpetit B, Schluter C, Wendland B, and Conibear E. (2008) Regulators of yeast endocytosis identified by systematic quantitative analysis. Journal of Cell Biology. 185 (6): 1097-1111. 1  27  2.1. Synopsis This chapter describes the screening approach used to identify genes involved in the Snc1 endocytic recycling pathway, and focuses on genes that were identified to play a role in yeast endocytosis, and the relationships between them. The main goal of this study was to explain why internalization defects have not been shown for many yeast homologs of mammalian endocytosis genes. This is a question that has puzzled many in the field, leading to the hypothesis that internalization in yeast and mammals utilizes fundamentally different mechanisms. Multiple explanations have been advanced to account for these differences. First, deletion of yeast endocytic homologs may have internalization defects that have not yet been detected, due to weak defects that cannot be measured by standard assays, which may be a result of functional redundancy. Second, many of these genes may be important only for a subset of endocytic cargo. To overcome these limitations, a quantitative approach was developed to systematically assess endocytic defects across the genome, using a novel enzymatic reporter based on Snc1. This revealed roles for many of these yeast endocytic homologs, as well as other previously uncharacterized genes. Genetic interaction mapping was used to place these genes into functional modules containing known and novel endocytic regulators. Hypothesis-driven downstream analysis was then used to functionally characterize novel regulators. In addition, an array-based comparison of two different reporters containing distinct internalization signals was carried out in order to evaluate cargo specificity on a large scale. This study demonstrates that clathrin and the yeast AP180 clathrin adaptor proteins have a cargo-specific role in Snc1 internalization. Additionally, low dye binding 17 (LDB17) is shown to be a novel conserved component of the endocytic machinery. Ldb17 is recruited to cortical actin patches before actin polymerization and regulates normal coat dynamics and actin assembly. Collectively, these findings highlight the conserved machinery and reveal novel mechanisms that underlie endocytic internalization. They also illustrate that parallel mechanisms regulate the internalization of Snc1 in yeast and mammalian cells.  2.2. Introduction Clathrin-mediated endocytosis selects proteins at the plasma membrane for internalization in membrane-bound vesicles. In mammalian cells, cargo is initially concentrated at endocytic sites by adaptor proteins that promote clathrin recruitment and assembly. A variety of adaptors, such AP2, AP180/CALM, epsins, arrestins and Dab2, recognize different endocytic motifs and select distinct classes of cargo (Maldonado-Baez  28  and Wendland, 2006). The subsequent recruitment of accessory factors including WASP, dynamin, and amphiphysin stimulates actin polymerization, resulting in membrane deformation and vesicle scission (Smythe and Ayscough, 2006). Studies in yeast have demonstrated conserved endocytic modules are recruited to sites of internalization in a similar temporal sequence (Kaksonen et al., 2005; Newpher et al., 2005; Perrais and Merrifield, 2005). Clathrin and adaptors are part of the early coat module that establishes the initial site of uptake, while the late coat module (Sla1/Sla2/End3/Pan1) couples coat formation to actin polymerization through recruitment and stimulation of yeast WASP (Las17) and Myo5. This activates the Arp2/3 complex to form an actin network that, stabilized by the actin regulatory module (Cap1/2, Sac6, Abp1), promotes rapid inward movement of the internalizing vesicle. Finally, the amphiphysins Rvs161/167 are thought to drive scission of endocytic vesicles from the plasma membrane. Recycling of the endocytic machinery by vesicle uncoating requires additional proteins, including the synaptojanin homolog Inp52. This sequence of events, defined by live cell imaging studies, suggests endocytic processes are largely conserved. However, striking differences in the functional requirement for certain regulatory proteins argue that internalization may be differentially regulated in yeast and mammalian cells. For example, the dynamic regulation of actin polymerization is essential for uptake in yeast but not in most mammalian cell types (Engqvist-Goldstein and Drubin, 2003; Smythe and Ayscough, 2006). Conversely, clathrin has an important role in higher cells yet loss of yeast clathrin causes only partial reduction in receptor internalization, and yeast homologs of major clathrin adaptor proteins such as AP2 and AP180 are not required for the uptake of known cargo (Huang et al., 1999; Wendland and Emr, 1998). A number of other yeast genes are homologous to components of the mammalian endocytic machinery, yet do not lead to observable endocytosis defects when mutated (Engqvist-Goldstein and Drubin, 2003; Kaksonen et al., 2005). Some of these factors may have functionally redundant homologs, or have cargo-specific roles. Additional, unrecognized factors may also regulate cargo uptake in yeast. To better understand the endocytic process in both yeast and mammalian cells, it will be important to identify the complete set of structural and regulatory proteins, and systematically define their specific roles. The yeast VAMP2/synaptobrevin homolog Snc1, which regulates the fusion of exocytic vesicles at the cell surface, is a widely studied endocytic cargo protein. After  29  delivery to the plasma membrane of the growing bud, it is rapidly internalized and transported to endosomal and Golgi compartments, where it is incorporated into new secretory vesicles (Lewis et al., 2000). Here, we perform a genome-wide analysis of Snc1 localization in yeast to uncover genes with functional roles in endocytosis, and quantify their relative contribution to this process. We identify functions for known and novel proteins not previously shown to be required for uptake, and demonstrate that the yeast AP180 homologs and clathrin have a cargo-specific role in Snc1 internalization. In addition, we describe an endocytic function for the previously uncharacterized protein Ldb17.  2.3. Results 2.3.1. A Snc1-based quantitative assay for endocytic recycling To quantify defects in endocytosis on a genome-wide scale, we developed an enzymatic assay based on the cell surface localization of a Snc1 reporter. The enzyme invertase (encoded by SUC2) was fused to the extracellular C-terminus of Snc1, and GFP was appended to its intracellular N-terminus, creating the chimeric protein GSS (GFP-Snc1-Suc2; Fig. 2.1 A). Cell surface GSS levels can be determined by a colorimetric invertase activity assay, using cell-impermeant reagents (Darsow et al., 2000). By fluorescence microscopy, GSS displayed a polarized distribution at the plasma membrane of small buds similar to Snc1 (Fig. 2.1 B; Lewis et al., 2000), which has been attributed to a cycle of localized exocytosis followed by rapid endocytosis that prevents diffusion away from the site of new growth (Valdez-Taubas and Pelham, 2003). In support of this model, when the Snc1 endocytosis signal was mutated, the polarization of GSS was lost and the level of invertase activity at the plasma membrane increased (Fig. 2.1 B and data not shown). Both Snc1 and GSS were mislocalized to the vacuole in vps51∆ mutants (data not shown), demonstrating that they follow a similar recycling pathway back to the cell surface (Conibear et al., 2003). We introduced the GSS reporter into two independent genome-wide collections of viable deletion mutants, and measured its cell surface level for each mutant in parallel, using a large-scale plate-based invertase assay (Fig. 2.1 C). Mutants were ranked according to their median invertase activity score, determined by automated image densitometry (Fig. 2.1 D and Table A2). Mutants with increased cell-surface GSS included well-characterized endocytosis-defective strains such as rvs161∆ and vrp1∆. Other predicted endocytosis genes were enriched in the top 400 hits (Table 2.1), including many not previously shown to cause defects in cargo uptake when deleted such as yeast homologs of AP180 (yap1801  30  and yap1802), epsin (ent1), tropomyosin (tpm1), type I myosin (myo5), synaptojanin (inp52), coronin (crn1), and syndapin (bzz1). We generated a “gold standard” list of 33 proteins that have been localized to sites of endocytosis by time-lapse live cell microscopy that were also represented in the haploid deletion collections we evaluated (Newpher et al, 2005; Maldonado-Baez et al., 2008; Toret et al., 2008; D. Drubin, personal communication). Of these, 23 were found in the top 400 hits, representing a significant degree of enrichment (p < 2.2 x 10-16, Fisher exact test). This set of ~400 top-scoring mutants was chosen for further analysis. Mutants with cell-surface GSS levels significantly lower than wildtype (e.g., snx4∆, vps51∆) were also identified in the screen. This class of mutants, which may have impaired endosomal recycling, will be described in more detail elsewhere. 2.3.2. Genetic interaction mapping clusters functionally related genes The large number of mutants with elevated surface invertase activity suggested many distinct processes influence GSS reporter distribution. To functionally dissect these processes, we analyzed GSS surface levels in 374 top-scoring deletion mutants in pairwise combination with a panel of 81 deletion mutants that affect a variety of protein trafficking pathways. Genes that co-function within individual complexes and pathways can be identified in large scale genetic interaction analyses based on two characteristic properties (Schuldiner et al., 2006). When loss of one gene disrupts a pathway or complex, loss of a second gene in the same pathway has no further effect, and the double mutant phenotype is less than would be expected under an additive model of genetic interaction. In addition, genes with related functions share similar patterns of genetic interactions and can be identified by cluster analysis. In typical applications, such as synthetic lethal analysis, the double mutant array is analyzed using cell viability as the phenotypic readout. Here, we quantified the GSS surface distribution to analyze the ~33,000 double mutants of our 374 x 81 array. Hierarchical clustering grouped genes according to their pattern of genetic interactions (Fig. 2.2 A and Fig. A1, see Materials and Methods for details). Strikingly, many clusters were enriched for genes that act together (Fig. 2.2 B). Endocytosis genes were found in two distinct clusters, whereas other gene groupings corresponded to distinct processes (see below). We observed that mutants deleted for two different endocytic genes (eg. vrp1 and inp52) often had lower cell surface levels of GSS than would be expected under an additive model of genetic interaction, consistent with a function in the same pathway or complex. In contrast, mutants disrupted for both endocytosis (vrp1, rvs167) and recycling (snx4, cvt20/snx42)  31  genes had cell surface GSS levels greater than expected under the additive null model. This is consistent with previous observations that endocytic defects are epistatic to defects in the Snx4-dependent recycling of Snc1 (Lewis et al., 2000). 2.3.3. Integration of genetic and physical interaction data identifies complexes required for Snc1 transport To comprehensively identify pathways that regulate Snc1 distribution, we integrated our genetic interaction results with genome-wide physical interaction datasets (see Materials and Methods for details). In a network analysis, genes are represented as “nodes” (circles) linked by “edges” (lines) that indicate an experimental observation such as a physical interaction. To generate a network from the genetic interaction data, pairwise correlation scores were calculated for all genes in the array, and genes were linked by an edge if their correlation score was greater than a threshold value. The resulting network connects genes that share similar patterns of genetic interaction, and thus may represent components of pathways and complexes. Physical interaction networks were then created for the same set of genes, where edges represent interactions observed in high throughput yeast two-hybrid or mass spectrometry experiments. Highly connected sets of genes in each network were identified and integrated, and significant gene clusters were assigned to subcellular compartments based on the localization of their constituent proteins (Huh et al., 2003). The resulting map (Fig. 2.3) provides a visual representation of the processes contributing to GSS localization and an indication of their relative importance. The size of each node is proportional to the phenotype of the corresponding deletion mutant, whereas edges represent either a physical interaction or a genetic correlation extracted from genome-wide data. Four major clusters, containing a total of 20 genes, mapped to the plasma membrane. The largest of these groupings was enriched for genes with known or predicted roles in endocytosis (SLA1, ABP1, INP52, EDE1, YAP1802, RVS161, RVS167, CRN1, and the INP52-overlapping ORF YNL105W). Additional genes implicated in endocytosis or actin regulation were found in three other clusters at the plasma membrane. These findings highlight the importance of known endocytic regulators in Snc1 trafficking, and predict an endocytic role for uncharacterized genes in these clusters, such as LDB17 (see below). A number of well-characterized protein complexes that function at intracellular organelles or regulate transcription or translation were also identified by our integrative network analysis (Fig. 2.3). Some have established roles in actin assembly or protein  32  transport, such as the cytosolic Prefoldin/GimC complex, which acts as a co-chaperone in actin folding (Siegers et al., 1999). ESCRT mutants are known to exhibit increased cell surface levels of receptors, which has been attributed to enhanced recycling from endosomal compartments (Davis et al., 1993). Chromatin remodeling complexes have also been implicated in regulating vesicle transport at endosomes (Schluter et al., 2008), although the mechanism remains unclear. Mutant strains lacking components of the Sin3/Rpd3 histone deacetylation complex, including Dep1 and Ume6, had particularly strong phenotypes. These proteins regulate the expression of phospholipid biosynthesis genes such as OPI3, INO1, and CHO2 (Elkhaimi et al., 2000; Lamping et al., 1994), which were among the top hits in our screen (Table S1). The regulation of plasma membrane lipid composition may also explain the identification of RIM pathway components, which mediate the cellular response to lipid asymmetry and other stimuli (Ikeda et al., 2008). 2.3.4. Clathrin and clathrin assembly proteins are required for Snc1 endocytosis Our network analysis suggested 20 genes, in four major clusters, are involved in Snc1 uptake at the plasma membrane. To confirm the results of the large-scale plate-based assay, cell-surface GSS levels in these 20 mutants were quantified by a liquid-based invertase activity assay (Fig. 2.4 A). Most had elevated GSS levels that correlated well with values derived from the large-scale densitometry data. The significant internalization defect of yap1802 mutants was surprising, as previous studies have not identified a clear role for AP180 homologs in yeast (Huang et al., 1999; Wendland et al., 1999). Yap1802 shares 43% sequence identity with Yap1801, which was also recovered by the genome-wide screen (Table A2). By microscopy, the polarized distribution of Snc1 at the bud was largely maintained in yap1801 or yap1802 single mutants, but Snc1 was evenly distributed at the cell surface of yap1801 yap1802 double mutants (Fig. 2.4 B). This strong defect in internalization is consistent with a functionally redundant role for the yeast AP180 proteins in Snc1 uptake. In mammalian cells, the ubiquitous AP180 homolog CALM participates in clathrinmediated endocytosis, and interacts with the AP-2 adaptor complex (Meyerholz et al., 2005). Deletion of clathrin heavy or light chain (CHC1 or CLC1), but not AP-2 (APL1), also caused a strong Snc1 localization defect (Fig. 2.4 B and data not shown). Surprisingly, no defect was seen when the major clathrin-binding site in Yap1802 (Wendland and Emr, 1998) was deleted in a strain lacking Yap1801 (Fig. 2.5 A), suggesting that Yap1802  33  contains additional clathrin binding sites, or that additional proteins can bridge the interaction. In contrast to Snc1 endocytosis, loss of Yap1801 and Yap1802 singly or in combination does not alter ligand-induced down-regulation of the a-factor/Ste2 complex (Huang et al., 1999). We found loss of Yap1801/2 had little effect on the internalization of a distinct endocytic cargo, Ste6-GFP (Fig. 2.5 B). Defects may be more apparent for Snc1, which depends on continuous rounds of internalization and recycling to maintain its polarized distribution, compared to Ste6, which is delivered to the vacuole following internalization (Kelm et al., 2004). To determine if Yap1801/2 are generally required for the uptake of proteins subject to endocytic recycling, we appended the well-characterized Sla1-dependent NPFxD endocytosis signal to the t-SNARE GFP-Sso1 to create a different recycling reporter, NPF-Sso1 (Valdez-Taubas and Pelham, 2003). Like Snc1, NPF-Sso1 has a polarized distribution that depends on endocytosis. Consistent with previous results, we found the localization of NPF-Sso1 to be similar to that of Snc1 in wild type cells, but completely depolarized in sla1 mutants (Fig. 2.4 B). Its polarized distribution was maintained in yap1801 yap1802 double mutants, suggesting Yap1801/2 are specifically required for Snc1 internalization. We created a chimeric version of the NPF-Sso1 reporter by fusing invertase to the NPF-GFP-Sso1 C-terminus and adding two additional NPFxD motifs to further enhance internalization (Fig. 2.4 C), and measured cell surface levels of the resulting NGSS (3xNPFGFP-Sso1-Suc2) and GSS reporters (Fig. 2.4 D). Internalization of GSS was blocked by mutation of the Snc1 endocytic signal, whereas NPF-dependent internalization of NGSS was abolished in a sla1 mutant, as expected. Combined deletion of YAP1801 and YAP1802 dramatically increased levels of GSS at the cell surface, whereas NGSS surface levels were unaffected. Taken together, these results indicate Yap1801 and Yap1802 act as cargospecific adaptors for the clathrin-mediated endocytosis of Snc1. 2.3.5. Systematic analysis of cargo specificity To determine if other genes identified in our genome-wide screen have cargospecific functions, we re-introduced the GSS and NGSS reporters in parallel into our array of top-scoring deletion mutants. Invertase activity values for each mutant, normalized to the array median, were determined from 6 independent mutant arrays per reporter and converted into relative sorting scores. The ranked differences between NGSS and GSS sorting scores for each mutant indicated that deletion of YAP1802 and INP52 had the  34  greatest specific effect on GSS (Fig. 2.6 A). By measuring cell surface reporter levels in liquid culture (Fig. 2.6 B), we confirmed inp52 mutants (rank=2) exhibited a highly significant degree of cargo specificity whereas a lower-ranking mutant (ldb17) that fell below the 2SD cutoff did not. A cargo-specific role for the synaptojanin homolog Inp52, a PI(4,5)P2 phosphatase required for recycling of coat proteins after vesicle budding is complete (Toret et al., 2008), was unexpected. Because the membrane association of Yap1801/2 is mediated by PI(4,5)P2-binding ANTH domains, we considered the possibility that elevated PI(4,5)P2 levels in inp52 mutants lead to retention of Yap1801/2 on budded vesicles. Consistent with this hypothesis, Yap1801/2-GFP were observed on cytosolic patches inp52 mutants, but not in wild type cells (Fig. 2.6 C). Likewise, loss of Inp52 resulted in cytoplasmic aggregates of the ANTH domain protein Sla2-GFP, but had no effect on Sla1-GFP localization, as previously reported (Fig. 2.6 C, (Toret et al., 2008)). Prolonged association of Yap1801/2 with budded vesicles would be expected to reduce levels available for GSS internalization, which may explain the apparent cargo specificity of the inp52 deletion. 2.3.6. Ldb17 is a new regulator of yeast endocytosis that is transiently recruited to cortical patches Mutation of the uncharacterized gene LDB17 (Low Dye Binding 17; Corbacho et al., 2005) caused a strong GSS localization defect (Fig 2.3 and Fig 2.4 A) that, in contrast to yap1801 yap1802 and inp52 mutants, was not strongly cargo-specific (Fig. 2.6 B). We confirmed both Snc1 and NPF-Sso1 lost their polarized distributions in ldb17 mutants (Fig. 2.6 D), and a weak internalization defect was also observed for Ste6-GFP (Fig. 2.5 B), suggesting Ldb17 is a general component of the endocytic machinery. Although an endocytic role for Ldb17 has not previously been described, it has a PTHR13357 domain also found in the mammalian protein SPIN90 (Fig. 2.6 E), which has been implicated in clathrin-mediated endocytosis in fibroblasts (Kim et al., 2007; Kim et al., 2006). Most organisms, including yeast and human, contain a single PTHR13357 family member, indicating Ldb17 and SPIN90 may have orthologous functions. To help define the role of Ldb17 in endocytosis, we analyzed its recruitment to sites of internalization using two-color time-lapse fluorescence microscopy. Although difficult to visualize due to its low abundance (Ghaemmaghami et al., 2003), GFP-tagged Ldb17 was observed in faint puncta at the cell surface where it showed partial overlap with mRFPtagged forms of the late coat component Sla1, the Type I myosin Myo5 and the F-actin-  35  binding protein Abp1 (Fig. 2.7 A-C), consistent with transient co-localization. Kymograph analysis showed Ldb17 joined pre-existing Sla1 patches just prior to the onset of Sla1 inward movement and Myo5 recruitment (Fig. A2 A-C). Myo5 patches appeared ~1-3 s after Ldb17 patches, whereas Ldb17 dissociated from the membrane ~4-6 s prior to Abp1 disassembly. These results allow us to place Ldb17 on the temporal map of the endocytic process (Fig. 2.7 D): Ldb17 is maximally recruited after assembly of the late coat components, immediately prior to the recruitment of the Myosin module, which precedes the actin-driven inward movement of the invaginating membrane. The coat module assembles at endocytic sites prior to the onset of actin polymerization, and its components are recruited before the appearance of F-actin. Treatment with the actin monomer-sequestering drug Latrunculin A (LatA) resulted in the expected complete delocalization of Abp1, whereas the membrane localization of Ldb17 was dramatically enhanced (Fig. 2.7 E). The endocytic coat components Sla1 and Sla2 show similar LatA-resistant cortical localization (Ayscough et al., 1997). This indicates actin is not required for the membrane recruitment of Ldb17, and is consistent with a role for Ldb17 at late stages of coat assembly. 2.3.7. Sla1 contributes to membrane recruitment of Ldb17 The F-actin-independent localization of Ldb17 suggests other factors regulate its recruitment to sites of endocytosis. We found the C-terminal Proline-Rich Domain (PRD) of Ldb17 was important for its localization at the cell periphery (Figs. A2 D, left panels, and 2.6 F). Many endocytic proteins contain SH3 domains that recognize specific PRD sequences and contribute to the assembly of the endocytic network. We confirmed by coimmunoprecipitation that the yeast syndapin homolog Bzz1 binds Ldb17 in a PRDdependent manner (Fig. A2 E), as predicted in a large-scale study (Tong et al., 2002). Surprisingly, membrane localization of Ldb17-GFP was enhanced in bzz1 mutants (Figs. A2 D and 6 F) and simultaneous loss of Bzz1 and the Ldb17 PRD had additive effects on cell surface GSS levels (Fig. A2 F), indicating Bzz1-PRD interactions are not involved in Ldb17 recruitment. To identify additional PRD binding partners, we used yeast two-hybrid analysis to screen a panel of 16 SH3 domains from proteins implicated in endocytosis or actin regulation (Tong et al., 2002). SH3 domains from Sla1, Lsb3, and Lsb4 all exhibited PRDdependent interactions with Ldb17 (Fig. A2 G and H). Because Lsb3 and Lsb4 are known binding partners of Sla1 (Dewar et al., 2002; Gavin et al., 2006), their interactions with  36  Ldb17 may be indirect. Interestingly, loss of Sla1 had an effect similar to loss of the PRD; by live-cell microscopy, the lifetime of Ldb17 at the cell surface was reduced from 9 sec to 5 sec (Fig. 2.7 F). Taken together, these results suggest Sla1 binds the Ldb17 PRD to enhance its membrane association at endocytic sites, whereas Bzz1 is important for its release. 2.3.8. Aberrant actin distribution in LDB17 mutants Based on the timing of its recruitment, we hypothesized Ldb17 may link coat formation to actin polymerization, as has been suggested for other components of the late coat module. Consistent with this, the actin module protein Abp1 was found in a smaller number of larger, brighter clusters in ldb17 cells compared to wildtype cells (Fig. 2.7 G), similar to the large actin clumps found in sla1 mutants (Kaksonen et al., 2005). Actin polymerization is required for internalization of the Sla1-containing late coat module; in cells treated with LatA or lacking the Arp2/3 activator Las17, Sla1 patches remain immobile at the cell surface (Ayscough et al., 1997; Sun et al., 2006). Sla1 was similarly stabilized at the membrane in the absence of Ldb17 (Fig. 2.7 H), suggesting Ldb17 plays a role in the dynamic regulation of actin polymerization required for the internalization of Sla1containing endocytic vesicles.  2.4. Discussion We have developed a genome-wide screening method that provides a systematic and quantitative analysis of genes required for the endocytic recycling of Snc1, the yeast VAMP2 homolog. In addition to well-established endocytic regulators, this screen uncovered phenotypes for deletion mutants of predicted endocytosis genes for which no cargo uptake defects had previously been observed. The mammalian homologs of many of these genes also have endocytic roles, underscoring the conserved nature of endocytic processes in eukaryotic cells (Engqvist-Goldstein and Drubin, 2003; Kaksonen et al., 2005; Smythe and Ayscough, 2006). Collectively, our results highlight the sensitive nature of our reporter assay and reinforce the importance of dynamic actin regulation for the endocytic process. Multiple regulators of the actin cytoskeleton were identified in our screen, including the actin binding proteins Abp1 and Crn1 (coronin homolog), the Actin Capping complex (Cap1/2), and the Cap1/2 binding protein twinfilin (Twf1). Lack of sufficiently sensitive assays and study of limited cargo may have prevented detection of endocytic phenotypes for these mutants in previous studies (Kubler and Riezman, 1993). In addition, redundancy of the  37  endocytic machinery may account for the weak but significant phenotypes resulting from loss of individual myosin-I (Myo5) and epsin (Ent1) isoforms (Lechler et al., 2000; Maldonado-Baez and Wendland, 2006; Wendland et al., 1999). 2.4.1. Role for clathrin adaptors in yeast endocytosis The severe defect in Snc1 uptake caused by loss of Yap1801 and Yap1802, homologs of the mammalian clathrin assembly factor AP180/CALM, represents the first demonstration of a unique role for these proteins in yeast endocytosis. Studies in mammalian cells, worms, and flies all support a requirement for AP180 and its homologs in the internalization of VAMP/Synaptobrevin. It mediates the selective uptake of this SNARE in worms, and appears to regulate the internalization of additional proteins in flies (Bao et al., 2005; Nonet et al., 1999). AP180-like proteins may cooperate with other adaptors for the uptake of certain cargo. For example, a redundant role with the epsins in the internalization of the yeast pheromone receptor Ste3 was recently reported (Maldonado-Baez et al., 2008). AP180/CALM contains multiple binding sites for AP2, Eps15, and clathrin (Maldonado-Baez and Wendland, 2006; Wendland and Emr, 1998). A quantitative analysis of synaptobrevin localization in selected C. elegans mutants using the pH-sensitive synaptobrevin-GFP fusion protein synaptopHluorin demonstrated its surface levels were enhanced in both AP180 and AP2 mutants (Dittman and Kaplan, 2006), suggesting the interaction between AP180 and AP2 is functionally important in worms. In contrast, our results show Yap1801/2 does not require AP2 to mediate Snc1 internalization in yeast, consistent with a recent study that saw little effect of mutating the AP2 binding site of CALM on synaptobrevin uptake in mammalian cells (Harel et al., 2008). The specific requirement for Yap1801/2 in Snc1 endocytosis suggests they may act as adaptors to recruit Snc1 into a clathrin-coated pit. Internalization signals recognized by AP180-like monomeric adaptors have yet to be described (Maldonado-Baez and Wendland, 2006) and, like most SNAREs, Snc1 lacks canonical adaptor sorting motifs such as YxxF or D/ExxxL/E. Instead, SNARE proteins appear to use specialized sorting signals that differ from those of other cellular cargo (Chidambaram et al., 2004; Miller et al., 2007). The V40A M43A mutations that reduce Snc1 endocytosis could impair a Yap1801/2 recognition site; however, this remains to be determined, as we have not been able to demonstrate a direct physical interaction between Yap1801/2 and Snc1.  38  2.4.2. Identification of novel components of the yeast endocytic machinery Our systematic analysis of cargo specificity did not identify additional adaptor proteins that were clearly specific for Snc1. Instead, we discovered loss of the syntaptojanin homolog Inp52 affected uptake of the Snc1 reporter more strongly than a reporter that uses a Sla1-dependent NPFxD signal. This can be explained by the differential recognition of PI(4,5)P2 by Yap1801/2 and Sla1. High levels of PI(4,5)P2 resulting from loss of Inp52 prevented efficient recycling of Yap1801/2, which is predicted to reduce its availability for further rounds of internalization. However, no such effect was seen for Sla1, consistent with previous reports (Toret et al., 2008). In contrast to the cargo-specific role of Yap1801/2, we found the previously uncharacterized gene LDB17 plays a broad role in regulating endocytosis. Ldb17 is transiently recruited to Sla1-containing cortical patches shortly before the onset of actin polymerization, and its membrane association is stabilized by interactions between its PRD and the Sla1 SH3 domain. The aberrant actin assembly and increased lifetime of Sla1 patches in ldb17 mutants suggest Ldb17 helps trigger the dynamic actin rearrangements that precede coat disassembly. Ldb17 may link coat assembly to the activation of the Arp2/3 complex through interactions with Sla1 and Arp2/3 activators, or act more directly. The C-terminal region of SPIN90 most similar to Ldb17 is sufficient to promote the WASP-independent activation of Arp2/3-mediated actin polymerization in vitro (Kim et al., 2007). The Arp2/3 and actin binding motifs present in SPIN90 are only partially conserved in Ldb17, and we did not observe binding of Ldb17 to the Arp2/3 complex and the yeast WASP homolog Las17 in vivo (unpublished results). However, we identified an interaction between the Ldb17 PRD and the Bzz1 SH3 domain that mirrors the PRD-dependent binding of the Ldb17-related protein SPIN90 to the mammalian Bzz1 homolog syndapin (Kim et al., 2006). The conserved interaction of syndapin-like proteins with proline-rich regions in Ldb17 and SPIN90, despite the different placement of the interacting domains, suggests these proteins may fulfill related roles. Further work will be needed to clarify the function of these conserved proteins in regulating actin polymerization and endocytosis. Overall, our results suggest that endocytic processes are generally more similar in yeast and higher cells than previously believed, and underscores Snc1 transport in yeast as a model for synaptobrevin recycling at the synapse. Characterization of other genes identified in our screen may yield additional endocytosis factors. Due to the functional  39  redundancy of the endocytic machinery in all eukaryotes, many regulatory proteins are likely to have weak mutant phenotypes that can only be discovered by more sensitive and quantitative approaches. Future application of our genome-wide analysis to different reporter proteins may identify alternative cargo-specific machinery and further define the complexity of endocytic systems.  2.5. Materials and methods Construction of plasmids and yeast strains Plasmids and yeast strains used in this study, and details of their construction, are listed in Table S2. Unless otherwise indicated, plasmids were constructed by homologous recombination after co-transforming yeast with linearized vector and DNA fragments bearing 50-55bp homology, and recovering recombinant plasmids in Escherichia coli. Oligonucleotide sequences are available upon request. The MATa yeast strain BY4741 and its gene deletion derivatives were obtained through Open Biosystems (Huntsville, AL). Unless otherwise stated, gene disruptions and tagging of genomic ORFs were carried out by transformation of a PCR product containing a drug selection marker, flanked by 50-55bp of homology to the region of interest.  Genome wide screening The GFP-Snc1-Suc2 (GSS) reporter was introduced into MATa and MATalpha knockout collections using the SGA procedure (Tong and Boone, 2006). Query strains containing the GSS reporter and a gene deletion marked with NATR were mated to a 384colony knockout array that contained 374 mutants identified in the genome-wide analysis (top 402 hits minus 28 mitochondrial mutants) and controls (wild type, blank, ent3, and rvs161). Double mutant haploid strains containing the GSS reporter were selected on –LEU – URA +G418 +clonNAT plates. Manipulation and screening of the resulting colony arrays was performed essentially as described (Burston et al., 2008). Colony arrays on 384-array stock plates were pinned four times to YPF plates creating 1536-arrays using a Virtek automated colony arrayer (BioRad, Hercules, CA). Digital images of colony arrays subjected to the invertase overlay assay (Darsow et al., 2000) were acquired using an Epson 2400 flat-bed scanner. Local normalization was carried out using National Institute of Health ImageJ software (http://rsb.info.nih.gov/ij/) with the local normalization plugin  40  (bigwww.epfl.ch/sage/soft/localnormalization) to correct for differences in intensity resulting from uneven distribution of reagents. Densitometry on corrected images used the spot-finding program GridGrinder (gridgrinder.sourceforge.net) and values were subjected to background subtraction and filtered to eliminate absent or very slow-growing strains. As densitometry values are inverted during analysis, reported values are inversely correlated with cell surface levels of the reporter. Therefore, these values were subtracted from the median value for all strains to give a score where a high value represents a high level of the reporter. The invertase overlay assay was carried out twice on each knockout collection; average values were used to generate the final ranking.  Genetic Interaction and Network Analysis In an additive model of genetic interaction, the phenotype of the double mutant strain (PXY) can be expressed as: PXY = PWT + EX + EY + EGI, where PWT is the wild type phenotype, EX is the effect of the first mutation, EY is the effect of the second mutation and EGI is the genetic interaction effect. In a genetic interaction matrix, all double mutants in a given row or column have a mutation in the same “array” gene (row) or “query” gene (column). Assuming genetic interactions are rare, and positive and negative interactions are equally common, dividing double mutant phenotype values (PXY) in each column by the mean of that column removes the effect of the query mutation (Pwt + EY). Subsequently dividing values in each row by the row-wise mean removes the effect of the array mutation (EX), leaving only the genetic interaction effect, EGI. We applied a modified version of this data pre-processing strategy to the matrix of raw double mutant invertase activity values. Rows with more than 20% missing observations were removed. Remaining values were divided by row- and column-wise medians, and values in each row and column were scaled so the sum of the squares of the values in each row or column was 1.0. Although these manipulations do not preserve the numerical value of EGI they optimize clustering of functionally related groups by emphasizing patterns of genetic interactions. Cluster 3.0 software (http://bonsai.ims.utokyo.ac.jp/~mdehoon/software/cluster/software.htm) was used to perform average linkage hierarchical clustering using an uncentered correlation similarity metric on both horizontal and vertical axes, and results were presented using Java Treeview (http://jtreeview.sourceforge.net/), where ordering of genes on both axes reflects the similarity of their genetic interaction profiles, and cluster relationships are indicated by  41  trees. The resulting heat map (Fig. A1) does not provide a true quantitative measure of genetic interaction, but instead approximates the direction of the genetic interaction effect for each double mutant where yellow is negative (less than expected under the additive null model), and blue positive (greater than expected). A network of correlated genetic interaction profiles was generated using the ARACNE algorithm (Margolin et al., 2006) with a threshold of 0.015. The MCODE plugin for Cytoscape (Bader and Hogue, 2003; Shannon et al., 2003) was used to identify denselyconnected gene clusters in synthetic genetic data and physical interaction networks from SGD (www.yeastgenome.org) and WI-PHI (Kiemer et al., 2007), which were then integrated. The size of each node (gene) was mapped to the strength of the endocytic defect in Cytoscape based on the results from the genome-wide analysis in Table A2.  Liquid invertase assay The liquid invertase assay was modified from (Darsow et al., 2000). Strains expressing GSS or NGSS reporters were grown for 20 h in 2ml YP fructose (OD600 ≈10). After dilution to 3 OD600/mL, 5μl of each culture was mixed with 55μl 0.1M NaOAc buffer pH4.9 in a 96 well microtiter plate. Subsequent steps were carried out with a VICTOR3 1420 multilabel plate reader system (PerkinElmer, Waltham, Massachusetts). 13μl of freshly prepared 0.5M ultra pure sucrose was added to each well, and the plate was incubated for 5min at 30°C. Next, 100μl of glucostat reagent (0.1M K2HPO4 pH7.0, 347.1 U Glucose Oxidase, 2.6 ng/ml Horseradish Peroxidase, 102.6 nM N-ethyl-maleimide, 0.15 mg/ml ODianisidine) was added to each well, and the plate incubated for 5 min at 30°C. Finally, 100μl of 6N HCl was added to each well to stop the reaction. The absorbance at 540nm was used to determine glucose concentration by comparison to a glucose standard curve (range from 5-50nM glucose). Results are reported as nM glucose produced per 1 OD600 of culture.  Live cell imaging Yeast cells expressing GFP-tagged proteins were grown to mid-log phase and were viewed directly in minimal selective media at room temperature using a Plan-Apochromat 100x 1.40 NA oil-immersion objective on a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Thornwood, NY). Images were captured with a CoolSNAP camera (Roper Scientific, Tuscan, AZ) using MetaMorph 6.1 software (Molecular Devices, Downingtown, PA) and adjusted using Adobe Photoshop CS. Alternatively, cells were grown to early log phase on  42  rich medium plates at 30°C and placed in 200 ml minimal media on Concavalin A-coated 8 well Lab-Tek coverglass bottom dishes (Nalge Nunc, Rochester, NY). Images were collected at room temperature with a 3i Marianas microscope (Intelligent Imaging Innovations, Denver, CO) equipped with an alpha Plan-Fluar 100x 1.46 NA objective and a Zeiss TIRF slider. Single color widefield GFP images were acquired using 488 nm laser excitation and a GFP dichroic and emission filter (Semrock, Rochester, NY). Two-color and RFP images were acquired with 488nm and/or 561nm laser excitation, with GFP and RFP emission split between two Cascade II 512 cameras with an Optical Insights Dual Cam (Photometrics, Germany) equipped with the D520/30m D630/50m splitting optics (Mag Biosciences). Exposure times varied from 600-750ms as follows: Ldb17-GFP single experiments, 750ms; two-color Ldb17/Myo5, 750ms; Ldb17/Sla1, 750ms; Lbd17/Abp1, 750ms for Ldb17 and 650 for Abp1. TetraSpeck 100nm beads (Invitrogen) were used to align the two channels and for subsequent registration in software. Slide-Book 4.2® software (Intelligent Imaging Innovations, Denver, CO) was used for image acquisition and dual channel image registration. Kymographs and montages were created using ImageJ software with Kymograph plugins (http://www.embl.de/eamnet/html/kymograph.html). Live-cell imaging experiments with LAT-A treatment were performed by incubating cells from early-log phase cultures in a final concentration of 200 mM LAT-A dissolved in DMSO for 30 min at 30°C.  Co-immunoprecipitation 30 OD600 cell pellets made from flash-frozen log phase cells were lysed by vortexing with glass beads in 200ul lysis buffer (100mM NaCl pH 8.0, 2mM EDTA, 0.5% Tween-20 + PMSF) and incubated with mouse αHA (1:1000 ABM) and 30ul Protein G-Sepharose (75% slurry in PBS). Co-precipitating proteins were identified by western blotting with mouse αGFP (1:1000, Roche) and HRP-labelled secondary antibodies (BioRad). Blots were developed with ECL (Pierce, Rockford, IL) and and luminescent images were captured with a Fluor S Max Multi-imager.  43  2.6. Figures and Tables  Figure 2.1. Genome-wide screen for endocytic recycling mutants. (A) Schematic of the GFP-Snc1-Suc2 (GSS) reporter. At the plasma membrane (PM), the Suc2 (invertase) portion is accessible to the extracellular space. (B) Localization of GFPSnc1, GSS, and an endocytosis-defective form of GSS containing snc1V40A,M43A mutations (Scale bar, 2μm). (C) Representative portion of the yeast knockout array tested using the invertase activity overlay assay. Mutants defective in Snc1 endocytosis exhibit increased surface invertase activity relative to wildtype cells, and appear dark. Mutants with high (tpm1 and rvs161) or low (snx4 and irs4) levels of cell surface invertase activity are indicated. (D) Relative cell surface invertase activities of top 1000 mutants. Red line indicates threshold used to designate top hits.  44  Table 2.1. Predicted yeast endocytic genes identified in the screen  ND, not determined aSee Table A1. bKaksonen et al., 2005 cInternalization of FM4-64 or cargo proteins. See Engqvist-Goldstein and Drubin (2003), Kaksonen et al. (2005), and Moseley and Goode (2006).  45  Figure 2.2. Endocytosis mutants have related genetic interaction profiles. (A) Hierarchical clustering was used to analyze the invertase activity values of double mutant strains generated by crossing top-scoring mutants from the primary screen (y-axis) to 81 diverse trafficking mutants (x-axis). Yellow indicates lower than expected levels of GSS at the cell surface, while blue indicates higher than expected levels (see Methods and Materials for details). (B) Detailed view of clusters showing names of genes and highly represented pathways/complexes. Clusters are numbered to indicate their relative position on the heatmap in (A). Red bar indicates gene clusters on the Y-axis enriched in endocytosis or recyling genes. See Fig. A1 for a detailed view of gene names and cluster relationships.  46  Figure 2.3. Integration of genome-wide genetic and physical interaction data identifies pathways and complexes. Significant genes clusters contributing to GSS localization were mapped based on integrated physical and genetic interaction networks. Array genes are represented as nodes (where relative size is proportional to GSS surface levels in the corresponding deletion mutant), and are connected by edges that represent either a physical interaction or a genetic correlation extracted from genome-wide data. Clusters were assigned to subcellular compartments based on the localization of their constituent proteins.  47  Figure 2.4. Yeast AP180 homologs have a conserved role in Snc1 internalization. (A) Quantification of cell surface GSS levels in WT and candidate endocytic mutants by the liquid invertase assay. Invertase activity represents relative surface levels of the GSS reporter, quantified as nmol glucose released/OD600 (mean of at least 3 experiments ± SD). (B) Localization of GFP-tagged Snc1 and NPF-Sso1 in WT and mutant strains (Scale bar, 2μm). (C) Schematic of the GFP-Snc1-Suc2 (GSS) and 3xNPF-GFP-Sso1-Suc2 (NGSS) reporters. (D) Relative cell surface levels of GSS and NGSS reporters measured by liquid invertase assay in mutant strains (mean of 3 experiments +/- SD). Results for each reporter are expressed as % invertase activity relative to WT. Different endocytosis defective controls (*EN-) were used for each reporter. GSS: endocytosis defective GSS (snc1V40A M43A) reporter in WT cells. NGSS: NGSS in sla1 mutant strain.  48  Figure 2.5. Cargo sorting defects of yap1801∆ yap1802∆ mutants. (A) The Yap1802 C-terminal clathrin-binding motif (CBM) is not required for Snc1 internalization. Localization of GFP-tagged Snc1 and NPF-Sso1 in WT and yap1801/ yap1802∆CBM mutants. (B) Deletion of YAP1801 and YAP1802 does not prevent internalization of Ste6-GFP, whereas loss of LDB17 results in a slight increase in cell surface Ste6-GFP. Localization of Ste6-GFP is shown in wild type (WT) and indicated mutants.  49  Figure 2.6. Cargo specificity of genes required for Snc1 endocytosis. (A) Array-based analysis of cargo specificity. The difference (DIFF) in cell surface levels of GSS and NGSS reporters was determined for each mutant by subtracting normalized invertase activity scores measured in parallel analyses of mutant arrays. Mutants with differences greater than 2SD (p<0.05) from the mean are shown. * indicates loss of gene function by deletion of an overlapping open reading frame (B) Relative cell surface levels of GSS and NGSS reporters measured by liquid assay in inp52 and ldb17 mutants. Results for each reporter are expressed as % invertase activity relative to appropriate WT control (*p < 0.05). (C) Yap180 and Sla2 are present on internal puncta in inp52 strains. Wild type and inp52 mutants expressing Sla1-GFP, Sla2-GFP or both Yap1801-GFP and Yap1802-GFP were examined by live cell microscopy. Arrows indicate internal patches. (D) Localization of GFP-tagged Snc1 and NPF-Sso1 in WT, ldb17, and end3 strains. Scale bar, 2μm. (E) Ldb17 shares homology with the mammalian SPIN90 and contains a PRD. The conserved PTHR13357 domain is indicated (blue region).  50  Figure 2.7. Ldb17 is required for proper coat and actin dynamics (A-C) Colocalization of Ldb17-GFP with Sla1-RFP, Myo5-RFP, and Abp1-RFP. Representative single frames from GFP and RFP channels are shown together with merged images. Arrows indicate cortical patches containing both Ldb17 and RFP-tagged proteins. (D) Schematic diagram, adapted from (Kaksonen et al., 2005), illustrating the timing of Ldb17 recruitment to cortical patches relative to markers of the late coat (Sla1), WASP/myo  51  (Myo5) and actin (Abp1) modules, based on kymograph analysis of 10 patches for each GFP/RFP pair (see Fig. S3 A-C). The average difference in the timing of Ldb17 arrival or disappearance (+/- SD, in seconds) relative to other markers is indicated, together with the total average lifetime of Ldb17, Myo5, and Abp1 patches. (E) Localization of Ldb17-GFP and Abp1-RFP in cells treated with DMSO (left panels), or 200 μM LAT-A (right panels), for 30 min at 30°C. (F) Lifetime of GFP-tagged Ldb17 and/or Ldb17∆PRD at cortical patches in WT, bzz1 and sla1. Lifetimes were measured for at least 10 patches/strain. (G) Reduced number and increased brightness of actin patches in ldb17 cells, as assessed by microscopy of Abp1-GFP. (H) Kymograph representation of time-lapse wide-field fluorescence microscopy showing extended lifetime of Sla1-RFP at endocytic patches in ldb17∆ mutants, as compared to wild type cells over 120s. Scale bar, 2μm.  52  CHAPTER 3: THE YEAST VARIANT AP-1R CLATHRIN ADAPTOR AND IMA1 ARE REQUIRED FOR SNC1 ENDOCYTIC RECYCLING1  1Burston  HE, Tam C, Davey M, Raghuram N, Maldonado-Báez L, Wendland B, and Conibear E. (2010). Chapter 2 is a version of a manuscript in preparation.  53  3.1. Synopsis In this chapter, the results of the primary genome-wide screen described in Chapter 2 were extended to identify factors required for Snc1 intracellular recycling. This identified a requirement for a previously uncharacterized variant of the AP-1 complex in this process, which we have named AP-1R (AP-1 related). AP-1R differs from the well-characterized AP1 complex only by the alternate inclusion of the medium subunit. The work presented in this chapter demonstrates that AP-1R and AP-1 have distinct functions in endosomal recycling, and the mechanisms regulating the specificity of AP-1R in Snc1 transport are investigated. Inclusion of alternate medium subunits has been shown to alter the sorting function of AP complexes (Gonzalez and Rodriguez-Boulan, 2009; Gan et al., 2002). However, it is not well understood how subunit exchange can confer novel functions to these complexes. Recent findings suggest that differential interactions between the medium subunit with cargo, regulatory proteins, and membrane lipids may underlie these differences (Fields et al., 2007 and 2010; Ang et al., 2003). Consistent with findings from these previous studies, this work demonstrates that AP-1 and AP-1R participate in different regulatory interactions. The previously uncharacterized gene YFL034w was identified as an AP-1Rspecific regulator, and has putative enzymatic activity required for AP-1R function. Yfl034w has been predicted to physically interact with AP-1R components in genome-wide studies (Krogan et al., 2006; Gavin et al., 2006), and here, it is shown to interact preferentially with AP-1R versus AP-1. Yfl034w is therefore referred to as Ima1 in this work (Interacts with  adaptin 1). Collectively, this study provides insight into the differential interactions that occur between alternate AP medium chains and regulatory proteins. It also reinforces the idea that SNARE transport is mediated by specialized components of the vesicle formation machinery.  3.2. Introduction The process of endocytic recycling is essential for maintaining the composition of the plasma membrane, and also in the regulation of nutrient uptake, cell polarity, and signal transduction (Jovic et al., 2010; Grant and Donaldson, 2009). The AP-1 adaptor complex plays a key role in endocytic recycling, and mediates sorting between the trans-Golgi network and endosomes (Reusch et al., 2002). AP-1 is a member of the heterotetrameric clathrin adaptor complexes, and consists of four adaptin subunits: γ1 and β1, which interact  54  with accessory proteins, and μ1 and σ1. The μ1 adaptin mediates cargo and membrane binding, while σ is involved both in complex stability, and cargo recognition (Mattera et al., 2011). AP-1 exists as variant isoforms, which differ through the alternate inclusion of one subunit. In many cases, these variants mediate distinct sorting functions. Mammalian cells express 3 isoforms of the small σ1 subunit (σ1A-C). Although deletion of the large subunits are embryonic lethal, specific deletion of σ1B in mice results in defects in the recycling of synaptic vesicles (Glyvuk et al., 2010). These mice exhibit a severe reduction in the number of synaptic vesicles at hippocampal boutons, and the endosomal accumulation of vesicle components. Most organisms also express AP-1 variants that differ by inclusion of alternate medium subunits. Mammalian polarized epithelial cells express the ubiquitous AP-1A (containing 1A), as well as the epithelial-specific AP-1B (containing 1B). While AP-1A mediates Golgi/endosomal sorting, AP-1B functions at recycling endosomes, and directs the transport of a distinct set of cargo to the basolateral domain (Gan et al., 2002). Although the mechanisms underlying this alternate specificity have not been well characterized, this subunit exchange may confer the ability of each complex to interact with a distinct set of cargo, lipids, and regulators (Fields et al., 2007 and 2010; Ang et al., 2003). Two alternate forms of the AP-1 complex have also been identified in yeast, which share the large γ and β (Apl2, Apl4) and small σ (Aps1) chains, differing only by the alternate inclusion of either Apm1 or Apm2 as the medium μ subunit (Stepp et al., 1995). The characterized AP-1 complex, which incorporates Apm1, has a well-established role in transport of cargo, including Chs3 (Chitin Synthase 3) and Ste13 between Golgi and endosomes (Valdivia et al., 2002; Foote and Nothwehr, 2006). No sorting role has previously been identified for the Apm2-containing complex. Early studies suggested that the Apm1 and Apm2-containing complexes are biochemically and functionally distinct; they co-fractionate with separate clathrin-coated vesicle populations, and while deletion of APM1 enhances the defects in growth and -factor processing in combination with a temperature-sensitive allele of clathrin light chain, deletion of APM2 does not (Stepp et al., 1995). A screening approach to identify regulators of the yeast Snc1/synaptobrevin endocytic recycling pathway was previously reported (Chapter II/Burston et al., 2009). Through this approach, a role for the alternate AP-1 clathrin adaptor complex containing Apm2 (AP-1R; AP-1-related) in the regulation of Snc1 cell surface levels was revealed.  55  The work reported in this chapter demonstrates that AP-1R is involved in Snc1 intracellular transport, and that AP-1 and AP-1R mediate different sorting pathways. Additionally, a requirement for the previously uncharacterized gene, YFL034w in AP-1Rmediated Snc1 recycling was identified. Yfl034w binds specifically to AP-1R vs. AP-1, and we have therefore named it IMA1 (interacts with mu Adaptin 1). Furthermore, this work demonstrates that Ima1 is a putative enzyme of the serine hydrolase family, conserved among eukaryotes, and its catalytic activity may be required for Snc1 transport. This finding is significant, as a growing body of research has identified interactions between AP medium subunits with various enzymatic regulators, although this is the first study to investigate this in yeast.  3.3. Results 3.3.1. A role for the variant AP-1R complex in endocytic recycling In the genome-wide screen to identify genes required for the intracellular recycling pathway of the yeast synaptobrevin homolog Snc1 (described in Chapter 2), deletion mutants of 3 subunits of the AP-1 complex resulted in increased Snc1 surface levels. These results were surprising, as the subunits that were identified (Apl4, Aps1, and Apm2, table A2) are components of the functionally uncharacterized AP-1 isoform, AP-1R. Interestingly, loss of the uncharacterized gene YFL034w/IMA1 also led to increased Snc1 surface levels. Ima1 has been predicted to bind AP-1R components in multiple high-throughput interaction studies (Krogan et al., 2006; Gavin et al., 2006). Furthermore, AP-1R components and IMA1 shared similar genetic interaction profiles with a panel of trafficking mutants representing various sorting pathways, and were highly-connected based on our network analysis (Burston et al., 2009). Collectively, these results suggest that Ima1 and AP-1R may share a related role in Snc1 transport. To verify that loss of AP-1R components leads to increased surface levels of GSS, these results were quantified in liquid culture (Fig. 3.1, A). Loss of APM2 and APL4 led to GSS surface levels significantly greater than WT. Although this phenotype was reproducible, it was weak. The increased GSS surface levels in apm2∆ mutants were lower than in yap1801∆ mutants. As described in chapter 2, yAP1801 is an endocytic adaptor required for Snc1 internalization, which is partially redundant with its homolog, yAP1802. This defect was specifically due to loss of AP-1R, as deletion of the AP-1 specific subunit, APM1, did not result in increased Snc1 surface levels.  56  3.3.2. AP-1R mediates intracellular recycling As Snc1 maintains a steady state surface localization through continuous rounds of endocytosis and redelivery through endosomal recycling, the increased surface levels of Snc1 in the absence of AP-1R could reflect either a defect in the rate of endocytosis or an increase in the rate of surface recycling. An endocytic role for AP-1R would be surprising, as AP-1 has a well-established role in intracellular sorting, and localizes to Golgi and endosomal compartments (Valdivia et al., 2002; Fernández and Payne, 2006). However, as no requirement for the endocytic adaptor AP-2 in Snc1 internalization was identified in the primary screen, it was considered possible that AP-1R may have a role in endocytosis. Whether AP-1R could be detected at endocytic sites was determined by comparing the localization of Apm2 with that of the endocytic AP-2 medium chain, Apm4 in cells treated with LAT-A, which permits the visualization of cortical actin patches. While Apm4 colocalized with Clc1 in coated pits at the plasma membrane, Apm2 did not. Instead, it localized to intracellular compartments (Fig. 3.1 B, upper). In very few cases, however, Apm2 was observed in close proximity to cortical patches. The kinetics of these surface patches was further investigated. While Apm4 retained co-localization with clathrin at the plasma membrane over the course of the TIRF experiment, Apm2 showed dynamic activity in relation to these cortical clathrin-containing patches (Fig. 3.1 B lower). These results suggest that Apm2 is mostly present in intracellular compartments, consistent with a role for AP-1R in the intracellular recycling of Snc1, and not in endocytosis. 3.3.3. AP-1R and Ima1 localize to Golgi/endosomal compartments As Apm2 localized to motile intracellular structures, the identity of these structures, and whether they contain clathrin was investigated. AP-1 has previously been shown to localize to Golgi/endosomal compartments (Valdivia et al., 2002). To determine if this is also the case for AP-1R, fluorescence microscopy was used to compare the localization of Apm2-GFP relative to Sec7-RFP, a marker of late Golgi and endosomal compartments (Franzusoff et al., 1991). Consistent with a role in Golgi/endosomal sorting, substantial overlap was observed between Apm2-GFP and Sec7-RFP (Fig. 3.2 A). Apm2-containing structures also showed overlap with intracellular clathrin patches (Fig. 3.2 B). The distinct sorting roles of AP-1 and AP-1R could reflect a different localization of the two complexes. To investigate whether AP-1 and AP-1R have non-overlapping distributions, the localization of Apm1 and Apm2 were compared. Consistent with previous findings, Apm1 localized to Golgi/endosomal membranes. Direct co-localization of Apm1-  57  RFP and Apm2-GFP revealed substantial overlap of Apm2-GFP and Apm1-RFP at these compartments (Fig. 3.2 C). Together with the functional invertase assays, AP-1 and AP-1R likely have non-overlapping sorting roles, and that this cannot likely be explained based purely on differential localization of the two complexes, as assessed by this study. As Ima1 was also identified by our screening approach, and is a predicted binding partner of AP-1R, this suggested that it may be involved in AP-1R function. If this were the case, Ima1 would likely localize to compartments consistent with AP-1 function. Although Apm2 and Ima1 could not be co-localized directly due to the low expression of both proteins, Ima1-GFP shared a similar extent of co-localization with both Apm1 and intracellular clathrin as did Apm2 (Fig. 3.2 D and E), suggesting that Ima1 and AP-1R localize to similar compartments, and that Ima1 may have a role in AP-1R-mediated transport. 3.3.4. Ima1 and AP-1R are not required for the transport of AP-1-dependent cargo Since Ima1 localized to compartments consistent with AP-1 function, the next question to be addressed is whether Ima1 functions specifically with AP-1R, or with both forms of AP-1. If Ima1 functions specifically with AP-1R, it could suggest a regulatory role, and may contribute to the specific sorting function of the complex. The relative importance of Ima1 in AP-1R versus AP-1-mediated trafficking was investigated by determining if Ima1 is required for sorting of AP-1-dependent cargo. AP-1 is required for the endosomal transport of the chitin synthase Chs3 (Valdivia, 2002), and when deleted, can restore the cell surface translocation of Chs3 from late Golgi/endosomes in chs6Δ cells, in which this pathway is blocked. It has previously been shown that Apm2 does not contribute to this sorting role (Valdivia et al., 2002). We therefore compared the requirement for AP-1R and Ima1 in Chs3 transport using a previously established assay to measure Chs3 surface levels, based on the fluorescence levels observed for cells grown on media containing Calcofluor white (CW). While deletion of AP-1 (APM1, APL2), restored WT cell surface levels of Chs3 in chs6Δ cells, deletion of either AP-1R (APM2) or IMA1 did not lead to an obvious restoration (Fig. 3.3, A). Furthermore deletion of APM2 did not affect Chs3 surface levels in combination with deletion of either APM1 or IMA1. These results suggest that Ima1 functions preferentially with AP-1R, and has a minor, if any role in AP-1 transport.  58  3.3.5. Ima1 specifically binds the AP-1R C-terminal domain If Ima1 localization overlaps with both AP-1 and AP-1R, how does Ima1 specifically contribute to AP-1R function? One explanation is that Ima1 may bind preferentially to AP1R vs. AP-1, as demonstrated in multiple genome-wide studies. To investigate this, the ability for Ima1-GFP to co-immunoprecipitate Apm2-HA versus Apm1-HA was assessed. Ima1-GFP co-precipitated exclusively with Apm2-HA, while no interaction was observed with Apm1-HA (Fig. 3.3, B). This confirmed that Ima1 interacts preferentially with the AP1R vs. the AP-1 complex, and may therefore play a role in AP-1R regulation. Most AP-1 regulators interact with the appendage domain formed by the Gamma subunit (GAE domain, Page et al., 1999; Hirst et al., 2003), although a few have been found to bind to the Beta appendage (Edeling et al., 2006). It was therefore evaluated whether Ima1 interacts with the AP-1 Gamma appendage. Interestingly, Ima1-GFP co-purified with Apl4-HA from yeast cell lysates, and this interaction was maintained in the absence of the Apl4 GAE appendage domain, suggesting that Ima1 binds to the AP-1R core complex (Fig. 3.3 C). Although the role of the Beta appendage in mediating this interaction was not tested, it is likely that Ima1 interacts with the AP-1R core complex, since Ima1 interacted preferentially with AP-1R vs. AP-1, and these complexes differ only by the alternate inclusion of Apm1 and Apm2. To better define the interaction between Ima1 and AP-1R, a Yeast Two-Hybrid assay was carried out between Ima1 and Apm2, which confirmed the interaction between the fulllength proteins. Based on structural homology to other  adaptin chains, Apm2 is divided into an N-terminal domain, required for incorporation into the AP complex, and a Cterminal domain, which contains regions for both membrane recruitment and tyrosinebased cargo binding (Collins et al., 2002). To potentially narrow down the role of Ima1 in AP-1R regulation, further yeast-two hybrid assays were used to identify the regions involved in this interaction. This demonstrated that the Ima1 N-terminal domain (residues 1-262) interacted with the Apm2 C-terminal domain (Fig. 3.3 D). As Ima1 binding does not require the presence of the Apm2 N-terminal domain, which mediates its assembly into the AP-1 complex, Ima1 likely interacts directly with Apm2, and not through another complex subunit. Based on structural analysis, the C-termini of adaptor  chains can be further divided into distinct subdomains (A and B). Subdomain A forms the tyrosine-binding pocket, while subdomain B contains regions shown to interact with specific membrane lipids and regulatory factors (Owen and Evans, 1998, Heldwein et al., 2004). Further Apm2  59  truncations revealed a minimal binding region of residues 389-562, which constitutes the B domain, and lacks subdomain A. This indicates that Ima1 does not interact with Apm2 as a typical cargo through the tyrosine-binding pocket, but may associate with it through a region which, by analogy to other  subunits, contains sites for multiple regulatory and lipid interactions (Fields et al., 2010; Owen and Evans, 1998; Heldwein et al., 2004). 3.3.6. Ima1 is not required for AP-1R recruitment or interaction with cargo The work described above demonstrates that Ima1 is both functionally and physically associated with AP-1R, and may regulate the sorting function of this AP complex. There are multiple mechanisms by which Ima1 may regulate AP-1R. Ima1 may be important for the recruitment of AP-1R to Golgi/endosomal membranes, or may be an alternate cargo adaptor, linking Apm2 to Snc1. To determine if Ima1 is required for AP-1R membrane recruitment, fluorescence microscopy was used to compare Apm2-GFP localization in WT and ima1Δ cells. No significant differences in the number of Apm2 membrane puncta in the absence of Ima1 were observed (Fig 3.4 A). To rule out any subtle defects in Apm2 membrane association in the absence of IMA1, subcellular fractionation was used to compare the distribution of Apm2-HA in WT and ima1Δ cells. Apm2 was enriched in the high-speed (P100) membrane fraction, containing vesicles and small organelles, in both WT and ima1Δ cells, consistent with the microscopy results (Fig. 3.4 B). These studies demonstrate that Ima1 is not important for the membrane recruitment or stabilization of AP-1R. An alternate possibility is that Ima1 may be important for AP-1R cargo recognition. AP medium subunits bind directly to signals within the cytosolic domain of their respective cargo (Robinson et al., 2004, Owen et al., 2004). Since Snc1 does not contain canonical APbinding tyrosine or dileucine motifs, Ima1 may be a non-conventional adaptor, bridging the interaction between Snc1 and Apm2. We sought to determine whether Apm2 physically interacts with Snc1 and subsequently, whether Ima1 is required for this interaction. A GSTtagged Snc1 cytosolic domain was expressed in bacterial cells, and it was determined whether Apm2 could be co-precipitated from yeast cell lysates on a GST-binding column. Apm2-TAP was pulled down by Snc1-GST, and this interaction was stronger than that between Apm2-TAP and GST alone (Fig. 3.4 C). This indicates that Apm2 binds to Snc1 through its cytosolic domain, typical of adaptor-cargo binding mechanisms. This interaction was maintained in the absence of IMA1. However, a very weak interaction was identified  60  between Ima1-TAP and GST-Snc1 (Fig. 3.4 C), suggesting that Ima1 may be physically associated with the Apm2-Snc1 adaptor-cargo complex. 3.3.7. Ima1 is a conserved protein with a consensus α/β hydrolase catalytic motif The results described above suggest that Ima1 is not critical for either AP-1R recruitment or cargo binding. In order to form alternate hypotheses regarding Ima1 function, its molecular properties were further investigated. Ima1 contains a C-terminal DUF726 domain, which is also present in a large number of uncharacterized eukaryotic proteins, from yeast to humans (Fig. 3.5 A). The DUF726 domain is predicted to conform to an / hydrolase fold, a catalytic domain found in a wide range of eukaryotic enzymes, including thioesterases, lipases, acetyltransferases, and peptidases (reviewed in Holmquist, 2000; Nardini and Dijkstra, 1999). A BLAST search and sequence alignment revealed that that this domain within Ima1 is highly conserved among eukaryotes. A characteristic feature of all / hydrolases is the presence of a conserved catalytic triad consisting of a nucleophile (generally a serine within the context of a G-X-S-X-G motif), an acidic residue (D or E), and a histidine. The residues surrounding the active site are also well conserved, to preserve its structural features. Based on these homology studies, a potential catalytic triad was identified within the Ima1 C-terminal domain, which was 100% conserved. The conserved residues consist of S759 (within the motif GFSIG), D817, and H858. Ima1is predicted to be a trans-membrane protein, according to its hydrophobicity profile (TMHMM Server v. 2.0 http://www.cbs.dtu.dk/services/TMHMM, accessed on January 17, 2011). If this were the case, however, the putative enzymatic domain would be contained within the predicted membrane-spanning region, which would not permit the association of this activity to potential substrates. To examine whether Ima1 is likely to be integral to the membrane, as predicted, subcellular fractionation was used. This showed that Ima1 is present mostly in the soluble S100 fraction (Fig. 3.5 B). The punctate membrane localization of Ima1 observed by microscopy suggests that it is likely peripherally membrane associated through weak or transient interactions with other membrane components. These studies were followed up by determining whether the membrane association of Ima1 is influenced by treatment with Brefeldin A (BFA). BFA is a fungal metabolite which blocks the GTP exchange and membrane recruitment of Arf1, therefore causing protein transport defects at the ER and Golgi, and the mislocalization of proteins whose recruitment to these membranes depend on Arf1 (Donaldson et al., 1992). Upon brief treatment of cells  61  with 100ug/mL BFA, Ima1 membrane localization was lost (Fig. 3.5 C). This suggests that Ima1, like many other clathrin coat-associated proteins, is localized either directly by Arf1 or indirectly through interaction with Arf1-dependent components, and that Ima1 is likely dynamically recruited from the cytosol to sites of AP-1R function. Together, the findings that the putative catalytic domain of Ima1 is not likely membrane spanning, and that Ima1 interacts with Apm2 through its N-terminal domain, supports that the putative catalytic residues may be accessible for binding to substrates and carrying out catalysis at these sites. 3.3.8. The Ima1 GXSXG catalytic motif is required for normal Snc1 surface levels To investigate whether the conserved predicted catalytic site identified in Ima1 is required for Snc1 sorting, the motif GFSIG containing the catalytic serine nucleophile was mutated to AFAIA by site-directed mutagenesis. The ability of this mutant construct (Ima1CM catalytic mutant) to restore normal Snc1 surface levels ima1Δ cells was assessed by a plate-based invertase assay (Fig. 3.5 D). Loss of IMA1 led to a significantly increased level of Snc1, as compared to WT cells in this assay. This increase was partially complemented by introduction of a plasmid expressing WT IMA1 (pIMA1), as Snc1 surface levels were lowered relative to ima1Δ. This complementation was not complete, likely because the final screening media (YPF) does not allow for plasmid selection. Introduction of a plasmid expressing the catalytic mutant (pIMA1CM) however, did not complement the Snc1 sorting defect to any degree. This suggests that Ima1 may have catalytic activity required for its role in Snc1 sorting by AP-1R. The serine hydrolases include thioesterases, which catalyze the depalmitoylation of proteins (Zeidman et al., 2009). Snc1 is palmitoylated at a single cysteine residue, adjacent to its transmembrane domain, although the effects of this modification are unknown (Couve et al., 1995; Valdez-Taubas and Pelham, 2005). Since cycles of protein palmitoylation and depalmitoylation have been shown to regulate the transport of SNARE proteins (ValdezTaubas and Pelham, 2005; He and Linder, 2009), one possibility is that Ima1 might have a role in Snc1 depalmitoylation, which may, in turn be required for its transport by AP-1R. No increase in Snc1 palmitoylation was observed when IMA1 was overexpressed, as assessed using the Acyl-biotin exchange assay (Fig. 3.6 A and B). This suggests that Ima1 does not likely have a direct role in its depalmitoylation.  62  As serine hydrolases catalyze a diverse array of reactions, future work should focus on identifying the biologically relevant substrates of Ima1, which will further our understanding of the role of Ima1 in AP-1R-mediated transport.  3.4. Discussion 3.4.1. AP-1R mediates Snc1 intracellular recycling In this study we identified a specific requirement for AP-1R in the endocytic recycling of the yeast synaptobrevin homolog Snc1, the first identified role for this complex. The co-localization of AP-1R with intracellular clathrin at Golgi/endosomal compartments supports a role for AP-1R in Snc1 recycling, and not internalization. Although the precise role of AP-1R in this pathway remains to be determined, the increase in cell-surface Snc1 in the absence of AP-1R may reflect re-routing of Snc1 along an alternate bypass pathway with faster recycling kinetics than that of the AP-1R-mediated pathway. AP-1 has also been shown to play a role in the transport of the S. pombe synaptobrevin homolog, Syb1 (Kita et al. 2004; Ma et al., 2009). Deletion of both the medium chain Apm1, homologous to yeast Apm1, and the small chain of the AP-1 complex resulted in the accumulation of Syb1 in Golgi⁄endosomes. In addition, loss of fission yeast Apm1 function causes defects in cellular processes such as secretion, cytokinesis, vacuole fusion, and cell wall integrity. Deletion of AP-1 in S. cerevisiae, however, results in no discernible phenotypes and does not cause the intracellular accumulation of Snc1. Despite these differences, both of these studies support the role of AP-1 in the endosomal sorting of synaptobrevin. While Ima1 is present in S. pombe, Apm2 does not have considerable homology to either of the two AP-1 medium subunits. In the future, it would be interesting to determine if the fission yeast Ima1 homolog plays a role in AP-1-mediated sorting of Syb1. 3.4.2. Apm2 binds to Snc1 as cargo A significant finding of this study is that Apm2 interacts with the Snc1 cytosolic domain, which is consistent with the well-established role of AP medium subunits in cargo recognition. Like most SNAREs, Snc1 does not contain tyrosine or di-leucine motifs. In mammalian cells, only VAMP4 and VAMP7 contain recognizable di-leucine-based motifs (Peden et al. 2001). Multiple adaptor complexes have now been shown to interact with SNAREs of the VAMP family, and a growing body of research suggests that most of these interactions are mediated by nonconventional mechanisms. The use of folded regulatory 63  domains, rather than the commonly used short, linear motifs, and specialized components of the transport machinery appears to be the paradigm for sorting SNAREs into post-Golgi transport vesicles (Miller et al., 2007; Chidambaram et al., 2004). As the fidelity of vesicle fusion processes in the cell requires appropriate amounts of SNAREs at their correct locations, these specialized mechanisms are likely in place to ensure that the transport of SNAREs are not vulnerable to competition from standard motif-containing cargo, and that sufficient amounts of the correct SNAREs are incorporated into CCVs for transport. The in vitro binding assays used in this study could not resolve whether Apm2 interacts directly with Snc1, or if additional factors are required to bridge this interaction. If this interaction is direct, it must be mediated by non-conventional sorting signals. Apm2 may recognize Snc1 through a folded epitope. Structural studies would therefore be insightful in the understanding of how this interaction is mediated. The primary screen identified a weak requirement for the epsins Ent1 and Ent4, suggesting the possibility that they bridge the interaction between Apm2 and Snc1. As discussed, epsins are known to function as alternate sorting adaptors, and have been shown to bridge the interactions between SNAREs and AP complexes. In yeast, Ent1 is partially redundant with Ent2 for internalization (Wendland et al., 1999). Ent3/5 are required for endosome to Golgi recycling of both Chs3 and Snc1, and bind to multiple SNAREs, including Vti1 and Pep12 (Chidambaram et al., 2004), while Ent4 mediates transport from the Golgi to the vacuole (Deng et al., 2009). Whether one or more epsins bridges the interaction between Apm2 and Snc1 should be a priority in future work. 3.4.3. Distinct sorting functions for AP-1/AP-1R These results support that the two alternate AP-1 complexes in yeast have largely non-overlapping functions. Deletion of AP-1R, but not AP-1, results in increased Snc1 surface levels. Conversely, deletion of AP-1R has no effect on the recycling of Chs3, which is mediated by AP-1 (Valdivia et al. 2002). As APM1 mutants, but not APM2 mutants display synthetic phenotypes in combination with a temperature sensitive allele of CLC1 (Stepp et al., 1995), it has been suggested that Apm2 may function in a non-clathrin-mediated pathway. This seems unlikely, however, as Apm2 co-fractionates with clathrin-coated vesicles (Stepp et al., 1995), and the results reported here demonstrated that Apm2 colocalizes with intracellular clathrin. As Apm1 and Apm2 have distinct co-fractionation profiles with clathrin, they likely function in different clathrin-mediated pathways.  64  Alternate forms of AP-1, formed by medium subunit exchange have been identified in other organisms, including C. elegans and mammalian polarized epithelial cells. In both examples, these complexes have largely non-overlapping roles. In C. elegans, disruption of UNC-101 (the 1 homolog), and mutation of the alternate medium chain Apm1 have distinct effects on viability (Shim et al., 2000). In polarized epithelial cells, the ubiquitously expressed AP-1A mediates TGN to endosome transport of the proprotein convertase furin, while the epithelial cell-specific AP-1B is required for the polarized targeting of the transferrin and LDL receptors from recycling endosomes. These complexes are not functionally interchangeable, as introduction of AP-1A cannot complement the sorting defects of AP-1B-deficient polarized cells, and vice versa. 3.4.4. AP-1/AP-1R localize to similar intracellular compartments The non-redundant cargo-specific functions of AP-1A and AP-1B in polarized cells have been partially explained by their differential localization to distinct membrane domains. Although mammalian AP-1A and AP-1B were first shown to share a similar localization, further studies revealed by immunoelectron microscopy that they define partially distinct subdomains, with AP-1A localizing to the Golgi, and AP-1B localizing to a perinuclear region immediately adjacent to the Golgi (Fölsch et al., 2003). Despite their specificity for distinct cargo, however, substantial overlap in the localization of Apm1 and Apm2 to Golgi/endosomal compartments was observed, suggesting that the two complexes may recognize distinct sets of cargo from a common membrane. This overlap was not complete, however. A small proportion of Apm2 puncta did not co-localize with Apm1, and it is therefore possible that AP-1R facilitates Snc1 recycling through a distinct endosomal population or membrane domain. Further studies may help to resolve whether there are small-scale differences in AP-1 and AP-1R localization. Currently, these studies are limited by the incomplete understanding of endosomal organization in yeast. It is not known if there are functionally distinct populations of early endosomes, or if endosomal membranes contain subdomains, with different compositions. 3.4.5. Role of Ima1 in AP-1R regulation In this study, Ima1 was identified as a specific regulator of AP-1R. Deletion of IMA1 resulted in increased Snc1 surface levels, and it shared a similar genetic interaction profile with AP-1R components based on E-MAP studies (Burston et al., 2009). Both Ima1 and AP-  65  1R localize to Golgi/endosomal compartments. Furthermore, a preferential and likely direct interaction between Ima1 with Apm2, but not Apm1 was demonstrated. The interaction between Apm2 with Ima1 was surprising, as most AP-regulatory proteins associate with the appendage domains of the large subunits (Page et al., 1999; Hirst et al., 2003). A few recent studies, however, have reported that the medium subunit also participates in regulatory interactions. Many µ-binding regulators are enzymes that have important roles in lipid regulation. Some of these regulators are themselves tyrosinebased cargo proteins, and bind to the canonical µ binding pocket. For example, the lipid kinase PIPKI gamma-p90 interacts with the AP-2 µ subunit. (Kahlfeldt et al., 2010). Binding to AP-2 µ stimulates the kinase activity of PIPKI gamma-p90, facilitating localized PI(4,5)P2 synthesis activity at endocytic sites. The increased PI(4,5)P2, in turn leads to enhanced AP-2 recruitment to these sites. This interaction is therefore cooperative, and enhances the activity of both the adaptor and the regulator in a positive feedback loop. The interaction between Ima1 and Apm2 is mediated through the N-terminal 162 amino acids of Ima1. Within the Apm2 µ2 C-terminal region, Ima1 binds subdomain B, and does not require its tyrosine motif-binding site. Interestingly, this region is quite divergent between Apm1 and Apm2, suggesting that it could contribute to the sorting specificity of the two complexes due to differential interaction with Ima1. In regions where µ1 homologs contain negatively charged residues, the corresponding residues in 2 homologs are often positively charged and vice-versa. A recent study identified a patch of three positively charged residues within the corresponding region of the mammalian AP-1B µ1-B subunit, which are conserved among µ1-B homologs, but not with those of µ1-A homologs (Fields et al., 2010). This patch was required for the differential recruitment of AP-1R complexes to PI(3,4,5)P3-rich recycling endosomes, and upon mutation of these residues to the corresponding residues in 1-A, this localization was lost, resulting in missorting of AP-1Bdependent cargo. Furthermore, AP-1B expression was required for the localization of PIPKI-gamma90, which generates PI(3,4,5)P3 at recycling endosomes. This indicates that there is interdependence in the function of both AP-1B, and PIPKI-gamma90. It was proposed that AP-1B either binds PI(3,4,5)P3 directly, or that it is recruited by an unidentified protein that requires PI(3,4,5)P3 for its localization. Since Ima1 binds to the region of Apm2 which by analogy to other µ subunits, contains residues important for lipid binding, it was hypothesized that Ima1 could be required for the membrane localization of AP-1R. However, no decrease in membrane  66  binding of AP-1R in ima1∆ cells was observed by either microscopy or subcellular fractionation. The possibility still remains that Ima1 is required for recruitment of AP-1R to the small population of compartments that do not contain Apm1. It is also possible that Ima1 is necessary for recruitment of AP-1R to specialized lipid domains within membrane compartments containing both complexes. This study also demonstrated that Ima1 is not required for Apm2 cargo binding. The in vitro approach that was taken, which assessed the binding of Apm2 from cell lysates to an immobilized GST-Snc1 construct, suggesed that Ima1 does not form a direct link between Apm2 and Snc1, and is not required for any modification of Apm2 necessary for this binding. It remains to be determined whether Ima1 is required for the interaction between Apm2 and Snc1 in vivo, as no reproducible interaction between these proteins was detected by coimmunoprecipitation. These results are consistent with a weak and transient interaction between Apm2 and Snc1 in vivo, as has been shown by multiple other studies for adaptors and their respective cargo. It remains possible that Ima1 is required for a later step in AP-1R function, such as vesicle formation or un-coating. Investigating these possibilities is of priority for future studies. 3.4.6. Ima1 has putative enzymatic activity The only previous study investigating the function of Ima1 was based on its interaction with the P0 subunit of the ribosomal stalk, which is translocated to the cell surface by unknown mechanisms (Aruna et al., 2004). The precise role of Ima1 in this process, however, has not been further investigated. Our homology studies revealed that Ima1 is conserved among eukaryotes, and is a member of the α/β-hydrolase family. This is a catalytic domain found in a wide-range of enzymes, including esterases, lipases, acetyltransferases, and peptidases, containing a unique structural fold, and encompassing a catalytic triad, consisting of the residues (Ser, Cys, or Asp), Asp/Glu, and His. We identified a putative catalytic site in Ima1, conforming to this consensus. This site is characteristic of serine hydrolases, and is 100% conserved among all considered sequences. Serine hydrolases are generally cytoplasmically localized, and show transient recruitment to their sites of activity. Although Ima1 is predicted to be a transmembrane protein, this work demonstrates that it is mainly cytosolic, and that it is likely has a dynamic localization to Golgi/endosomal compartments. The conserved Ima1 catalytic residues appear to be required for Snc1 sorting, as a mutant version in which these  67  residues were mutated to alanine was unable to complement Snc1 surface levels in ima1∆ cells. An important challenge in the future will be to characterize the precise enzymatic activity of Ima1 and to identify its biologically relevant substrates. Based on its binding to the Apm2 B domain, Ima1 may play a role in lipid remodeling at Golgi/endosomal compartments. A full understanding of Ima1 function will require the identification of its substrates, and a better understanding of the organization of endosomal membranes in yeast. In conclusion, this work has identified a role for the previously uncharacterized AP1R complex in Snc1 recycling, a function distinct from AP-1. Furthermore, Ima1 was specifically required for AP-1R-mediated transport, and has putitive enzymatic activity related to this function. These findings fit with a growing body of research suggesting that alternate isoforms carry out distinct sorting functions through participating in differential interactions with sorting determinants, and provide new evidence that AP medium subunits are the target of interactions by enzymatic regulators.  3.5. Materials and methods Construction of plasmids and yeast strains Plasmids and yeast strains constructed for this study, and details of their construction, are listed in Table B1. Unless otherwise indicated, plasmids were constructed by homologous recombination after cotransforming yeast with linearized vector and DNA fragments bearing 50–55-bp homology, and recovering recombinant plasmids in E. coli. The MATa yeast strain BY4741 and its gene deletion derivatives were obtained through Thermo Fisher Scientific. Unless otherwise stated, gene disruptions and tagging of genomic ORFs were performed by transformation of a PCR product containing a drug selection marker, flanked by 50–55 bp of homology to the region of interest. Liquid invertase assay The liquid invertase assay used in this study is modified from that previously described (Darsow et al., 2000). Strains expressing either the GSS construct were inoculated into 2ml YP fructose cultures and allowed to grow for 20 hours to saturation (OD 600 ≈10). Cultures were diluted to 3OD/mL, and 5μl was mixed with 55μl 0.1M NaOAc buffer pH4.9 inside a well of a 96 well microtiter plate. With the use of a Victor3 multilabel counter and injectors (PerkinElmer, Waltham, Massachusetts), 13μl of freshly prepared 0.5M ultra pure sucrose was added to each well, and the plate was incubated for 5min. at 30°C. Following  68  sucrose incubation, 100μl of glucostat reagent (0.1M K2HPO4 pH 7.0, 347.1 U Glucose Oxidase, 2.6 ng/ml Horseradish peroxidase (HRP), 102.6 nM N-ethylameide (NEM), 0.15 mg/ml O-Dianisidine) was added to each well, and the plate incubated for an additional 7min. at 30°C. Finally, 100μl of 6N HCL was added to each well to stop the reaction. The absorbance of each well at 540nm was measured and used to determine glucose concentration. Results are reported as nM glucose produced per 1 OD600 of culture. Glucose standards (ranging from 5-50nM) and blanks were included in each experiment to ensure accurate measurements that were within the linear range.  Calcofluor white screening Knockout strains were plated in 1,536-array format onto YPD plates containing 50 μg/ml CW (Sigma-Aldrich), using a Virtek automated colony arrayer (Bio-Rad Laboratories). After incubation at 30°C for 3 d, white-light images were acquired using a flat-bed scanner (model 2400; Epson), and fluorescent-light images were captured with a Fluor S Max MultiImager (Bio-Rad Laboratories) using the 530DF60 filter and Quantity One software (version 4.2.1; Bio-Rad Laboratories). The open-source spot-finding program GridGrinder (http://gridgrinder.sourceforge.net) was used for the densitometry of digital images. Average growth and fluorescence values from two independent screens were calculated for each strain using Excel (Microsoft).  Fluorescence microscopy Indirect immunofluorescence microscopy, fluorescent microscopy of yeast cells expressing GFP-tagged proteins were carried out as described (Conibear and Stevens, 2000, 2002). Cells were viewed using a 100x oil-immersion objective on a Zeiss Axioplan2 fluorescence microscope, and images were captured with a CoolSnap camera using MetaMorph software and adjusted using Adobe Photoshop. Immunofluorescence experiments involving BFA were carried out by incubating cells from early-log phase cultures in a final concentration of 100ug/mL BFA for 10 minutes. Two color widefield images were acquired and processed as described in chapter 2. Exposure times varied from 600-750ms. TIRF images were collected with a 3i Marianas microscope (Intelligent Imaging Innovations, Denver, CO) equipped with an alpha Plan-Fluar 100x 1.46 NA objective and a Zeiss TIRF slider (Carl Zeiss, Thornwood, NY). Images were acquired with 488nm and/or 561nm laser excitation, with GFP and RFP emission split between two Cascade II 512  69  cameras with an Optical Insights Dual Cam with an exposure time of 750ms. TetraSpeck 100nm beads (Invitrogen) were used to align the two channels and for subsequent registration in software. Slide-Book 4.2® software (Intelligent Imaging Innovations, Denver, CO) was used for image acquisition and dual channel image registration. Montages were created using the National Institute of Health ImageJ software (http://rsb.info.nih.gov/ij/) with the Kymograph plugins installed (http://www.embl.de/eamnet/html/kymograph.html). Live-cell imaging experiments involving LAT-A treatments were performed by incubating cells from early-log phase cultures in a final concentration of 200 μM LAT-A dissolved in DMSO for 30 min at 30°C. LAT-A treated early-log phase cultures were used for the total internal reflection microscopy (TIRF) experiments. For each TIRF experiment 200 μL of treated cells were spotted onto Conconavalin A-coated 8 well Lab-Tek dishes (Nalge Nunc, Rochester, NY).  Co-immunoprecipitation Log phase cells were converted to spheroplasts and stored at -85°C (Conibear and Stevens, 2000). Spheroplasts from 20OD600 of cells were resuspended in 1mL lysis buffer (600mM Sorbitol, 50mM KH2PO4 pH7.5, 50mM NaCl, 1.5%TX-100), on ice for 10 minutes. Lysates were incubated with 40ul IgG sepharose (75% slurry) for 2.5 hours, rotating at 4°C. The pellets were washed two times with 1ml lysis buffer (50mM KH2PO4 pH 7.5, 75mM NaCl, 1.5% TX100), and were subjected to SDS-PAGE. Co-precipitated proteins were detected by Western blotting with antibodies to GFP (mouse mαGFP 1:1000, Roche #1814460, monoclonal) HA (mouse mαHA 1:1000, Abcam #ab16918, monoclonal) or TAP (rabbit α-TAP 1:5000, Open biosystems #cab1001 polyclonal) followed by HRP-labelled secondary antibodies (GAR-HRP Biorad #170-6515 or GAM-HRP Biorad #170-5047). Blots were developed with ECL and exposed to X-ray film (X-OMAT LS, Kodak).  In vitro Snc1-GST binding assays Truncated Snc1 lacking its transmembrane domain cloned into vector pETGEXCT (Sharrocks, 1994, gift from Anne Spang). Snc1-GST was expressed and purified from in E. coli BL21 using standard procedures. Bacterial Induction of GST proteins and immobilization on glutathione: Cells were grown at 37°C to mid-log phase, and expression was induced at 30°C by addition of 1mM IPTG for 3 hours. 3 OD pellets were frozen at  70  stored at -85°C. Cell pellets were lysed by sonication in PBS, on ice (in the presence of protease inhibitors). Bacterial lysates were incubated with 20 ul of 50% glutathione Sepharose 4B for 1 hour at 4°C, while rotating. Unbound proteins were removed with three washes in PBS. Incubation of yeast lysates with immobilized GST proteins: Yeast lysates were prepared as described above. Lysates were incubated with GST proteins immobilized on glutathione sepharose beads for 2.5 hours at 4°C, while rotating (total volume of 1mL; NaCl adjusted to 150mM). Following incubation, beads were washed two times with cold lysis buffer. Samples were heated to 70°C in SDS sample buffer, and eluted proteins were analyzed by SDS-PAGE and Western Blotting, as described above.  Yeast two-hybrid screening PJ694a strains carrying pOAD-based vectors were mated to PJ694alpha strains carrying pOBD2-based plasmids expressing the indicated full length and truncation proteins (Tong et al. 2002). Positive two-hybrid interactions were scored on minimal media lacking histidine (containing 5mM 3-AT) or adenine after growth at 30°C for 5 days.  Subcellular fractionation Fractionation of organelles was performed by differential centrifugation of cell lysates as described by Conibear and Stevens (2000). 20 OD600 of exponentially growing cells were spheroplasted, lysed and centrifuged for 5 min at 500 g to remove unbroken cells. The supernatant was centrifuged for 10 min at 13,000 g to generate the membrane pellet fraction (P13), and the supernatant (S13) was centrifuged at 100,000 g for 60 min generating the P100 pellet fraction and S100 supernatant. The membrane pellets were resuspended in 300ul sample buffer. Proteins in the S100 fraction were precipitated by the addition of TCA to a final concentration of 5%, collected by centrifugation for 5 min at 13,000 g, washed twice with acetone, air dried and resuspended in 300ul sample buffer. The proteins in 10ul of each fraction were separated by 15% SDS-PAGE, transferred to nitrocellulose, and were subjected to western blotting, as described above.  Invertase plate assay Colony arrays of strains containing an integrated version of the GSS reporter plus the indicated pRS415-based plasmids were spotted on SC-LEU plates in 96-array format. This array was then replicated to 384-array and then 1536-array formats on the same  71  media. For screening, the 1536 array was replicated to YPF plates. The invertase assay was carried out as described in chapter 2.  Snc1 palmitoylation assay Snc1 palmitoylation was assessed by acyl-biotin exchange (Politis et al., 2005), using a modified version of the Drisdel and Green method (Drisdel and Green, 2004). Ima1 overexpression was carried out from GAL-based plasmids with a 4-hour galactose-induction period. Overnight cultures grown in SC-URA+2% raffinose, and were then grown to early log phase, at which point they were transferred to either SC-URA +2% galactose or SC-URA +2% raffinose media for the 4 hour induction period. Denatured protein extracts were prepared from Snc1-GFP–expressing cells by glass bead lysis either with or without IMA1 overexpression. Extracts were subjected to the three steps of acyl-biotinyl exchange protocol. The epitope-tagged Snc1 was immunoprecipitated with anti-GFP antibodies, and the resulting samples were run on a 10% SDS gel. Western blotting was then carried out with either anti–biotin-HRP or anti-GFP antibodies.  72  3.6. Figures  A  * * *  B  WT  apm1∆  apm2∆  apl4∆  yap1801∆  Figure 3.1. AP-1R is required for correct sorting of Snc1 (A) Quantification of cell surface levels of the Snc1 reporter (GSS) at the cell surface in AP-1R deletion strains by invertase assay of liquid cultures. Invertase activity is reported as nmol glucose release/OD600, and the mean +/-SD of 5 independent experiments is shown. * P<0.001 by student t-test, as compared with WT. (B) Co-localization of Apm4-GFP and Apm2-GFP with Clc1-RFP at cortical actin patches in LAT-A-treated cells by wide-field microscopy (upper panels). Lower panels show TIRF microscopy and kymograph analysis of Apm2-GFP or Apm4-GFP relative to a single cortical clathrin patch marked by Clc1-RFP (sla2∆ cells)  73  Apm2-GFP  Sec7-RFP  Apm2-GFP  Clc1-RFP  Apm2-GFP  Apm1-RFP  Ima1-GFP  Apm1-RFP  Ima1-GFP  Clc1-RFP  A Apm2-GFP  B  C  D  E  Figure 3.2. AP-1R and Ima1 localize to Golgi and endosomal compartments Co-localization of Apm2-GFP or Ima1-GFP with the RFP-tagged markers Sec7-RFP (late Golgi), Clc1-RFP (clathrin), and Apm1-RFP (AP-1).  74  Median Fluorescence intensity  A  chs6∆  WT  C  chs6∆ apl2∆  ∆  chs6∆ chs6∆ apm1∆ apm2  ∆  B  chs6∆ apm1∆ apm2∆  chs6∆ ima1∆  chs6∆ ima1∆ apm2∆  Ima1-GFP:  +  +  +  Ima1-GFP:  +  +  +  Apm1-HA:  +  -  -  Apm2-HA:  -  +  -  Apl4-HA: Apl4Dear-HA:  + -  +  -  -HA  -HA  Lysate  Lysate -GFP  -GFP -HA  -HA  IP  IP -GFP  -GFP  D  Apm2-BD constructs AP-Binding 1  AD constructs  Cargo (A)/lipid (B) binding 246  1-605 (FL) 246-605  Apl4  Ima1  Ima1-N  605 A  B  A  + ++  +++  +++  A  B  A  N/A  +  +++  B  A  N/A  +++  +++  N/A  +++  + ++  391-605 389-562  B  Figure 3.3. Ima1 associates functionally and physically with AP-1R (A) Chs3 surface levels were quantified in chs6Δ cells in combination with mutations in AP-1 or AP-1R components and IMA1, based on Calcoflour White fluorescence assay. Results are reported as spot median pixel intensity, as assessed by densitometry.  75  (B) Ima1 preferentially interacts with Apm2 versus Apm1 by co-immunoprecipitation. Cells were grown to midlog phase and were processed for immunoprecipitation (see Materials and Methods). Immunoprecipitation (IP) was performed with anti-GFP antibodies, whereas Western blotting was performed with both anti-HA and anti-GFP antibodies. (C) Ima1 interaction does not require the Apl4 GAE domain. Immunoprecipitation was carried out as in (B). (D) Yeast two-hybrid assay between fulllength Apm2-GAD and Ima1-GBD, and between full-length and N-terminal domain of Ima1 (1-262) with the indicated Apm2 truncations. Apl4-GBD was used as a positive control for the expression of Full length Apm2. The relative strength of interaction is indicated, as qualitatively assessed by growth on SC-HIS supplemented with 5mM 3AT.  76  im a1 ∆  S100 WT  im a1 ∆  P100 WT  WT  Apm2-GFP  P13 im a1 ∆  Lysate  WT  B  im a1 ∆  A  Apm2-HA Vps10  WT  ima1∆  ALP PGK  SNC1-GST  Ap Ap m2m2 HA -H A A impm a1 2 ∆-H A Im a1 -H A  -GST  Ap m Ap 2-H m2 A -H A iA mpam 1∆ 2-H A Im a1 -H A  C  WB: TAP  Ima1-TAP Apm2-TAP  Figure 3.4. Ima1 is not required for Apm2 membrane recruitment or cargo binding (A) Localization of Apm2-GFP in WT vs. ima1Δ cells. (B) Membrane association of Apm2-3HA in WT versus ima1Δ cells, assessed by subcellular fractionation. Apm2 is enriched in the high speed membrane fraction (P100). Markers of subcellular fractions are Vps10 (Golgi), ALP (vacuole), and PGK (cytosol). (C) Apm2-3HA binds to immobilized GSTSnc1 in vitro, and does not require IMA1. Purified recombinant GST-Snc1 or GST alone were incubated with lysates prepared from Apm2-3HA WT or Apm2-3HA ima1Δ cells, and a GSTpull down assay was performed. Samples were subjected to SDS-PAGE and analyzed by immunoblotting with anti-HA antibodies. Ima1 interacts weakly with GST-Snc1, as assessed on the same blot.  77  A  C A  ap m2 ∆  S100  WT  ap m2 ∆  P100  WT  ap m2 ∆  WT  P13  ap m2 ∆  Lysate  WT  B A  Ima1  Ima1GFP  EtOH  BFA (100ug/mL)  Ima1GFP  Vps10 ALP  Apl2 -GFP  PGK  D A  WT pRS415  Ima1∆ pRS415  Ima1∆ pIMA1  Ima1∆ pIMA1CM  Figure 3.5. Ima1 contains putative catalytic residues, required for Snc1 sorting (A) Schematic of Ima1, indicating N-terminal Apm2-binding domain, and DUF726 domain (top) Sequence alignment of Ima1 C-terminal domain with conserved homologs, highlighting the conserved catalytic triad residues (bottom) (B) Subcellular fractionation of  78  Ima1). Control markers for other subcellular fractions are Vps10 (P100; light membrane fraction), ALP (P13; heavy membrane fraction), and PGK (S100; soluble fraction). (C) Microscopy of Ima1-GFP in cells treated with BFA (100ug/mL, 10 min). Apl2-GFP is included as a control for peripheral membrane association. (D) Quantification of GSS surface levels in WT versus ima1Δ cells, containing plasmids for the expression of WT IMA1, IMACM (catalytic mutant) or empty vector (pRS415). (invertase overlay assay; n=7 replicates per group). pRS415-based plasmids were introduced into a background strain expressing the GFP-Snc1-Suc2 (GSS) reporter, and were selected for based on growth on –LEU plates. Strains were arrayed onto YPF plates in 1536 format, and were assessed for GSS surface levels as described in chapter 2 (genome-wide screening, materials and methods). Boxes represent upper and lower quartiles.  79  A  B  -  +  -  +  Figure 3.6. Overexpression of Ima1 does not affect Snc1 palmitoylation (A) IMA1 GAL-induced overexpression. GAL-inducible plasmids expressing Ima1 (pIMA1GAL) or empty vector control (pBG1805) were introduced into WT cells. Cells grown overnight in SC + 2% raffinose were diluted and grown to mid-log phase (T0), at which point they were grown for 4 hours in SC + 2% galactose for 4 hours. Overexpression of Ima1 was detected by immunoblotting to HA. (B) Snc1 palmitoylation status was assessed by AcylBiotin Exchange (ABE) in strains expressing GFP-Snc1 (endogenously tagged), and overexpressing either IMA1 (pIMA1GAL) or empty vector control (pBG1805) (4h gal induction, as described in A). Cells were were subjected to the acyl-biotin exchange reaction. Protein extracts were treated with (+) or without (-) hydroxylamine (NH2OH). Anti-GFP precipitates were blotted with anti-biotin and anti-GFP to detect palmitoylated and total Snc1, respectively.  80  CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS  81  4.1. Overview The overall goal of this work was to identify the requirements for the endocytic recycling pathway of Snc1, the yeast VAMP2 homolog. Towards this goal, a sensitive and quantitative assay was used to systematically identify genes required for this pathway, on a genome-wide level. Genetic and bioinformatic approaches were used to infer functional relationships between top candidates, and to guide downstream molecular analysis of these genes. Through this work, multiple genes were identified that were predicted to function in endocytosis and recycling, but have not previously been shown to have sorting defects. A model that encompasses the findings from this work is proposed in illustration 4.1 Chapter 2 investigated the mechanisms regulating Snc1 endocytosis. This study identified roles for many yeast homologs of mammalian endocytosis genes, including clathrin and the yeast AP180 clathrin adaptors. The results showed that yAP1801 and yAP1802 are partially redundant in Snc1 internalization. Furthermore, they have a cargoselective function: combined deletion selectively blocked internalization of Snc1, but not other endocytic cargo. Similarly, the synaptojanin Inp52 also had a cargo-selective function, likely through its role in the uncoating of AP180 from endocytic vesicles. This study also identified a requirement for the previously uncharacterized protein Ldb17 in the regulation of coat and actin dynamics at endocytic sites. Chapter 3 focused on characterizing components required for Snc1 intracellular recycling, and demonstrated a role for the AP-1 complex variant, AP-1R in this process, and for the previously uncharacterized protein Ima1. The results support that the closely related AP-1 and AP-1R have cargo-specific functions in Golgi/endosomal transport. Ima1 was found to be a regulator of AP-1R function, and is specifically associated with AP-1R both functionally and physically, but not AP-1. It is therefore likely to contribute to the specificity of the AP-1R complex. Ima1 is highly conserved in eukaryotes, and has predicted enzymatic residues that are required for normal Snc1 surface levels. Collectively, these studies show that considerable functional redundancy and weak endocytic defects at least partially explain the lack of observable defects for these components in previous studies, and demonstrate that endocytic mechanisms in yeast and man are more similar than previously appreciated. These studies are also consistent with the view that sorting of SNARE proteins is subject to highly specific mechanisms, and that the use of unconventional adaptors may be widespread for these recognition events.  82  Although this work has generated insight into mechanisms regulating Snc1 endocytic recycling, it also encourages new questions. How is AP180 recruited to endocytic sites, and what is the mechanism by which Inp52 contributes to AP180 uncoating? The signals in Snc1 that mediate its interaction with the endocytic machinery and with AP-1R also remain to be identified. These questions, methods to address them, and ways to apply the insight generated here to future studies, are discussed in this chapter.  4.2. Mechanisms regulating Snc1 internalization 4.2.1. Role of clathrin and yeast AP180 homologs in Snc1 endocytosis A requirement for clathrin and its adaptors in the internalization of Snc1 was a highly significant finding. This study revealed that the yeast AP180 clathrin adaptor homologs yAP1801/1802 have a partially redundant role in Snc1 internalization. While individual deletion of yAP1801 or yAP1802 led to a relatively weak endocytic defect, combined deletion of yAP1801/1802 completely blocked internalization. Furthermore, yAP180 was not required for the uptake of other commonly studied endocytic cargo. The partial redundancy and cargo-specificity of these adaptors serve to explain why endocytic defects have not previously been identified for yAP1801 and yAP1802. This was significant because although clathrin contributes to the total number of endocytic sites and to the stability of endocytic complexes, no cargo has previously been shown to be dependent on clathrin or its adaptors for internalization in yeast (Newpher et al., 2005; Drubin et al., 2005). This discrepancy has remained unexplained in the field for decades, as it contrasts with the essential role for clathrin in mammalian cells. There are many important questions raised by our work on yAP180 that should be addressed in future studies. What are the mechanisms regulating its recruitment to endocytic sites, and release from endocytic vesicles? Does AP180 bind to cargo directly, or through additional bridging interactions? In this study, some of these questions were addressed. In the context of our findings, future approaches that would be useful in further addressing these questions are discussed below. 4.2.2. How is yAP180 recruited to endocytic sites? We identified a strong requirement for clathrin in Snc1 uptake, but found that AP180 does not need to bind to clathrin directly to carry out internalization, as deletion of its clathrin binding site did not prevent Snc1 uptake. This suggested that AP180 might be  83  linked to clathrin through additional proteins. Although CLASPs generally bind to AP-2 for recruitment to endocytic sites (Maldonado-Báez and Wendland, 2006), we did not identify a requirement for AP-2 in Snc1 uptake. It thus remains unclear how yAP180 is recruited. The requirement for clathrin and AP-2 for AP180 recruitment appears to differ between organisms, and thus remains unclear. In C. elegans, it is likely that AP180 works with AP-2 to direct internalization, as a similar increase in surface levels was shown in AP180 and AP2 mutants (Dittman and Kaplan, 2006). In mammalian cells, however, the AP-2 binding sites within AP180 have only a minor contribution, suggesting that other factors regulate the recruitment of AP180 to endocytic sites (Harel et al., 2008). Although mutation of the known yAP180 clathrin-binding motif did not prevent Snc1 uptake, it remains possible that yAP180 contains additional clathrin binding motifs yet to be identified. Mammalian AP180/CALM contains multiple clathrin binding motifs (Legendre-Guillemin et al., 2004). Whether the yAP180 clathrin binding mutant actually blocks interaction with clathrin has not been determined, and should be assessed in future studies. Is yAP180 recruited to sites of internalization through association with proteins other than clathrin and AP-2? AP180 proteins contain binding sites for not only clathrin and AP-2, but also for PI(4,5)P2 (the ANTH domain), and for EH domain motif-containing proteins (NPF motifs) (Maldonado-Báez and Wendland, 2006). It is therefore possible that yAP180 is recruited by lipid binding, or by EH-domain proteins. Interaction between the AP180 NPF motifs with EH-domain-containing scaffolding proteins seems to be important in mammalian cells: AP180 interacts with the EH-domain proteins eps15 and intersectin. In yeast, the yAP180 NPF motifs bind to the EH-containing epsins (Ent1 and Ent2), and to yeast homologs of eps15 (Ede1) and intersectin (Pan1) (Maldonado-Báez L, and Wendland, B., 2006; Wendland and Emr, 1998; Aguilar et al., 2003; Miliaras and Wendland, 2004). These EH domain proteins interact with multiple components of the endocytic machinery, and may be important in AP180 recruitment. Additionally, Ent1, Ent2, and Ede1 all contain clathrin binding sites, and may therefore link AP180 to clathrin. How could the role of Ent1/2, Ede1, and Pan1 in the recruitment of yAP180 be determined? A first approach could be to investigate whether mutation of the AP180 NPF motifs causes defects in Snc1 internalization using the invertase assay, or microscopy. If strong defects were observed, this would suggest an important function for association between the AP180 NPF motifs and one or more of these EH-motif containing proteins.  84  Further studies could help determine which of these proteins are important. This could be accomplished by assessing the effects of mutating the EH-motifs in each of these proteins on Snc1 internalization. Our screen results demonstrated that ede1∆ mutants have a relatively weak defect in Snc1 internalization, similar to that of the yap1801∆ and yAP1802∆ single mutants. This demonstrates that Ede1 alone is not likely to be the only requirement for yAP180 recruitment. Ent1 mutants had an even weaker phenotype, but Ent1 has been shown to be partially redundant with Ent2. Although ent1∆/ent2∆ mutants are inviable (Wendland et al., 1999), assessment of Snc1 sorting in ent2∆ mutants in combination with a temperature sensitive ENT1 allele could reveal a role for these proteins in AP180 function. PAN1 is an essential gene and was not assessed in our primary screen. The role of Pan1 could therefore also be assessed through the use of temperature sensitive alleles, overexpression studies, or through mutation of its EH motifs. If mutation of any of these proteins is found to result in a strong endocytic defect, their effects on yAP180 endocytic patch recruitment could be evaluated by TIRF microscopy. AP180 recruitment could alternatively be mediated though PI(4,5)P2 binding through its ANTH domain. A similar strategy could therefore assess the effects of ANTH domain deletion on Snc1 internalization. Although we may find that individual deletion of the NPF motifs, or the ANTH domain impairs the AP180 Snc1 sorting function, mutation of these domains may not be enough. Since adaptors bind to multiple components simultaneously for membrane recognition, often mutations in multiple motifs are required to observe recruitment defects (Motley et al., 2006). Therefore, the combinatorial effects of these mutations on yAP180 recruitment may also need to be assessed by the methods described above. 4.2.3. Role of synaptojanin in yAP180 vesicle un-coating By screening for additional cargo-specific factors, a requirement for the PI(4,5)P2 lipid phosphatase, synaptojanin (Inp52) was identified. Loss of INP52 resulted in a cargo-specific internalization defect that was similar to the yap1802∆ mutant. Further work showed that deletion of INP52 caused the accumulation of yAP180 on cytosolic vesicles, likely explaining the similar cargo-specific defect in Snc1 internalization that we observed for both yAP180 and INP52 mutants. Inp52 has previously been shown to localize to endocytic patches, and to specifically control the dissociation of proteins that bind PI(4,5)P2 through ANTH or ENTH domains, including Sla1, and Ent1/2, respectively (Sun, Y., 2007). The ability of Inp52 to hydrolyze PI(4,5)P2 is important to release these ANTH/ENTH domain proteins from the vesicle,  85  following fission. This led us to propose that the similar cargo-specific defects observed in the absence of either INP52 or yAP180 is a result of failed disassembly of yAP180 from internalized vesicles. An alternate explanation is that this accumulation is due to inappropriate recruitment of yAP180 to internal compartments. To distinguish between these possibilities, yAP180-GFP patches could be tracked in inp52∆ mutants by TIRF microscopy. A similar approach was used to demonstrate that in inp52∆ mutants, Sla2 patches are internalized normally, but that Sla2 remains associated with these vesicles following scission (Sun et al., 2007). This showed that loss of Inp52 causes prolonged association of Sla2 with endocytic vesicles, and does not cause inappropriate recruitment of Sla2 to internal compartments. Loss of INP52 did not completely block internalization of Snc1, as it showed a similar defect as a yap1802∆ single mutant, which is considerably weaker than the defect observed in yap1801∆/1802∆ double mutants. This suggests that Inp52 may be partially redundant, and that other factors may facilitate PI(4,5)P2 hydrolysis in its absence. In addition to Inp52, yeast expresses the related synaptojanin Inp51, which also localizes to endocytic sites, and Inp53, which is involved in sorting at the TGN (Bensen et al., 2000). Inp53 is recruited to cortical actin patches under conditions of hyperosmotic stress (Ooms et al., 2000). None of these genes are essential for growth; however a triple deletion of INP51, INP52, and INP53 is lethal (Stolz et al., 1998). Although loss of Inp52, but not Inp51, has previously been shown to result in the accumulation of ENTH/ANTH domain containing proteins, this could be due to the lower expression level of Inp51 relative to Inp52. Our results suggest that Inp52 function may be partially substituted by Inp51 and possibly Inp53 for Snc1 internalization. To investigate this, it could be tested whether combined deletion of INP51/INP52 or INP51/INP53 results in a more severe internalization defect than a single INP52 deletion. The effect of the triple mutant could also be tested in inp51∆/inp53∆/inp52ts cells at a non-permissive temperature (Stefan et al., 2002). It will be important to carry out these studies to better understand how AP180 and synaptojanin may function together in higher cells. The synaptojanins are evolutionarily conserved from yeast to humans, and their regulation of PI(4,5)P2 turnover at the cell surface is critical for cellular function, and relevant to human disease (Liu Y, Bankaitis VA, 2010). Synaptojanin 1 null mice die shortly after birth and present neurological defects and accumulate clathrin-coated vesicles at nerve endings, due to failed disassembly of these coats (Cremona O., 1999, Kim WT., 2002). Interestingly, increased synaptojanin 1 activity is  86  directly associated with the brain dysfunction and cognitive defects manifested in some Down’s sydrome patients. Synaptojanin1 resides in a genomic region present in trisomic rearrangement in Ts65Dn mice, a model for Down’s syndrome. In these mice, homoeostasis of PI(4,5)P2 is disrupted. Restoring the Synaptojanin gene to disomy in these mice is alone is sufficient to correct these altered levels of PI(4,5)P2, and the neurological defects observed in this model (Voronov SV, 2008). Synaptojanin 2 functions in non-neuronal cells, and its disruption inhibits internalization of the epidermal growth factor and transferrin receptors, and has been shown to disrupt the formation of clathrin coated vesicles in lung carcinoma cells (Hill E., 2001, Malecz N., 2000, Rusk N., 2003). Although defects in both synaptojanin and AP180 result in coated vesicle accumulation and neurological defects in multiple model organisms, it is unknown if synaptojanin contributes directly to the uncoating of AP180 proteins at the synapse. The studies in yeast described here, could contribute important insight into the coordinated roles of synaptojanin and AP180 proteins, and guide future studies in mammalian cells. 4.2.4. How does yAP180 recognize Snc1 for internalization? Another outstanding question regarding yAP180 function is whether it interacts directly with Snc1, or indirectly through additional factors. An interaction between yAP180 and Snc1 could not be detected in vivo. This is consistent with other studies, which have generally failed to detect interactions between adaptors and their cargo in vivo, due to their weak and transient nature. Generally, interactions between adaptors and their cargo have been more successful by in vitro methods using recombinant proteins. An in vitro approach may therefore aid in determining whether yAP180 interacts directly with Snc1, or whether other proteins bridge this interaction. If an interaction between AP180 and Snc1 were to be identified though these studies, the next step would be to identify the Snc1 sorting signal, as the signal recognized by AP180 has not yet been identified in any organism. Two cytosolic residues have been defined that are required for internalization of both Snc1 and Vamp2. In Snc1, mutation of V40A and M43A cause a complete endocytic block (Grote E., 1995, 2000; Lewis et al., 2000). Although these residues are located on the same face of the amphipathic helix that binds to t-SNAREs, they are not required for SNARE complex assembly or function. Currently, no sorting adaptor has been shown to bind to this motif. It is also unknown whether these residues independently mediate internalization, or if they are part of a larger conformational epitope required for recognition by AP180 or other adaptors.  87  Whether the V40M43A mutation blocks the interaction between Snc1 and AP180 could be tested in vitro. An alternate approach would be to identify putative Snc1 sorting signals by error-prone PCR to introduce random mutations in its cytosolic domain. The effects of these mutations on Snc1 internalization could be tested systematically using our GSS invertase assay. If candidate endocytic mutants are identified, they could then be tested individually for binding to yAP180 in vitro. If a non-continuous set of mutations is found to block internalization, this could suggest that Snc1 is recognized by a conformational epitope. To further investigate this, structural studies, like those carried out to determine the interaction surface of the SNARE Vti1 recognized by the epsin adaptor, would likely be required. Currently, there is no evidence from our studies, or those of others, that yAP180 binds directly to Snc1; it is possible that other proteins could bridge this interaction. yAP180 interacts with multiple endocytic proteins, as previously discussed. It would therefore be useful to test if deletion of yAP180 binding partners, such as the epsins, blocks its interaction with Snc1. A larger scale approach could also be used to identify binding partners of the Snc1 endocytic signal in vitro. For example, total cell lysates could be passed through a GST-Snc1 column, and the purified components separated by SDS-PAGE. The identity of each co-purifying protein could be identified by mass spectroscopy. To identify proteins that bind to the endocytic signal, the profiles of binding partners could be compared between GST-Snc1 and GST-Snc1V40M43A mutant. For example, if a protein is present in the WT, but not the mutant purification, it may be a candidate bridging protein. A similar approach was recently used to identify and compare the profiles of effectors for all 60 mammalian Rab proteins (Kanno et al., 2010). 4.2.5. Does AP-2 mediate endocytosis in yeast? A requirement for AP-2 in yeast endocytosis has not yet been identified, in contrast to the essential role for this complex in mammalian endocytosis. Targeted disruption of AP-2 leads to embryonic lethality in mice (Mitsunari T, 2005), and has been shown to block the uptake of multiple cargo including the transferrin receptor (Conner, S. D., and S. L. Schmid. 2003), as well as a population of lysosome-associated membrane proteins that traffic via the plasma membrane (Janvier and Bonifacino, 2005). Yeast AP-2 deletion mutants, however, are viable, and have no clear defects in the internalization of commonly studied endocytic cargo. This has led to the suggestion that AP-2 is unimportant for endocytosis in yeast (Sorkin, 2004; Yeung et al., 1999). Could considering a broader range of cargo help us  88  identify an endocytic role for AP-2? One recent study has shown that AP-2 also shows cargo-specific internalization defects, which may serve to explain this discrepancy. A large-scale screen demonstrated that mutants lacking any of the four AP-2 subunits are resistant to K28, a virally encoded yeast killer toxin that enters the cells via endocytosis by binding to its unidentified receptor. AP-2 mutants have a strong defect in uptake of K28 from the media, but do not affect the uptake of other endocytic markers, including the peptide cargo α-factor and of the lipophilic dye FM4-64 (Carroll et al., 2006). The authors thus proposed that AP-2 has a strong but cargo-specific effect on K28 endocytosis. This study also showed that AP-2 is recruited to endocytic sites concurrent with clathrin and other early coat components, suggesting that it likely has a direct role in uptake. In contrast to what was previously believed, both this study and our screen for regulators of Snc1 internalization demonstrate that clathin and its associated adaptors are important in both yeast and mammalian cells. Our understanding of these similarities has only become clear through the study of cargo proteins that have not been previously studied by genome-wide methods in yeast. Further large-scale functional screens using additional non-conventional cargo will aid in the identification of proteins recognized by AP-2 and cargo-specific adaptors, and of new adaptor-related proteins required for internalization. 4.2.6. Role of actin and its regulators in Snc1 internalization The results from our screen re-enforced the importance of dynamic actin regulation in the yeast endocytic process. We identified multiple regulators of the actin cytoskeleton, for which no endocytic defects have previously been shown. Abp1 and Crn1 are actinbinding proteins, and promote the activity of the Arp2/3 complex, a key regulator of actin polymerization. The actin capping proteins Cap1/2 are thought to function in polymerization by limiting monomer addition to recently assembled ends, and increasing local polymerization rates (Kim K., 2004; 1990; Kaksonen M., 2005). We also identified a novel regulator of actin dynamics, Ldb17, and demonstrated that it is an important link between cargo selection and the regulation of actin polymerization. The absence of Ldb17 resulted in defects in both vesicle internalization and actin morphology. The mechanism by which Ldb17 promotes actin polymerization, however, is currently undefined. Does Ldb17 promote actin polymerization through direct regulation of the Arp2/3 complex, or does it function indirectly in this regulation? Like Ldb17, its mammalian  89  homolog, SPIN90, interacts with multiple components of the vesicle coat, and with the actin regulatory machinery. SPIN90 has been proposed to carry out a similar role as Ldb17, linking coat and actin dynamics (Kim et al., 2005, 2006 (1), 2007, 2009). SPIN90 stimulates actin polymerization both directly and indirectly, through binding to components of the Arp2/3 complex, as well as to its activators, including N-WASP (Kim et al., 2006; Lee et al., 2006). The results of this study suggest that Ldb17 does not likely have a direct effect in Arp2/3 regulation. Ldb17 did not bind to Las17, the yeast homolog of N-WASP or the Arp2/3 complex, likely because the regions important for binding to N-WASP and Arp2/3 in SPIN90 are only partially conserved in Ldb17. This suggests that Ldb17 interacts with additional regulators to stimulate actin polymerization in yeast. Interestingly, interactions were identified between the Ldb17 PRD with the SH3 domain-containing proteins Sla1, Lsb3, and Lsb4. These proteins bind to Las17, and are important in stimulating actin polymerization. Additionally, the PRD of Ldb17 interacts with the SH3 domain of Bzz1, the yeast syndapin homolog, and this interaction is required for membrane release of Ldb17. Bzz1 interacts directly with Las17 via its SH3 domain, and with other components of the actin polymerization machinery (Soulard et al., 2002 and 2005). These factors could thus be important in forming a link between Ldb17 and Arp2/3 regulation. Whether Ldb17 can promote actin polymerization and the requirement for its interacting factors in this process could be assessed in future studies. The role for actin polymerization factors is often tested in vitro, and there are a variety of commercially available in vitro actin polymerization assays. One of these assays is based on the enhanced fluorescence of pyrene-conjugated actin that occurs during polymerization, which can be detected using fluorimetry (Blader et al., 1999). In this assay, purified Ldb17 could be added to the reaction mixture in the presence of Arp2/3, to determine if it has a direct effect on actin polymerization. Ldb17 could also influence actin polymerization indirectly, through its association with Bzz1, Sla1, or Lsb3/4, which could be tested by addition of these factors in combination with Ldb17.  90  Yeast is an important model for understanding protein sorting in higher cells, and the underlying mechanisms that govern vesicle formation are generally well conserved. Our work and that of other groups has provided explanations for some of the apparent discrepancies between endocytic processes in yeast and man. This study has highlighted that specific mechanisms are in place for the endocytosis of different cargo proteins, which is true in both mammalian and yeast systems. Each internalization pathway shows distinct requirements for clathrin, actin regulators, and lipids, and the use of these distinct pathways may also change in response to the context of the cellular environment. Different cell types in mammals also appear to use specialized sorting mechanisms to suit their specific functions. Elucidating the unique and conserved features of these processes will be essential in understanding the fundamental principles that underlie cargo selection, membrane deformation, and vesicle scission in all organisms and cell types.  4.3. Regulators of Snc1 recycling: AP-1R and Ima1 4.3.1. What is the role of AP-1R in Golgi/endosomal recycling? In addition to the clathrin adaptor AP180, we identified a requirement for the alternate AP-1 isoform, AP-1R in Snc1 transport. In contrast to the endocytic role of yAP180, AP-1R is involved in the intracellular recycling of Snc1. Deletion of AP-1R components leads to a weak, yet reproducible increase in cell-surface localized Snc1. Based on its co-localization with intracellular clathrin, and Golgi/endosomal markers, we have concluded that AP-1R likely functions in Snc1 intracellular recycling, and not in endocytosis. How does loss of AP-1R lead to increased levels of Snc1 at the surface? As all trafficking pathways in the cell are part of a network, and their interconnected nature allows cargo to follow alternate “bypass” routes, the increase in cell surface levels suggests that deletion of AP-1R allows Snc1 to transit to the plasma membrane by an alternate recycling bypass pathway, with faster recycling kinetics than the normal route. We considered different mechanisms by which this might occur, which are discussed below and shown in illustrations 4.2 and 4.3.  91  Model 1: AP-1R mediates sorting to an intermediate compartment prior to surface delivery. In this first model (Illustration 4.2), AP-1R is present on endosomes, and promotes Snc1 recycling at these compartments. The recycling pathway regulated by AP-1R may be a ‘slow’ route, whereby AP-1R may transport Snc1 from endosomes to either the Golgi, or perhaps another intermediate compartment prior to cell surface delivery. In the absence of AP-1R, Snc1 may be diverted along to a more rapid and direct recycling route, in which it bypasses the Golgi/intermediate compartment, and is recycled directly from endosomes. As discussed in chapter 3, this bypass ability has been observed for other recycling cargo in yeast, including Chs3, which is able to recycle directly to the cell surface from early endosomes or the Golgi in the absence of AP-1 (Valdivia et al., 2002). In mammalian cells, there are at least two main types of recycling pathways to the cell surface, each of which has different kinetics. The rapid recycling route mediates transport of cargo such back to the plasma membrane from either early endosomes or an earlier stage in the endocytic pathway. The so-called slow recycling route transports cargo from the early endosome to an intermediate endocytic recycling compartment (ERC), before redelivery to the plasma membrane. Kinetic studies measuring the recycling rates of the transferrin receptor (TfR) show that it follows both pathways. Whether TfR follows the fast or slow pathway appears to be determined largely at the early endosome. This endosomal compartment contains several Rab GTPases, including Rab4 and Rab5. For TfR to undergo fast recycling, it must be quickly sorted away from Rab5, into a specialized Rab4-containing microdomain on the early endosome. These GTPases likely recruit different regulators that sort cargo either directly back to the cell surface or to the recycling endosome (Grant and Donaldson, 2009). The characterization of the mammalian endocytic system raises important questions about the nature of endosomal organization in yeast. Currently, it is not clear whether yeast have kinetically distinct recycling pathways, or different populations of endosomes like those in mammals. The current view of protein recycling in yeast is much simpler, in that cargo is thought to pass only through one type of endosome, and is returned to the plasma membrane following direct retrieval to the Golgi. Furthermore, it is not known whether the early endosome contains distinct microdomains that are important for directing proteins to different locations. Differential inclusion of AP-1 and AP-1R into distinct microdomains of a common membrane would be consistent with the overlap of these complexes at endosomal  92  compartments, and could thus explain their different sorting function. Snc1 has previously been shown to recycle to the plasma membrane through bypass pathways in the absence of components required for it normal recycling itinerary. Mutation of certain components of the COPI B coat (sec27-1 and sec28), for example, results in increased surface levels of GFP-Snc1, and enrichment at bud sites. This implies that Snc1 retrieval and recycling through early endosomes to the Golgi may require COP1B (Robinson et al. 2006), and that loss of this complex results in retargeting of Snc1 to the plasma membrane. Despite the established role of the COPI coat in intra-Golgi and Golgi-ER retrograde transport, studies in both yeast and mammalian cells also describe a post-Golgi transport role for COPI (Whitney et al., 1995; Aniento et al., 1996; Gu and Gruenberg, 2000; Faure et al., 2004). It would be interesting to test the relationship between AP-1R and COP1B in Snc1 intracellular recycling. Although most components of this coat are essential, and therefore would not have been present in our screen, a strong requirement was identified for Sec28, the only non-essential COP1B component. Genetic interaction studies between AP-1R and COPIB components could be used to determine whether they work in the same or parallel recycling pathways. Snc1 surface levels could be compared in double mutant AP-1R/COP1B strains, using the invertase assay, but would require the use of temperature-sensitive alleles of most COP1B components. Model 2: AP-1R sorts Snc1 into a specific class of secretory vesicles In contrast to an endosomal sorting role, AP-1R may function at the Golgi, and mediate inclusion of Snc1 into secretory vesicles (Illustration 4.3). There are two populations of vesicles that are targeted to the plasma membrane in yeast, each of which is enriched for distinct cargo. As these vesicles have distinct densities (dense and light), they can be distinguished based on subcellular gradient separation in sec6∆ mutants, which prevent their fusion with the plasma membrane. Snc1 is normally contained in the lighter vesicle fraction, as is the SNARE Tlg1, which partners with Snc1 in the fusion of vesicles derived from early endosomes with the Golgi (Harsay E., and Bretscher A., 1995). AP-1 is involved in the sorting of cargo into the light fraction, and in the absence of AP-1, both Chs3 and Tlg1 are missorted from the light to the dense fraction (Valdivia et al., 2002). It would be interesting to test whether AP-1R is required for Snc1 incorporation into light vesicles, which could be done by determining if deletion of AP-1R shifts the incorporation of Snc1 from the light to the dense exocytic vesicle population by subcellular fractionation. How  93  can this model explain the increased levels of Snc1 in the absence of AP-1R? This could occur if the dense vesicle fraction can carry relatively more cargo than the light fraction over a given time frame. Dense vesicles may have faster kinetics of transport. One way that this could occur is if dense vesicles are transported directly to the cell surface, while light vesicles require an intermediate transport through endosomes prior to cell surface delivery. Alternatively, dense and light vesicles could both arise from the Golgi: dense vesicles, however, could carry a higher concentration of cargo, have or have higher fusion efficiency at the cell surface than the lighter fraction. Model 3: AP-1R has indirect effects on Snc1 recycling Although localization studies suggest a role for AP-1R at Golgi/endosomal compartments, this is not sufficient to conclude that AP-1R has a direct role in Snc1 intracellular recycling. Indirect effects resulting in increased Snc1 surface levels could reflect a variety of indirect mechanisms. For example, a partial defect in endocytosis could be a result of the mislocalization of a component required for Snc1 internalization in the absence of AP-1R. It is also possible that the total cellular levels of Snc1 are increased in AP-1R mutants. Deletion of AP-1R may lead to either increased transcription or decreased degradation of Snc1. These indirect effects should be considered in future studies. 4.3.2. What is the Snc1 recycling signal recognized by AP-1R? In this study, an interaction between AP-1R and GST-Snc1 was identified vitro, but the Snc1 signal recognized by AP-1R for recycling remains to be determined. Whether AP-1R recognizes Snc1 directly or indirectly is also unknown. Like the signal required for Snc1 endocytosis the Snc1 recycling signal could be a yet unrecognized short cytosolic motif or a conformational epitope, consisting of a combination of folded residues. Furthermore, Snc1 is both palmitoylated and ubiquitinated, posttranslational modifications that have been found to affect the sorting of multiple other SNARE proteins (Valdez-Taubas and Pelham; He and Linder, 2009). It is unlikely that the Snc1 sorting signal is a post translational modification, however, as the GST-Snc1 construct used for our in vitro assay was isolated from E. Coli, which lacks the machinery required for these modifications. This suggests that the Snc1 sorting signal is likely an intrinsic part of the Snc1 cytosolic domain. To identify residues in Snc1 that are important for AP-1R recognition, alanine-scanning mutagenesis could be used to systematically replace regions of Snc1 with alanine. The effects of these mutations on preventing the interaction between Apm2 and Snc1 could then be tested in vitro. 94  It will also be important to determine whether the interaction between Apm2 and Snc1 is direct or indirect. The identification of a putative Snc1 recycling signal could help resolve this. To identify potential bridging proteins, for example, the co-purification profiles of GST-tagged WT Snc1 vs. Snc1 containing a mutated recycling signal could be compared by mass spectroscopy. An outstanding question in the field is whether Snc1 is transported as part of a trans or cis-SNARE complex. It is possible that Apm2 may bind to other SNAREs in complex with Snc1 at the Golgi/endosomes, including the functionally redundant t-SNAREs Tlg1 and Tlg2. Our in vitro Snc1 binding assay could be used to determine if Tlg1/2 is also present in the pulldown between Snc1 and Apm2, by blotting with anti-Tlg1 antibodies. If this is found to be the case, it could be interesting to test whether Snc1 and Tlg need to be in a SNARE complex to bind Apm2. Snc1 and Tlg interact though critical residues in their SNARE domain. While Snc1 provides an arginine residue, Tlg1/2 provide a glatamine. Mutation of either the Snc1 arginine or the Tlg glutamine prevents assembly of these proteins. Our in vitro assay could therefore be used to determine if Snc1 binding to Apm2 is prevented in a TlgQ>A mutant strain, and if mutation of the Snc1 arginine shows the same effects. If these mutations prevent interaction between Apm2 and Snc1, this would provide evidence that AP-1R sorts Snc1 as part of a trans-SNARE complex. 4.3.3. How do AP-1 and AP-1R regulate distinct sorting pathways? As AP-1 and AP-1R show considerable co-localization to Golgi/endosomal compartments, it is unclear how they mediate largely non-overlapping transport functions. In mammalian cells, AP-1A localizes to the TGN, wheras AP-1B localizes to the perinuclear recycling endosomes, adjacent to the TGN. This is not clearly the case for AP-1 and AP-1R in yeast, although, they may localize to slightly different membrane domains. Studies in mammalian cells suggest that exchange of the medium subunit may confer alternate specificity to AP-1A and AP-1B by providing a novel interaction surface, which is able to associate with distinct sets of lipids and also with regulatory proteins. AP1B specifically facilitates the recruitment of exocyst subunits Exo70 and Sec8, which mediate tethering and fusion from recycling endosomes to the cell surface (Fölsch et al., 2003). The SNARE protein cellubrevin has also recently been shown to be required for AP1B-dependent sorting, and co-localizes with AP-1B within recycling endosomes (Fields et al., 2007).  95  Although these alternate complexes are not analogous to AP-1 and AP-1R in yeast based strictly on homology, important insights about the differential functions of AP-1 and AP-1R may be gained from these mammalian studies. Apm2 is considerably larger than Apm1, and is only 30% identical on the amino acid level. Inclusion of Apm2 therefore may confer alternate specificity to AP-1 through facilitating differential interaction with regulators, including Ima1, and possibly different membrane lipids. These ideas are expanded upon in the following sections. 4.3.4. Do AP-1 and AP-1R localize to different lipid domains? Although we found that Apm1 and Apm2 show considerable overlap at Golgi/endosomal compartments, this overlap was not complete. This could suggest that AP1R mediates Snc1 transport at these unique sites, or that the localization of these complexes may be more different than can be appreciated based on the resolution of techniques used in this study. The differential localization of mammalian AP-1A and AP-1B has been at least explained by the differential preferences of the medium subunits for membrane lipids. While 1A binds the Golgi-enriched PI(4)P, 1B binds PI(3,4,5)P3, enriched in recycling endosomes. Interestingly, a patch of only three amino acids within the 1B C-terminal B domain was found to be necessary for its recruitment to recycling endosomes. Switching this patch with the analogous residues of 1A both inhibited the ability for AP-1B to localize to recycling endosomes, and resulted in the mis-targeting of AP-1B-dependent cargo. This suggests that differences in the lipid binding domains of these similar  subunits are important for their differential targeting and sorting function (Fields et al., 2010). The analogous regions in Apm1 and Apm2 differ substantially, suggesting that this site may contribute to the distinct sorting functions of AP-1 and AP-1R. This may also be explained by the ability of this region in Apm2 to associate with specific phospholipid domains. In the future, it would be interesting to determine whether AP-1 and AP-1R localize to slightly different Golgi/endosomal sub-domains. These studies will rely on a better understanding of membrane domains and their corresponding lipids within these compartments in yeast. Powerful microscopy techniques and novel assays to detect these small-scale localization differences will likely be required. It could, however, be investigated whether replacing these residues in Apm2 with the analogous residues in Apm1 result in the mis-sorting of Snc1. If this is the case, this would be evidence that these complexes may be differentially targeted, by analogy to the mammalian AP-1 complexes.  96  4.3.5. Ima1 is a putative enzyme and binds the Apm2 C-terminal domain This study demonstrated that AP-1 and AP-1R participate in differential regulatory interactions, and the previously uncharacterized protein Ima1 was identified as a specific regulator of AP-1R. Deletion of IMA1, which shares a similar genetic interaction profile with AP-1R components, also resulted in increased Snc1 surface levels. Furthermore, Ima1 binds specifically to AP-1R vs. AP-1, and co-localizes to similar intracellular compartments. Ima1 is not critical for the early steps of AP-1R vesicle formation, including membrane recruitment or cargo binding. It may act at a later stage of AP function, possibly as a mediator of vesicle formation, or AP-1R un-coating. As Apm2 has previously been shown to co-fractionate with clathrin-coated vesicles by gel filtration (Stepp et al., 1995), the role of Ima1 in Apm2 vesicle inclusion could be tested by this method. This would involve comparing the quantity of Apm2 included in vesicles in the presence or absence of Ima1. Decreased abundance of Apm2 vesicles in ima1∆ cells would provide evidence for a role for Ima1 in Apm2 vesicle formation. Conversely, an increased abundance of these vesicles would suggest a role for Ima1 in AP-1R vesicle dissociation. An interesting finding is that Ima1 interacts with the Apm2 C-terminus, but not through the Apm2 tyrosine-binding pocket (subdomain A). Rather, it binds to subdomain B, which has been shown by recent studies to associate with membrane lipids lipids and may be a target of regulatory interactions (Fields et al., 2010; Owen and Evans, 1998; Heldwein et al., 2004). Evidence was provided that Ima1 may have enzymatic function related to AP1R function. The Ima1 C-terminal domain is remarkably conserved among eukaryotes, and contains a putative enzymatic motif, which is characteristic of serine hydrolases. The Ima1 catalytic mutant failed to complement Snc1 surface levels in ima1∆ cells, suggesting that this motif may have catalytic activity required for AP-1R-mediated sorting. Subcellular fractionation revealed that this enzymatic domain is not integral to the membrane, as predicted. Together with the finding that the Ima1 N-terminal region binds to Apm2, it is conceivable that the Ima1 C-terminal enzymatic domain may be accessible to its substrates at sites of AP-1R function. The finding that Ima1 is a candidate enzyme that binds to the Apm2 B-domain is interesting, as recent studies in mammalian cells have shown that the  subunit is an important target of regulation. These studies are changing the canonical view of  subunit function, in which their primary role is to bind tyrosine-based cargo. Interaction between the  subunit and specific lipids and lipid-regulatory enzymes is one recurrent theme of  97  these studies. While some of these regulatory enzymes bind to the tyrosine cargo-binding site in a canonical manner, others bind to regions that are distinct from this site, like Ima1. Interestingly, the subdomain B has been shown to regulate some of these interactions. For example, differences in the B domain of AP-1A 1 and AP-1B 2 confer differential localization of these complexes in polarized cells (Fields et al., 2010). Understanding the mechanisms by which these -binding proteins regulate AP function could thus inform us how Ima1 may contribute to AP-1R sorting, and how we can best direct future experiments to identify this role. These mechanisms are reviewed below. AP complexes – Interactions with lipids and lipid-regulatory enzymes AP complexes can influence lipid metabolism, and can specifically participate in the generation of lipids that recruit them to their target membranes, through interaction with lipid regulatory enzymes. APs and lipid regulators have been shown to interact in positive feedback loops; binding of an enzyme to its target AP has been shown to stimulate enzymatic activity, which can result in the localized generation of phospholipids (Fields et al., 2010; Kahlfeldt et al., 2010). In turn, the increased lipids generated through this binding leads to further AP recruitment and membrane stabilization. The  subunit appears to be an important target for these interactions, and multiple  subunits have now been shown to bind lipid kinases. PI(3,4,5)P3 enrichment at recycling endosomes is required for recruitment of AP-1B. In turn, PI(3,4,5)P3 accumulation is dependent on the recruitment of the lipid kinase PIPKI-p90 to recycling endosomes by AP-1B (Fields et al., 2010). PIPKIp90 interacts directly with 1B via a tyrosine-based motif, and this interaction stimulates the kinase activity of the enzyme. Furthermore, -binding enzymes can have additional roles in AP regulation. PIPKI-p90, for example, also binds to E-cadherin, an AP-1B-specific cargo. The dual interaction supports that PIPK serves as both a scaffold, linking E-cadherin to AP-complexes, and a regulator of spatially restricted lipid generation (Ling et al., 2007). PIPKI-p90 also binds directly to AP-2 2, and is required for the generation of PI(4,5)P2 during endocytosis (Bairstow et al, 2006; Krauss et al., 2006). The mechanism of this interaction is unclear. One study has suggested that PIPKI-p90 interacts with the cargobinding pocket of 2 through a tyrosine motif (Bairstow et al, 2006). In contrast, a second study has shown that this interaction is not dependent on the tyrosine motif, and may therefore involve a site of 2 that does not include the cargo-binding pocket (Krauss et al., 2006). Endocytic cargo protein binding to 2 leads to a potent stimulation of PIPK activity. These data thus identify a positive feedback loop consisting of endocytic cargo proteins, AP-  98  2µ, and PIPK type I, which may provide a specific pool of PI(4,5)P2 dedicated to clathrin/AP2-dependent receptor internalization. The mechanism by which binding of PIPKI to the  subunit stimulates its kinase activity remains to be determined. The AP-2  subunit has also recently been shown to interact directly with phospholipase D (PLD1), a receptor-associated signaling protein and this facilitates the membrane recruitment of AP2 and the endocytosis of epidermal growth factor receptor (EGFR). PLD catalyzes the hydrolysis of phosphatidylcholine (PC) to choline and phosphatidic acid (PA) (Lee et al., 2009). Interestingly, like Ima1, PLD is also a serine hydrolase. The PLD1-2 interaction requires the binding of PLD1 with phosphatidic acid, its own product. A model was proposed, in which auto-regulatory interactions beween PLD1 and PA promote 2 binding, which subsequently facilitates EGFR endocytosis in response to receptor activation. It has been hypothesized that PA, in turn, stimulates a phosphoinositide kinase to generate PI(4,5)P2 at the plasma membrane. It was therefore suggested that PA generation by PLD1 activation is a key molecular event that causes the local accumulation of PI(4,5)P2 through direct activation of a kinase and enhancing the AP-2/kinase pathway by placing these two molecules in close proximity to facilitate EGFR endocytosis. Members of the Dishevelled family of proteins have been found to bind directly to AP2 2 in both mammals and C. elegans. Dishevelled (Dvl) functions as an adaptor between AP-2 and other components of the Wnt signaling pathway. This interaction is required for internalization of the Wnt signaling protein Frizzled. The interaction between Dvl and AP2  is bipartite, and requires simultaneous association of the Dvl DEP domain and a tyrosinebased signal. The tyrosine-based signal interacts with the 2 cargo binding pocket, while the DEP domain binds at one end of the elongated, C-terminal domain of 2. This domain:domain interface further reinforces that parts of the 2 surface distinct from the tyrosine-motif pocket can help recruit specific receptors or adaptors into a clathrin coated pit. It is also considered likely that other parts of this surface may also have partners, as yet unidentified. The incorporation of two interaction modes into one cargo molecule further suggests that combinatorial recognition (and combinatorial cargo addressing) may be more widespread than previously suspected (Yu et al., 2007 and 2010). Wnt signaling stimulates the production of PI(4,5)P2 through Frizzled and Dvl, which activates the lipid kinases phosphoinositide kinases 4 and 5. (MacDonald et al., 2009; Qin et al., 2009). This further supports the direct role for lipids and lipid-regulatory enzymes in driving the membrane recruitment and function of AP complexes.  99  4.3.6. Is Ima1 important for the generation of lipids required for AP-1R function? What is the enzymatic function of Ima1, and could it regulate the generation of lipids important in AP-1R-mediated sorting? The finding that Ima1 binds to the Apm2 B domain suggests this possibility, in light of the work described in the previous section. The serine hydrolase family includes many proteins that can influence lipid metabolism, including esterases and lipases. Confirming that Ima1 is a serine hydrolase and identifying its potential substrates could help elucidate how it contributes to AP-1R function. Experimental strategies to dissect its enzymatic function are discussed below. The first step will be to verify that Ima1 is a serine hydrolase, as predicted based on homology. To address this central question, we can take advantage of a chemical strategy referred to as activity-based protein profiling (ABPP) that uses active site-directed probes to profile the functional state of enzymes. ABPP probes label active enzymes by binding covalently to the active site. They bind enzymes only in their active state, but not their inactive precursor or inhibitor-bound forms. ABPP probes remain covalently bound to the active site, and in this way impede catalytic activity. These probes contain a tag or label, such as biotinylation, providing a means to detect them (Barglow and Cravatt, 2007; Heal et al., 2011). Fluorophosphonate (FP) probes have been developed that target active serine hydrolases (Simon and Cravatt., 2010). In order to determine if Ima1 is a serine hydrolase, the FP probe would be added to cell lysates. Ima1 would then be affinity purified, and western blotting would be carried out to determine if the probe was incorporated by detection with anti-biotin. As the probe only labels active enzymes, we could test whether mutation of the potential catalytic residues prevents probe incorporation. Other questions that could be addressed by ABPP are whether AP-1R binds the active or inactive form of Ima1, and whether binding to AP-1R stimulates Ima1 enzymatic activity. Since FP probes inhibit enzymatic activity, whether Apm2 preferentially coimmunoprecipitates with the labeled or non-labeled form of Ima1 could be investigated. Whether binding to AP-1R stimulates Ima1 enzymatic activity could also be evaluated, by comparing the amount of probe that is incorporated into Ima1 upon deletion or overexpression of APM2. 4.3.7. Determination of Ima1 substrates If Ima1 were confirmed to be a serine hydrolase by the studies described above, it would next be important to identify its biologically relevant substrate. Serine hydrolases comprise a large class of enzymes, incuding proteases, esterases, lipases, peptidases, and  100  aminases, and therefore have diverse substrate specificity. In recent years high-throughput screening assays have been developed to identify substrates of hydrolytic enzymes. The majority of these assays are based on screening an enzyme in question against a library of structurally diverse synthetic substrates. Upon reaction with the enzyme, its substrates release a colored or fluorescent product, permitting their identification. Many of these substrates are now commercially available, and can be tested in parallel in 96-well microtiter plates. This method, called enzyme activity fingerprinting (Reymond, 2008), has been used to classify different types of enzymes, including lipases, proteases, and peptidases, and to differentiate between closely related enzymes within these groups (Wahler et al., 2002; Konarzycka-Bessler and Bornscheuer, 2003). By screening a diverse library of these compounds in the presence of Ima1, we may be able to refine the class of enzyme to which Ima1 belongs, by identifying which of these substrates it reacts with. This could help us to identify potentially biologically relevant substrates. By doing so, we may be able to decipher whether Ima1 has a role in the remodeling of lipid membranes, and which lipids are potential substrates. This would be beneficial in understanding the role of lipid regulation in endosomal sorting, and could help us formulate directed hypotheses about how Ima1 activity may regulate the AP-1R sorting pathway. 4.3.8. Investigating the role of TMC04 in higher cells Identifying the enzymatic activity and substrates of Ima1 could help us understand the potential role of its mammalian homolog TMCO4. Like most other mammalian serine hydrolases, the role of TMCO4 remains uncharacterized. Although serine hydrolases make up 1% of the mammalian proteome, nearly half of them remain completely uncharacterized with respect to substrates and functions. Of the enzymes that have been characterized, many have been shown to have important biological functions. They are important targets of several pharmaceutical agents, including proteases involved in the coagulation cascade, and amidases required for the metabolism of signaling molecules. Widely used drugs targeted against specific human serine hydrolases include Angiomax for cardiovascular disease, Xenical for obesity, and Aricept and Cognex for Alzheimer’s disease, as well as drugs in development for diabetes, arthritis, and cancer (Baxter et al., 2004). It is of great interest in the field to gain a more complete understanding of these enzymes, and our work on Ima1 may contribute important insight into the function of TMCO4. One perplexing issue is that while Ima1 is highly conserved in all organisms, Apm2 is not. Apm2 is unique as a medium chain, as it is considerably larger than, and shares only  101  30% homology to other  subunits. This may suggest that although AP-1R is not conserved strictly based on similarity, it may carry out an analogous role to an AP complex in higher cells. Alternatively, Ima1 may have evolved to carry out function unrelated to AP-mediated sorting in multi-cellular organisms. To investigate these possibilities, methods such as mass spectroscopy could be used to identify TMCO4-interacting factors in mammalian cells. A more directed approach could also be used to determine if Ima1 interacts with specific AP complex components by co-immunoprecipitation.  4.4. Genetic interaction profiling to identify complexes and pathways required for Snc1 transport 4.4.1. Phenotypic profiling and the construction of genetic interaction networks Genetic interaction profiling was used to screen the top candidates from the primary screen against a panel of query mutants in multiple trafficking pathways. This analysis allowed these candidates to be classified based on shared genetic interactions with these query genes, and enabled discovery of a set of genes enriched for functions in Snc1 endocytic transport. In this case, the cell surface levels of the GSS reporter in each double mutant served as a quantitative phenotypic readout. These results re-enforce the power of genetic interaction profiling in identifying genes that are highly influential to a given biological process after performing a genomic screen, and how these genes are interrelated. Many previous studies have used genetic interaction profiling to infer functional relationships between genes required for a given process (reviewed in Boone et al., 2007). In the vast majority of these studies, however, growth is used as a phenotypic readout. The resulting networks from these studies therefore leave out processes that have no effect on cell viability or growth (Dixon et al., 2009). The quantitative nature of our assay was beneficial in overcoming this limitation, allowing for more precise determination of phenotypes directly related to Snc1 transport, and the accurate assessment of their genetic relationships. This study took advantage of recent advances in technology, which have made it possible to measure large numbers of genetic interactions systematically and in parallel (Dixon et al., 2009). An E-Mapping approach was used to determine the genetic interaction profile between the top candidates from the primary screen, and a selected panel of mutants representing a variety of intracellular transport pathways, instead of the whole genome. This approach has several advantages beyond decreasing the number of  102  interactions that require analysis. It also increases the signal to noise ratio because the frequency of genetic interactions is higher between genes acting in related pathways, and provides a richer set of patterns for analysis. In previous studies, this subset of genes has been selected based on multiple criteria, including shared protein localization (Schuldiner et al., 2005), or protein-protein interaction data (Collins et al., 2007). This selection process has multiple limitations. Selecting genes based purely on either of these criteria would miss important regulatory factors, such as signaling proteins, which may mediate strong effects on spatially distant processes. As large-scale protein interaction data suffers from a high degree of false positive and false negative interactions (Szilágyi et al., 2005), starting with this dataset may miss important factors, or may include factors that are not relevant to the process in question. For example, the interactions found in two independent genome-wide Y2H studies in S. cerevisiae show little overlap and are estimated to have a 50% falsepositive rate (von Mering et al., 2002; Ito et al., 2001). We took advantage of the E-Mapping approach to investigate the profile of genetic interactions of our top candidates, in combination with a panel of query mutants with established function in diverse intracellular transport pathways. In our approach, we selected our gene set based on a sensitive and quantitative assay. By selecting our gene set based on this criteria, we avoided many of the limitations that have been observed in other less stringent methods such as protein localization or interaction data. Furthermore, an integrative approach, which incorporated both genetic and physical interaction data, was used to refine the network of genes important for regulating Snc1 surface levels into discrete complexes and pathways. Integration of multiple data sets have generally been shown to add to the confidence of inferred related functions, and reduce the noise associated with individual genome-scale approaches. By using this integrative approach, we were able to conceive strong hypotheses about the function of uncharacterized genes, based on both a shared genetic interaction profile, and physical interaction with other wellstudied genes. Despite the insight generated by this study, this screening approach had important limitations. First, there could have been errors in the deletion collections. It is widely known that these mutant arrays are subject to genetic changes as a result of manipulation and selective pressure, including aneuploidy and second site mutations (Rancati et al., 2008; Yuen et al., 2007). As a multi-step selection process was required to generate double mutants for the interaction analysis, undesired mutations and rearrangements could have  103  been introduced. This would lead to false interpretation of the phenotypic data for some of these genes. A gene involved in Snc1 transport could have been missed, for example, if a second site mutation suppressed the defect caused by deletion of the gene in question (false negative). A gene with no role in this process could also be falsely interpreted to perturb this process if a second site mutation occurs in a functionally relevant gene (false positive). Screening two independent genome wide collections and averaging the results for both helped address this issue. Furthermore, many candidates were also verified by independent knockout or complementation. 4.4.2. Endocytic processes are part of a broader cellular context The systems-level approach allowed us to identify factors involved in Snc1 endocytic recycling, and also to determine how these factors are interrelated in a broad cellular context. This work revealed surprising diversity among the processes involved. In addition to cellular transport, we identified proteins involved in roles including signaling, chromatin modification, transcription and translation, and lipid regulation. The role of some of these components can be readily explained by the current knowledge of the regulation of intracellular transport pathways, such as the critical role of actin polymerization and phospholipid distribution. We identified a strong requirement for the cytosolic prefoldin-GimC complex, which acts as a co-chaperone in actin folding (Siegers et al., 1999). Some of the transcripional/translational factors identified regulate the expression of specific phospholipid species known to be important for endocytic processes Ikeda et al., 2008). Some identified genes, however, have molecular functions that are not immediately reconcilable with membrane trafficking, including various signaling proteins. Recent studies have uncovered a wealth of evidence that endocytic transport and signaling are deeply integrated in the cellular program, and that these processes are closely connected. (Scita and Di Fiore, 2010; Sorkin and von Zastrow, 2009). Despite evidence for connections between transport and other cellular processes, a caveat of our study is that the increased surface levels of our Snc1 reporter could be an indirect result of increased expression in some mutant strains. Although we did not directly address this issue, the effect of our candidates on GSS reporter expression will be important to address in future studies. This could be accomplished by creating a transcriptional reporter, in which the upstream regions of the GSS integration site are fused with LacZ. Expression of this reporter could be evaluated by a B-galactosidase assay in candidate mutants. Increased B-galactosidase levels in these mutants could inform us whether these  104  mutants are involved in the transciption of Snc1. 4.4.3. Assessing the role of essential genes in Snc1 transport Another important limitation of this screening approach is that it considered the ~5000 non-essential genes, but not the ~1000 essential genes in yeast. Therefore, our view of Snc1 endocytic recycling is incomplete. Essential genes, by definition, encode critical cellular functions that are not buffered by redundant functions or pathways (Ben-Aroya et al., 2010). Inclusion of essential genes would be expected to have a significant impact on the size and complexity of our Snc1 sorting network, as multiple studies have demonstrated that they exhibit significantly more genetic interactions than nonessential genes, and the essential network is therefore significantly denser than the nonessential network. (Davierwala et al, 2005). Furthermore, essential genes tend to be more highly conserved in evolution; 38% of essential yeast proteins have easily identifiable counterparts in humans, versus 20% for nonessential genes (Ben-Aroya et al., 2010). Inclusion of essential genes could have therefore improved our study by adding to the connectivity between relevant pathways, and by expediting the analysis of critically important genes involved in endocytic recycling. As many genome-wide studies have faced this limitation, this issue has prompted the development of genome-wide collections that facilitate the investigation of essential gene function. Most notably, temperature-sensitive (ts) mutants have been used with great success to analyze essential genes, and current work is in progress to develop ts alleles covering the yeast genome. Ts mutations are typically missense mutations, which retain the function of a specific essential gene at standard (permissive) temperatures, but lack that function at a defined high (non permissive) temperature. These mutants therefore facilitate analysis of physiologic changes that follow controlled inactivation of a gene by shifting cells to a non-permissive temperature. A subset of 250 ts mutants, including alleles for all uncharacterized essential genes and genes with human counterparts, is now ready for distribution (Ben-Aroya et al., 2008 and 2010). A complete set of these genes is expected to be available shortly, and will be tested for a role in Snc1 endocytic transport. Another approach involves the replacement of the native promoter of each gene with one that can be rapidly repressed. This includes the use of the tetracycline (tet)-regulatable promoter (Mnaimneh et al., 2004). In this system, the expression of each gene is controlled by a promoter that can be shut off by adding the tetracycline analog doxycyline, to the growth medium. Currently, TetO-promoter alleles have been created for over two-thirds of all  105  essential yeast genes. A major advantage of this system is that the native open reading frame of each gene is maintained. The addition of doxycycline to the growth media has also been shown to have little effect on yeast physiology at concentrations used for promoter shutoff. These genes have been developed for two-thirds of all yeast essential genes. 4.4.4. Relationships between endocytic recycling factors; implications in disease A better understanding of the machinery regulating the selective transport of proteins within the endosomal system, and their relationships is critical for deciphering the genetic causes of diseases relating to trafficking malfunction. One feature common among diseases of intracellular transport is the considerable underlying genetic heterogeneity, which is a direct result of the complex regulatory mechanisms involved in these pathways. Many of these transport diseases present common clinical phenotypes, but are due to defects in seemingly unrelated gene products. As a result of this heterogeneity, it is difficult to carry out studies based purely on clinical phenotypes because of variable underlying mutations. More complete knowledge of how candidate genes could be related will therefore aid in the interpretation of the results from studies investigating diseases of intracellular transport (Howell et al., 2006; Olkkonen and Ikonen, 2006). This study demonstrated unanticipated links between factors required for endocytic recycling. One example is the coordinated roles of yAP180 and synaptojanin in Snc1 internalization. Neurological defects are observed in the absence of both AP180 and synaptojanin in mouse models, although these defects have not previously been linked (Cao et al., 2010; Kim et al., 2002). Altered levels of clathrin regulatory proteins, including dynamin I, AP180, and synaptophysin have been observed in AD patients and mouse models, as compared to controls, suggesting that malfunctioning of clathrin dynamics may contribute to disease pathology. This example illustrates how a better understanding of relationships between transport factors could generate a more complete picture of the mechanisms underlying disorders of intracellular transport. Therapeutic intervention for the manipulation of endocytic transport pathways could provide a means for treating diseases resulting from defects in these transport processes. In Alzheimer’s patients, for example, defective transport of amyloid precursor protein (APP) in axons leads to the generation of AB plaques and the progression of neurodegenerative disease (Mayeux and Hyslop, 2008). Manipulating the transport of ABcontaining vesicles in the endosomal system may therefore facilitate clearance of accumulated APP. There has been some significant progress towards this goal. It has been  106  found that APP processing to its plaque producing form is enhanced by inhibition of cholesterol transport, and that treatments that restore normal cholesterol levels and enhance APP transport to rab7-positive late endosomes may reduce aberrant APP processing (Stein et al., 2003; Huttunen et al., 2009). Furthermore, many cancers result from the increased recycling of growth factor receptors to the cell surface (Hyun and Ross., 2004; Rao et al., 2003). Implementing therapies that result in receptor down-regulation through shifting the balance between recycling and degradation may therefore positively impact outcomes. It is therefore of interest to develop gene therapies that effectively target the specific pathway in question, without significantly affecting other important transport steps. Current work in the field aims to develop therapies that selectively block or enhance Rab protein function, or stimulate cofactors that shunt cargo along the desired pathway, including increased expression at the cell surface, or down-regulation via enhancing degradation. This approach would benefit significantly from a greater understanding of cargo-specific cellular transport pathways, which was an important issue addressed by this study. 4.4.5. Future perspectives The main theme that has been highlighted by this work is that many genes involved in endocytic recycling remain unidentified, due to functional redundancy, or cargo-specific effects. These findings highlight the importance of considering multiple cargo proteins, and the development of highly sensitive quantitative assays to detect defects in this transport process. Future application of our genome-wide analysis to different reporter proteins is expected to aid in the identification of additional cargo-specific machinery. Furthermore, the effects of different environmental conditions should also be considered. The endocytosis of some cargo is directly regulated by environmental conditions, including many nutrient and amino acid transporters, whose cell surface expression is regulated based on external nutrient availability (Nikko and Pelham, 2009). Studies of the same cargo under different conditions may help us determine if genes involved in their uptake are context-dependent. Although the vast majority of functional genetic screens have been carried out in yeast, with the advent of RNAi and other knockdown techniques, it is expected that it will not be long before these types of screens could be applied to multi-cellular systems. Our quantitative phenotypic and genetic interaction approaches will provide considerable guidance in the identification of transport components in higher cells, and the relationships between them. Together, the emerging development of technology to study genetic  107  interactions in mammalian cells, combined with the analytical methods used in this study, could provide us with considerable insight into how defects in cellular transport pathways can contribute to human disease. Furthermore, application of these studies to different cell types could provide insight into the similarities and differences in endocytic transport in cell types that contain specialized organelles and pathways, such as neurons and polarized epithelial cells.  108  4.4. Illustrations  Plasma Membrane  1 2 4 7  PI(4,5)P2  3 PI(3)P  TGN 5  AP-1  EE AP-1R/ IMA1  LEGEND Snc1  Clathrin  6  WASP/Myo NPF-Sso1 Actin yAP180  Chs3  Amphyphysin Ldb17  Ldb17  Inp52/Synaptojanin  Illustration 4.1. New factors required for Snc1 endocytic recycling. A proposed model summarizing the findings from this study regarding the functional requirements for clathrin and the clathrin adaptors yAP180s and Ldb17 in Snc1 internalization and AP-1R/Ima1 in Snc1 recycling. (1) yAP180 is recruited to the plasma membrane by combinatorial recognition of the membrane lipid PI(4,5)P2 and cargo. Snc1, but not all endocytic cargo (including NPF-Sso1) is recognized by yAP180 for internalization. Additional components of the early coat complex are subsequently recruited and clathrin assembly begins (2) Late coat components are then recruited, including components of the actin polymerization machinery. Ldb17 joins the late coat complex, just before the onset of actin polymerization, and is required for both normal coat and actin dynamics. (3) Actin polymerization begins, and the vesicle begins to internalize. (4) Inp52/synaptojanin mediates the hydrolysis of PI(4,5)P2 to PI(3), leading to uncoating of yAP180. (5) The free vesicle fuses with the early endosome to which it delivers Snc1. (6) Snc1 is recognized by AP-1R, possibly at the early endosome. Ima1 associates with the AP1R-specific medium subunit Apm2. AP-1R/Ima1 mediate Snc1 recycling to the Trans Golgi Network (TGN), from which it is Snc1 is subsequently re-delivered to the plasma membrane.  109  Illustration 4.2. AP-1R transport function: Model 1 In this model, AP-1R is present on early endosomes (EE), and directs internalized Snc1 to either the TGN or an intermediate recycling endosome (RE), from which Snc1 recycles back to the cell surface. In the absence of AP-1, Snc1 may avoid transport through these intermediate compartments and recycle back to the surface direcly from EEs (hatched arrow).  Illustration 4.3. AP-1R transport function: Model 2 In this model, AP-1R may function at the TGN to incorporate Snc1 into the light vesicle fraction, which may arise either directly from the TGN or from early endosomes (EE) (L: red arrows). In the absence or AP-1R, Snc1 may be missorted into the dense vesicle fraction (D: blue arrows).  110  BIBLIOGRAPHY Aghamohammadzadeh and Ayscough. Differential requirements for actin during yeast and mammalian endocytosis. Nat Cell Biol (2009) vol. 11 (8) pp. 1039-42 Aguilar et al. The yeast Epsin Ent1 is recruited to membranes through multiple independent interactions. J.Biol.Chem. (2003) vol. 278 (12) pp. 10737-43 Andersen et al. Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein. Proc.Natl.Acad.Sci.U.S.A. (2005) vol. 102 (38) pp. 13461-6 Ang et al. The Rab8 GTPase selectively regulates AP-1B-dependent basolateral transport in polarized Madin-Darby canine kidney cells. J.Cell Biol. (2003) vol. 163 (2) pp. 339-350 Aniento et al. An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes. J.Cell Biol. (1996) vol. 133 (1) pp. 29-41 Aridor and Hannan. Traffic jams II: an update of diseases of intracellular transport. Traffic (2002) vol. 3 (11) pp. 781-90 Aridor and Hannan. Traffic jam: a compendium of human diseases that affect intracellular transport processes. Traffic (2000) vol. 1 (11) pp. 836-51 Aruna et al. Identification of a hypothetical membrane protein interactor of ribosomal phosphoprotein P0. J Biosci (2004) vol. 29 (1) pp. 33-43 Ascough. Endocytosis: Actin in the driving seat. Curr.Biol. (2004) vol. 14 (3) pp. R124-6 Ayscough et al. High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A. J.Cell Biol. (1997) vol. 137 (2) pp. 399-416 Bader and Hogue. An automated method for finding molecular complexes in large protein interaction networks. BMC Bioinformatics (2003) vol. 4 pp. 2 Bairstow et al. Type Igamma661 phosphatidylinositol phosphate kinase directly interacts with AP2 and regulates endocytosis. J Biol Chem (2006) vol. 281 (29) pp. 20632-42 Bankaitis et al. Isolation of yeast mutants defective in protein targeting to the vacuole. Proc.Natl.Acad.Sci.U.S.A. (1986) vol. 83 (23) pp. 9075-9 Bao et al. AP180 maintains the distribution of synaptic and vesicle proteins in the nerve terminal and indirectly regulates the efficacy of Ca2+-triggered exocytosis. J Neurophysiol (2005) vol. 94 (3) pp. 1888-903 Barglow and Cravatt. Activity-based protein profiling for the functional annotation of enzymes. Nat Methods (2007) vol. 4 (10) pp. 822-7 Barr. Vesicular transport. Essays Biochem. (2000) vol. 36 pp. 37-46  111  Baxter et al. Synergistic computational and experimental proteomics approaches for more accurate detection of active serine hydrolases in yeast. Mol Cell Proteomics (2004) vol. 3 (3) pp. 209-25 Becherer et al. Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast. Mol.Biol.Cell (1996) vol. 7 (4) pp. 579-94 Behnia and Munro. Organelle identity and the signposts for membrane traffic. Nature (2005) vol. 438 (7068) pp. 597-604 Ben-Aroya et al. Making temperature-sensitive mutants. Meth Enzymol (2010) vol. 470 pp. 181-204 Ben-Aroya et al. Toward a comprehensive temperature-sensitive mutant repository of the essential genes of Saccharomyces cerevisiae. Mol Cell (2008) vol. 30 (2) pp. 248-58 Bensen et al. Synthetic genetic interactions with temperature-sensitive clathrin in Saccharomyces cerevisiae. Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network. Genetics (2000) vol. 154 (1) pp. 83-97 Berman and Greenamyre. Update on Huntington's disease. Curr Neurol Neurosci Rep (2006) vol. 6 (4) pp. 281-6 Bishop. Dynamics of endosomal sorting. Int.Rev.Cytol. (2003) vol. 232 pp. 1-57 Black and Pelham. A selective transport route from Golgi to late endosomes that requires the yeast GGA proteins. J.Cell Biol. (2000) vol. 151 (3) pp. 587-600 Blader et al. GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitro. Mol.Biol.Cell (1999) vol. 10 (3) pp. 581-96 Bock et al. A genomic perspective on membrane compartment organization. Nature (2001) vol. 409 (6822) pp. 839-41 Boehm and Bonifacino. Genetic analyses of adaptin function from yeast to mammals. Gene (2002) vol. 286 (2) pp. 175-86 Bonangelino et al. Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae. Mol Biol Cell (2002) vol. 13 (7) pp. 2486-501 Bonifacino and Glick. The mechanisms of vesicle budding and fusion. Cell (2004) vol. 116 (2) pp. 153-66 Bonifacino. The GGA proteins: adaptors on the move. Nat Rev Mol Cell Biol (2004) vol. 5 (1) pp. 23-32  112  Bonifacino and Lippincott-Schwartz. Coat proteins: shaping membrane transport. Nat Rev Mol Cell Biol (2003) vol. 4 (5) pp. 409-14 Bonifacino and Traub. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu.Rev.Biochem. (2003) vol. 72 pp. 395-447 Boone et al. Exploring genetic interactions and networks with yeast. Nat Rev Genet (2007) vol. 8 (6) pp. 437-49 Botos and Wlodawer. The expanding diversity of serine hydrolases. Curr Opin Struct Biol (2007) vol. 17 (6) pp. 683-90 Bouché et al. The cellular fate of glucose and its relevance in type 2 diabetes. Endocr Rev (2004) vol. 25 (5) pp. 807-30 Brett and Traub. Molecular structures of coat and coat-associated proteins: function follows form. Curr Opin Cell Biol (2006) vol. 18 (4) pp. 395-406 Brodsky et al. Biological basket weaving: formation and function of clathrin-coated vesicles. Annu.Rev.Cell Dev.Biol. (2001) vol. 17 pp. 517-568 Brunger. Structure and function of SNARE and SNARE-interacting proteins. Q.Rev.Biophys. (2005) vol. 38 (1) pp. 1-47 Burston et al. Regulators of yeast endocytosis identified by systematic quantitative analysis. J.Cell Biol. (2009) vol. 185 (6) pp. 1097-110 Cao et al. Changed clathrin regulatory proteins in the brains of Alzheimer's disease patients and animal models. J Alzheimers Dis (2010) vol. 22 (1) pp. 329-42 Carlton and Cullen. Coincidence detection in phosphoinositide signaling. Trends Cell Biol. (2005) vol. 15 (10) pp. 540-7 Carroll et al. A yeast killer toxin screen provides insights into a/b toxin entry, trafficking, and killing mechanisms. Dev Cell (2009) vol. 17 (4) pp. 552-60 Caviston and Holzbaur. Huntingtin as an essential integrator of intracellular vesicular trafficking. Trends Cell Biol. (2009) vol. 19 (4) pp. 147-55 Chen et al. Ypt31/32 GTPases and their novel F-box effector protein Rcy1 regulate protein recycling. Mol Biol Cell (2005) vol. 16 (1) pp. 178-92 Chen and Davis. Ubiquitin-independent entry into the yeast recycling pathway. Traffic (2002) vol. 3 (2) pp. 110-23 Chen and Davis. Recycling of the yeast a-factor receptor. J.Cell Biol. (2000) vol. 151 (3) pp. 731-8 Chidambaram et al. ENTH domain proteins are cargo adaptors for multiple SNARE proteins at the TGN endosome. J.Cell.Sci. (2008) vol. 121 (Pt 3) pp. 329-38  113  Chidambaram et al. Specific interaction between SNAREs and epsin N-terminal homology (ENTH) domains of epsin-related proteins in trans-Golgi network to endosome transport. J.Biol.Chem. (2004) vol. 279 (6) pp. 4175-9 Collins et al. Quantitative genetic interaction mapping using the E-MAP approach. Meth Enzymol (2010) vol. 470 pp. 205-31 Collins et al. Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map. Nature (2007) vol. 446 (7137) pp. 806-10 Collins et al. Molecular architecture and functional model of the endocytic AP2 complex. Cell (2002) vol. 109 (4) pp. 523-35 Conibear E. Converging views of endocytosis in yeast and mammals. Curr Opin Cell Biol (2010) vol. 22 (4) pp. 513-18 Conibear et al. Vps51p mediates the association of the GARP (Vps52/53/54) complex with the late Golgi t-SNARE Tlg1p. Mol Biol Cell (2003) vol. 14 (4) pp. 1610-23 Conner and Schmid. Differential requirements for AP-2 in clathrin-mediated endocytosis. J.Cell Biol. (2003) vol. 162 (5) pp. 773-9 Corbacho et al. A genome-wide screen for Saccharomyces cerevisiae nonessential genes involved in mannosyl phosphate transfer to mannoprotein-linked oligosaccharides. Fungal Genet Biol (2005) vol. 42 (9) pp. 773-90 Couve et al. Yeast synaptobrevin homologs are modified posttranslationally by the addition of palmitate. Proc.Natl.Acad.Sci.U.S.A. (1995) vol. 92 (13) pp. 5987-91 Cremona et al. Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell (1999) vol. 99 (2) pp. 179-88 D'Angelo et al. The multiple roles of PtdIns(4)P -- not just the precursor of PtdIns(4,5)P2. J Cell Sci (2008) vol. 121 (Pt 12) pp. 1955-63 Darsow et al. Invertase fusion proteins for analysis of protein trafficking in yeast. Meth Enzymol (2000) vol. 327 pp. 95-106 Davierwala et al. The synthetic genetic interaction spectrum of essential genes. Nat Genet (2005) vol. 37 (10) pp. 1147-52 Davis et al. Cis- and trans-acting functions required for endocytosis of the yeast pheromone receptors. J.Cell Biol. (1993) vol. 122 (1) pp. 53-65 De Matteis and D'Angelo. The role of the phosphoinositides at the Golgi complex. Biochem.Soc.Symp. (2007) (74) pp. 107-16 De Matteis and Godi. PI-loting membrane traffic. Nat.Cell Biol. (2004) vol. 6 (6) pp. 487-492  114  Dell'Angelica et al. Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor. Mol Cell (1999) vol. 3 (1) pp. 11-21 Deng et al. Gga2 mediates sequential ubiquitin-independent and ubiquitin-dependent steps in the trafficking of ARN1 from the trans-Golgi network to the vacuole. J.Biol.Chem. (2009) vol. 284 (35) pp. 23830-41 Derby and Gleeson. New insights into membrane trafficking and protein sorting. Int.Rev.Cytol. (2007) vol. 261 pp. 47-116 Dewar et al. Novel proteins linking the actin cytoskeleton to the endocytic machinery in Saccharomyces cerevisiae. Mol Biol Cell (2002) vol. 13 (10) pp. 3646-61 Dickman et al. Altered synaptic development and active zone spacing in endocytosis mutants. Curr.Biol. (2006) vol. 16 (6) pp. 591-8 Dietrich et al. The SNARE Ykt6 is released from yeast vacuoles during an early stage of fusion. EMBO Rep (2005) vol. 6 (3) pp. 245-50 Dittman and Kaplan. Factors regulating the abundance and localization of synaptobrevin in the plasma membrane. Proc.Natl.Acad.Sci.U.S.A. (2006) vol. 103 (30) pp. 11399-404 Dixon et al. Systematic mapping of genetic interaction networks. Annu Rev Genet (2009) vol. 43 pp. 601-25 Donaldson et al. Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature (1992) vol. 360 (6402) pp. 350-2 Drees et al. A protein interaction map for cell polarity development. J.Cell Biol. (2001) vol. 154 (3) pp. 549-71 Edeling et al. Molecular switches involving the AP-2 beta2 appendage regulate endocytic cargo selection and clathrin coat assembly. Dev Cell (2006) vol. 10 (3) pp. 329-42 Edeling et al. Life of a clathrin coat: insights from clathrin and AP structures. Nat.Rev.Mol.Cell Biol. (2006) vol. 7 (1) pp. 32-44 Elkhaimi et al. Combinatorial regulation of phospholipid biosynthetic gene expression by the UME6, SIN3 and RPD3 genes. Nucleic Acids Res (2000) vol. 28 (16) pp. 3160-7 Engqvist-Goldstein and Drubin. Actin assembly and endocytosis: from yeast to mammals. Annu.Rev.Cell Dev.Biol. (2003) vol. 19 pp. 287-332 Evans and Owen. Endocytosis and vesicle trafficking. Curr.Opin.Struct.Biol. (2002) vol. 12 (6) pp. 814-821 Fauré et al. ARF1 regulates Nef-induced CD4 degradation. Curr.Biol. (2004) vol. 14 (12) pp. 1056-64  115  Fernández and Payne. Laa1p, a conserved AP-1 accessory protein important for AP-1 localization in yeast. Mol.Biol.Cell (2006) vol. 17 (7) pp. 3304-17 Fields et al. Phosphatidylinositol 3,4,5-trisphosphate localization in recycling endosomes is necessary for AP-1B-dependent sorting in polarized epithelial cells. Mol.Biol.Cell (2010) vol. 21 (1) pp. 95-105 Fields et al. Phosphatidylinositol 3,4,5-trisphosphate localization in recycling endosomes is necessary for AP-1B-dependent sorting in polarized epithelial cells. Mol Biol Cell (2010) vol. 21 (1) pp. 95-105 Fields et al. v-SNARE cellubrevin is required for basolateral sorting of AP-1B-dependent cargo in polarized epithelial cells. J.Cell Biol. (2007) vol. 177 (3) pp. 477-88 Fölsch et al. The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains. J.Cell Biol. (2003) vol. 163 (2) pp. 351-62 Fölsch et al. Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells. J.Cell Biol. (2001) vol. 152 (3) pp. 595-606 Foote and Nothwehr. The clathrin adaptor complex 1 directly binds to a sorting signal in Ste13p to reduce the rate of its trafficking to the late endosome of yeast. J.Cell Biol. (2006) vol. 173 (4) pp. 615-26 Franzusoff et al. Localization of components involved in protein transport and processing through the yeast Golgi apparatus. J.Cell Biol. (1991) vol. 112 (1) pp. 27-37 Gabriely et al. Involvement of specific COPI subunits in protein sorting from the late endosome to the vacuole in yeast. Mol Cell Biol (2007) vol. 27 (2) pp. 526-40 Galan et al. Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast. Mol Cell Biol (2001) vol. 21 (9) pp. 3105-17 Gan et al. The epithelial-specific adaptor AP1B mediates post-endocytic recycling to the basolateral membrane. Nat.Cell Biol. (2002) vol. 4 (8) pp. 605-609 Gavin et al. Proteome survey reveals modularity of the yeast cell machinery. Nature (2006) vol. 440 (7084) pp. 631-6 Ghaemmaghami et al. Global analysis of protein expression in yeast. Nature (2003) vol. 425 (6959) pp. 737-41 Ghosh and Kornfeld. The GGA proteins: key players in protein sorting at the trans-Golgi network. Eur.J.Cell Biol. (2004) vol. 83 (6) pp. 257-62 Giaever et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature (2002) vol. 418 (6896) pp. 387-91  116  Girard et al. Non-stoichiometric relationship between clathrin heavy and light chains revealed by quantitative comparative proteomics of clathrin-coated vesicles from brain and liver. Mol Cell Proteomics (2005) vol. 4 (8) pp. 1145-54 Glyvuk et al. AP-1/sigma1B-adaptin mediates endosomal synaptic vesicle recycling, learning and memory. EMBO J. (2010) vol. 29 (8) pp. 1318-30 Gonzalez and Rodriguez-Boulan. Clathrin and AP1B: key roles in basolateral trafficking through trans-endosomal routes. FEBS Lett. (2009) vol. 583 (23) pp. 3784-3795 Grant and Donaldson. Pathways and mechanisms of endocytic recycling. Nat Rev Mol Cell Biol (2009) vol. 10 (9) pp. 597-608 Greaves et al. The fat controller: roles of palmitoylation in intracellular protein trafficking and targeting to membrane microdomains (Review). Mol Membr Biol (2009) vol. 26 (1) pp. 67-79 Grote et al. A snc1 endocytosis mutant: phenotypic analysis and suppression by overproduction of dihydrosphingosine phosphate lyase. Mol Biol Cell (2000) vol. 11 (12) pp. 4051-65 Grote et al. A targeting signal in VAMP regulating transport to synaptic vesicles. Cell (1995) vol. 81 (4) pp. 581-9 Gruenberg. Lipids in endocytic membrane transport and sorting. Curr Opin Cell Biol (2003) vol. 15 (4) pp. 382-8 Gu and Gruenberg. ARF1 regulates pH-dependent COP functions in the early endocytic pathway. J Biol Chem (2000) vol. 275 (11) pp. 8154-60 Guarente. Synthetic enhancement in gene interaction: a genetic tool come of age. Trends Genet (1993) vol. 9 (10) pp. 362-6 Harel et al. Evidence for CALM in directing VAMP2 trafficking. Traffic (2008) vol. 9 (3) pp. 417-29 Harsay and Bretscher. Parallel secretory pathways to the cell surface in yeast. J.Cell Biol. (1995) vol. 131 (2) pp. 297-310 Hatakeyama et al. Endocytosis of the aspartic acid/glutamic acid transporter Dip5 is triggered by substrate-dependent recruitment of the Rsp5 ubiquitin ligase via the arrestinlike protein Aly2. Mol Cell Biol (2010) vol. 30 (24) pp. 5598-607 He and Linder. Differential palmitoylation of the endosomal SNAREs syntaxin 7 and syntaxin 8. J Lipid Res (2009) vol. 50 (3) pp. 398-404 Heal et al. Activity-based probes: discovering new biology and new drug targets. Chem Soc Rev (2011) vol. 40 (1) pp. 246-57  117  Heilker et al. Recognition of sorting signals by clathrin adaptors. Bioessays (1999) vol. 21 (7) pp. 558-67 Heldwein et al. Crystal structure of the clathrin adaptor protein 1 core. Proc.Natl.Acad.Sci.U.S.A. (2004) vol. 101 (39) pp. 14108-13 Hettema et al. Retromer and the sorting nexins Snx4/41/42 mediate distinct retrieval pathways from yeast endosomes. EMBO J (2003) vol. 22 (3) pp. 548-57 Hirst et al. EpsinR: an ENTH domain-containing protein that interacts with AP-1. Mol.Biol.Cell (2003) vol. 14 (2) pp. 625-41 Holmquist. Alpha/Beta-hydrolase fold enzymes: structures, functions and mechanisms. Curr.Protein Pept.Sci. (2000) vol. 1 (2) pp. 209-35 Hong. SNAREs and traffic. Biochim.Biophys.Acta (2005) vol. 1744 (3) pp. 493-517 Horvath et al. Epsin: inducing membrane curvature. Int J Biochem Cell Biol (2007) vol. 39 (10) pp. 1765-70 Howell et al. Cell biology of membrane trafficking in human disease. Int.Rev.Cytol. (2006) vol. 252 pp. 1-69 Huang et al. Tyrosine phosphorylation of the beta2 subunit of clathrin adaptor complex AP2 reveals the role of a di-leucine motif in the epidermal growth factor receptor trafficking. J.Biol.Chem. (2003) vol. 278 (44) pp. 43411-7 Huang et al. Clathrin functions in the absence of heterotetrameric adaptors and AP180related proteins in yeast. EMBO J (1999) vol. 18 (14) pp. 3897-908 Huh et al. Global analysis of protein localization in budding yeast. Nature (2003) vol. 425 (6959) pp. 686-91 Huizing et al. Hermansky-Pudlak syndrome: vesicle formation from yeast to man. Pigment Cell Res (2002) vol. 15 (6) pp. 405-19 Huizing and Gahl. Disorders of vesicles of lysosomal lineage: the Hermansky-Pudlak syndromes. Curr Mol Med (2002) vol. 2 (5) pp. 451-67 Huttunen et al. Inhibition of acyl-coenzyme A: cholesterol acyl transferase modulates amyloid precursor protein trafficking in the early secretory pathway. FASEB J (2009) vol. 23 (11) pp. 3819-28 Hyun and Ross. HIP1: trafficking roles and regulation of tumorigenesis. Trends Mol Med (2004) vol. 10 (4) pp. 194-9 Ikeda et al. The Rim101 pathway is involved in Rsb1 expression induced by altered lipid asymmetry. Mol Biol Cell (2008) vol. 19 (5) pp. 1922-31  118  Ito et al. A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc.Natl.Acad.Sci.U.S.A. (2001) vol. 98 (8) pp. 4569-74 Itoh and De Camilli. BAR, F-BAR (EFC) and ENTH/ANTH domains in the regulation of membrane-cytosol interfaces and membrane curvature. Biochim Biophys Acta (2006) vol. 1761 (8) pp. 897-912 Itzen and Goody. GTPases involved in vesicular trafficking: Structures and mechanisms. Semin.Cell Dev.Biol. (2010) Jackson et al. A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex. Cell (2010) vol. 141 (7) pp. 1220-9 Janvier and Bonifacino. Role of the endocytic machinery in the sorting of lysosomeassociated membrane proteins. Mol Biol Cell (2005) vol. 16 (9) pp. 4231-42 Jovic et al. The early endosome: a busy sorting station for proteins at the crossroads. Histol Histopathol (2010) vol. 25 (1) pp. 99-112 Jung and Haucke. Clathrin-mediated endocytosis at synapses. Traffic (2007) vol. 8 (9) pp. 1129-36 Kahlfeldt et al. Molecular basis for association of PIPKI gamma-p90 with clathrin adaptor AP-2. J.Biol.Chem. (2010) vol. 285 (4) pp. 2734-2749 Kaiser and Schekman. Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell (1990) vol. 61 (4) pp. 723-33 Kaksonen et al. Harnessing actin dynamics for clathrin-mediated endocytosis. Nat Rev Mol Cell Biol (2006) vol. 7 (6) pp. 404-14 Kaksonen et al. A modular design for the clathrin- and actin-mediated endocytosis machinery. Cell (2005) vol. 123 (2) pp. 305-20 Kaksonen et al. A pathway for association of receptors, adaptors, and actin during endocytic internalization. Cell (2003) vol. 115 (4) pp. 475-87 Kanno et al. Comprehensive screening for novel rab-binding proteins by GST pull-down assay using 60 different mammalian Rabs. Traffic (2010) vol. 11 (4) pp. 491-507 Karylowski et al. GLUT4 is retained by an intracellular cycle of vesicle formation and fusion with endosomes. Mol.Biol.Cell (2004) vol. 15 (2) pp. 870-82 Katzmann et al. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell (2001) vol. 106 (2) pp. 145-55 Kawasaki et al. Membrane recruitment of effector proteins by Arf and Rab GTPases. Curr.Opin.Struct.Biol. (2005) vol. 15 (6) pp. 681-689  119  Kelm et al. The internalization of yeast Ste6p follows an ordered series of events involving phosphorylation, ubiquitination, recognition and endocytosis. Traffic (2004) vol. 5 (3) pp. 165-80 Kiemer et al. WI-PHI: a weighted yeast interactome enriched for direct physical interactions. Proteomics (2007) vol. 7 (6) pp. 932-43 Kim et al. Regulation of dendritic spine morphology by SPIN90, a novel Shank binding partner. J.Neurochem. (2009) vol. 109 (4) pp. 1106-17 Kim et al. F-actin binding region of SPIN90 C-terminus is essential for actin polymerization and lamellipodia formation. Cell Commun Adhes (2007) vol. 14 (1) pp. 33-43 Kim et al. Interaction of SPIN90 with syndapin is implicated in clathrin-mediated endocytic pathway in fibroblasts. Genes Cells (2006) vol. 11 (10) pp. 1197-211 Kim and Chang. Ever-expanding network of dynamin-interacting proteins. Mol Neurobiol (2006) vol. 34 (2) pp. 129-36 Kim et al. Interaction of SPIN90 with dynamin I and its participation in synaptic vesicle endocytosis. J Neurosci (2005) vol. 25 (41) pp. 9515-23 Kim et al. Capping protein binding to actin in yeast: biochemical mechanism and physiological relevance. J.Cell Biol. (2004) vol. 164 (4) pp. 567-80 Kim et al. Delayed reentry of recycling vesicles into the fusion-competent synaptic vesicle pool in synaptojanin 1 knockout mice. Proc.Natl.Acad.Sci.U.S.A. (2002) vol. 99 (26) pp. 17143-8 Kirchhausen. Three ways to make a vesicle. Nat.Rev.Mol.Cell Biol. (2000) vol. 1 (3) pp. 187198 Kita et al. Loss of Apm1, the micro1 subunit of the clathrin-associated adaptor-protein-1 complex, causes distinct phenotypes and synthetic lethality with calcineurin deletion in fission yeast. Mol.Biol.Cell (2004) vol. 15 (6) pp. 2920-31 Konarzycka-Bessler and Bornscheuer. A high-throughput-screening method for determining the synthetic activity of hydrolases. Angew Chem Int Ed Engl (2003) vol. 42 (12) pp. 1418-20 Krauss et al. Stimulation of phosphatidylinositol kinase type I-mediated phosphatidylinositol (4,5)-bisphosphate synthesis by AP-2mu-cargo complexes. Proc.Natl.Acad.Sci.U.S.A. (2006) vol. 103 (32) pp. 11934-9 Krogan et al. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature (2006) vol. 440 (7084) pp. 637-43 Kübler and Riezman. Actin and fimbrin are required for the internalization step of endocytosis in yeast. EMBO J (1993) vol. 12 (7) pp. 2855-62  120  Lamping et al. Isolation and characterization of a mutant of Saccharomyces cerevisiae with pleiotropic deficiencies in transcriptional activation and repression. Genetics (1994) vol. 137 (1) pp. 55-65 Lechler et al. Direct involvement of yeast type I myosins in Cdc42-dependent actin polymerization. J.Cell Biol. (2000) vol. 148 (2) pp. 363-73 Lee et al. Determination of EGFR endocytosis kinetic by auto-regulatory association of PLD1 with mu2. PLoS ONE (2009) vol. 4 (9) pp. e7090 Lee et al. SPIN90/WISH interacts with PSD-95 and regulates dendritic spinogenesis via an N-WASP-independent mechanism. EMBO J (2006) vol. 25 (20) pp. 4983-95 Legendre-Guillemin et al. ENTH/ANTH proteins and clathrin-mediated membrane budding. J.Cell.Sci. (2004) vol. 117 (Pt 1) pp. 9-18 Lewis and Pelham. A new yeast endosomal SNARE related to mammalian syntaxin 8. Traffic (2002) vol. 3 (12) pp. 922-9 Lewis et al. Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes. Mol.Biol.Cell (2000) vol. 11 (1) pp. 23-38 Lin and Guttman. Hijacking the endocytic machinery by microbial pathogens. Protoplasma (2010) vol. 244 (1-4) pp. 75-90 Linder and Deschenes. Palmitoylation: policing protein stability and traffic. Nat Rev Mol Cell Biol (2007) vol. 8 (1) pp. 74-84 Ling et al. Type I gamma phosphatidylinositol phosphate kinase modulates adherens junction and E-cadherin trafficking via a direct interaction with mu 1B adaptin. J.Cell Biol. (2007) vol. 176 (3) pp. 343-53 Liu and Bankaitis. Phosphoinositide phosphatases in cell biology and disease. Prog Lipid Res (2010) vol. 49 (3) pp. 201-17 Loerke et al. Cargo and dynamin regulate clathrin-coated pit maturation. PLoS Biol (2009) vol. 7 (3) pp. e57 Lundmark and Carlsson. Driving membrane curvature in clathrin-dependent and clathrinindependent endocytosis. Semin.Cell Dev.Biol. (2010) vol. 21 (4) pp. 363-70 Ma et al. Deletion mutants of AP-1 adaptin subunits display distinct phenotypes in fission yeast. Genes Cells (2009) vol. 14 (8) pp. 1015-28 MacDonald et al. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell (2009) vol. 17 (1) pp. 9-26 Maldonado-Baez and Wendland. Endocytic adaptors: recruiters, coordinators and regulators. Trends Cell Biol. (2006) vol. 16 (10) pp. 505-513  121  Maldonado-Báez et al. Interaction between Epsin/Yap180 adaptors and the scaffolds Ede1/Pan1 is required for endocytosis. Mol.Biol.Cell (2008) vol. 19 (7) pp. 2936-48 Malecz et al. Synaptojanin 2, a novel Rac1 effector that regulates clathrin-mediated endocytosis. Curr.Biol. (2000) vol. 10 (21) pp. 1383-6 Margolin et al. Reverse engineering cellular networks. Nat Protoc (2006) vol. 1 (2) pp. 66271 Mattera et al. Conservation and Diversification of Dileucine Signal Recognition by Adaptor Protein (AP) Complex Variants. J.Biol.Chem. (2011) vol. 286 (3) pp. 2022-30 Mayeux and Hyslop. Alzheimer's disease: advances in trafficking. Lancet Neurol (2008) vol. 7 (1) pp. 2-3 Mercer et al. Virus entry by endocytosis. Annu.Rev.Biochem. (2010) vol. 79 pp. 803-33 Meyerholz et al. Effect of clathrin assembly lymphoid myeloid leukemia protein depletion on clathrin coat formation. Traffic (2005) vol. 6 (12) pp. 1225-34 Miller et al. A SNARE-adaptor interaction is a new mode of cargo recognition in clathrincoated vesicles. Nature (2007) vol. 450 (7169) pp. 570-4 Mitsunari et al. Clathrin adaptor AP-2 is essential for early embryonal development. Mol Cell Biol (2005) vol. 25 (21) pp. 9318-23 Mnaimneh et al. Exploration of essential gene functions via titratable promoter alleles. Cell (2004) vol. 118 (1) pp. 31-44 Moseley and Goode. The yeast actin cytoskeleton: from cellular function to biochemical mechanism. Microbiol Mol Biol Rev (2006) vol. 70 (3) pp. 605-45 Motley et al. Functional analysis of AP-2 alpha and mu2 subunits. Mol Biol Cell (2006) vol. 17 (12) pp. 5298-308 Munro. Organelle identity and the organization of membrane traffic. Nat.Cell Biol. (2004) vol. 6 (6) pp. 469-472 Nakatsu et al. Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor. J.Cell Biol. (2004) vol. 167 (2) pp. 293-302 Nakatsu and Ohno. Adaptor protein complexes as the key regulators of protein sorting in the post-Golgi network. Cell Struct Funct (2003) vol. 28 (5) pp. 419-29 Nardini and Dijkstra. Alpha/beta hydrolase fold enzymes: the family keeps growing. Curr Opin Struct Biol (1999) vol. 9 (6) pp. 732-7 Newell-Litwa et al. Roles of BLOC-1 and adaptor protein-3 complexes in cargo sorting to synaptic vesicles. Mol.Biol.Cell (2009) vol. 20 (5) pp. 1441-53  122  Newpher et al. In vivo dynamics of clathrin and its adaptor-dependent recruitment to the actin-based endocytic machinery in yeast. Dev Cell (2005) vol. 9 (1) pp. 87-98 Nikko and Pelham. Arrestin-mediated endocytosis of yeast plasma membrane transporters. Traffic (2009) vol. 10 (12) pp. 1856-67 Nonet et al. UNC-11, a Caenorhabditis elegans AP180 homologue, regulates the size and protein composition of synaptic vesicles. Mol Biol Cell (1999) vol. 10 (7) pp. 2343-60 Novick et al. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell (1980) vol. 21 (1) pp. 205-15 Ohmura-Hoshino et al. A novel family of membrane-bound E3 ubiquitin ligases. J.Biochem. (2006) vol. 140 (2) pp. 147-54 Ohno. Physiological roles of clathrin adaptor AP complexes: lessons from mutant animals. J.Biochem. (2006) vol. 139 (6) pp. 943-948 Ohno. Overview: membrane traffic in multicellular systems: more than just a housekeeper. J.Biochem. (2006) vol. 139 (6) pp. 941-2 Olkkonen and Ikonen. When intracellular logistics fails--genetic defects in membrane trafficking. J Cell Sci (2006) vol. 119 (Pt 24) pp. 5031-45 Ooms et al. The yeast inositol polyphosphate 5-phosphatases inp52p and inp53p translocate to actin patches following hyperosmotic stress: mechanism for regulating phosphatidylinositol 4,5-bisphosphate at plasma membrane invaginations. Mol Cell Biol (2000) vol. 20 (24) pp. 9376-90 Owen et al. Adaptors for clathrin coats: structure and function. Annu.Rev.Cell Dev.Biol. (2004) vol. 20 pp. 153-191 Owen. Linking endocytic cargo to clathrin: structural and functional insights into coated vesicle formation. Biochem.Soc.Trans. (2004) vol. 32 (Pt 1) pp. 1-14 Owen and Evans. A structural explanation for the recognition of tyrosine-based endocytotic signals. Science (1998) vol. 282 (5392) pp. 1327-32 Padrón et al. Phosphatidylinositol phosphate 5-kinase Ibeta recruits AP-2 to the plasma membrane and regulates rates of constitutive endocytosis. J.Cell Biol. (2003) vol. 162 (4) pp. 693-701 Page et al. Gamma-synergin: an EH domain-containing protein that interacts with gammaadaptin. J.Cell Biol. (1999) vol. 146 (5) pp. 993-1004 Peden et al. The Di-leucine motif of vesicle-associated membrane protein 4 is required for its localization and AP-1 binding. J.Biol.Chem. (2001) vol. 276 (52) pp. 49183-7 Pelham. Insights from yeast endosomes. Curr Opin Cell Biol (2002) vol. 14 (4) pp. 454-62  123  Pelham. SNAREs and the specificity of membrane fusion. Trends Cell Biol. (2001) vol. 11 (3) pp. 99-101 Peña-Castillo and Hughes. Why are there still over 1000 uncharacterized yeast genes?. Genetics (2007) vol. 176 (1) pp. 7-14 Pereira-Leal and Seabra. Evolution of the Rab family of small GTP-binding proteins. J Mol Biol (2001) vol. 313 (4) pp. 889-901 Perrais and Merrifield. Dynamics of endocytic vesicle creation. Dev.Cell. (2005) vol. 9 (5) pp. 581-592 Prescott et al. Palmitoylation of the synaptic vesicle fusion machinery. J Neurochem (2009) vol. 110 (4) pp. 1135-49 Pryor et al. Molecular basis for the sorting of the SNARE VAMP7 into endocytic clathrincoated vesicles by the ArfGAP Hrb. Cell (2008) vol. 134 (5) pp. 817-27 Qin et al. Regulation of phosphatidylinositol kinases and metabolism by Wnt3a and Dvl. J Biol Chem (2009) vol. 284 (34) pp. 22544-8 Rancati et al. Aneuploidy underlies rapid adaptive evolution of yeast cells deprived of a conserved cytokinesis motor. Cell (2008) vol. 135 (5) pp. 879-93 Rao et al. Altered receptor trafficking in Huntingtin Interacting Protein 1-transformed cells. Cancer Cell (2003) vol. 3 (5) pp. 471-82 Raymond et al. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol.Biol.Cell (1992) vol. 3 (12) pp. 1389-402 Reusch et al. AP-1A and AP-3A lysosomal sorting functions. Traffic (2002) vol. 3 (10) pp. 752-61 Reymond. Substrate arrays for fluorescence-based enzyme fingerprinting and highthroughput screening. Ann N Y Acad Sci (2008) vol. 1130 pp. 12-20 Ritter and Wendland. Clathrin-Mediated Endocytosis. Trafficking Inside Cells: Pathways (2009) Robinson et al. The Gcs1 Arf-GAP mediates Snc1,2 v-SNARE retrieval to the Golgi in yeast. Mol Biol Cell (2006) vol. 17 (4) pp. 1845-58 Robinson. Adaptable adaptors for coated vesicles. Trends Cell Biol. (2004) vol. 14 (4) pp. 167-174 Rönty et al. Palladin interacts with SH3 domains of SPIN90 and Src and is required for Srcinduced cytoskeletal remodeling. Exp Cell Res (2007) vol. 313 (12) pp. 2575-85  124  Roth et al. Proteomic identification of palmitoylated proteins. Methods (2006) vol. 40 (2) pp. 135-42 Rusk et al. Synaptojanin 2 functions at an early step of clathrin-mediated endocytosis. Curr.Biol. (2003) vol. 13 (8) pp. 659-63 Russell et al. Molecular mechanisms of late endosome morphology, identity and sorting. Curr Opin Cell Biol (2006) vol. 18 (4) pp. 422-8 Scherens and Goffeau. The uses of genome-wide yeast mutant collections. Genome Biol (2004) vol. 5 (7) pp. 229 Schluter et al. Global analysis of yeast endosomal transport identifies the vps55/68 sorting complex. Mol Biol Cell (2008) vol. 19 (4) pp. 1282-94 Schu. Vesicular protein transport. Pharmacogenomics J. (2001) vol. 1 (4) pp. 262-271 Schuldiner et al. Quantitative genetic analysis in Saccharomyces cerevisiae using epistatic miniarray profiles (E-MAPs) and its application to chromatin functions. Methods (2006) vol. 40 (4) pp. 344-52 Schuldiner et al. Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell (2005) vol. 123 (3) pp. 507-19 Scita and Di Fiore. The endocytic matrix. Nature (2010) vol. 463 (7280) pp. 464-73 Seaman. Endosome protein sorting: motifs and machinery. Cell Mol Life Sci (2008) vol. 65 (18) pp. 2842-58 Segev. GTPases in Intracellular Trafficking: An Overview. Semin.Cell Dev.Biol. (2010) pp. Selkoe. Alzheimer's disease: genes, proteins, and therapy. Physiol Rev (2001) vol. 81 (2) pp. 741-66 Seong et al. Genetic analysis of the neuronal and ubiquitous AP-3 adaptor complexes reveals divergent functions in brain. Mol.Biol.Cell (2005) vol. 16 (1) pp. 128-40 Shah and Yu. sorLA: sorting out APP. Mol Interv (2006) vol. 6 (2) pp. 74-6, 58 Shannon et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res (2003) vol. 13 (11) pp. 2498-504 Sheng and Kim. Postsynaptic signaling and plasticity mechanisms. Science (2002) vol. 298 (5594) pp. 776-80 Shim et al. Distinct and redundant functions of mu1 medium chains of the AP-1 clathrinassociated protein complex in the nematode Caenorhabditis elegans. Mol.Biol.Cell (2000) vol. 11 (8) pp. 2743-56  125  Siegers et al. Compartmentation of protein folding in vivo: sequestration of non-native polypeptide by the chaperonin-GimC system. EMBO J (1999) vol. 18 (1) pp. 75-84 Simon and Cravatt. Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study. J Biol Chem (2010) vol. 285 (15) pp. 11051-5 Smaczynska-de Rooij et al. A role for the dynamin-like protein Vps1 during endocytosis in yeast. J.Cell.Sci. (2010) vol. 123 (Pt 20) pp. 3496-506 Smythe and Ayscough. Actin regulation in endocytosis. J.Cell.Sci. (2006) vol. 119 (Pt 22) pp. 4589-98 Sorkin and von Zastrow. Endocytosis and signalling: intertwining molecular networks. Nat Rev Mol Cell Biol (2009) vol. 10 (9) pp. 609-22 Sorkin. Cargo recognition during clathrin-mediated endocytosis: a team effort. Curr.Opin.Cell Biol. (2004) vol. 16 (4) pp. 392-399 Soulard et al. The WASP/Las17p-interacting protein Bzz1p functions with Myo5p in an early stage of endocytosis. Protoplasma (2005) vol. 226 (1-2) pp. 89-101 Soulard et al. Saccharomyces cerevisiae Bzz1p is implicated with type I myosins in actin patch polarization and is able to recruit actin-polymerizing machinery in vitro. Mol Cell Biol (2002) vol. 22 (22) pp. 7889-906 Spang. The life cycle of a transport vesicle. Cell Mol.Life Sci. (2008) vol. 65 (18) pp. 27812789 Stefan et al. The yeast synaptojanin-like proteins control the cellular distribution of phosphatidylinositol (4,5)-bisphosphate. Mol.Biol.Cell (2002) vol. 13 (2) pp. 542-57 Stein et al. Rab proteins and endocytic trafficking: potential targets for therapeutic intervention. Adv Drug Deliv Rev (2003) vol. 55 (11) pp. 1421-37 Stepp et al. A late Golgi sorting function for Saccharomyces cerevisiae Apm1p, but not for Apm2p, a second yeast clathrin AP medium chain-related protein. Mol Biol Cell (1995) vol. 6 (1) pp. 41-58 Stolz et al. Identification and characterization of an essential family of inositol polyphosphate 5-phosphatases (INP51, INP52 and INP53 gene products) in the yeast Saccharomyces cerevisiae. Genetics (1998) vol. 148 (4) pp. 1715-29 Sun et al. PtdIns(4,5)P2 turnover is required for multiple stages during clathrin- and actindependent endocytic internalization. J.Cell Biol. (2007) vol. 177 (2) pp. 355-67 Sun et al. Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity. Dev.Cell. (2006) vol. 11 (1) pp. 33-46  126  Sun et al. The yeast casein kinase Yck3p is palmitoylated, then sorted to the vacuolar membrane with AP-3-dependent recognition of a YXXPhi adaptin sorting signal. Mol.Biol.Cell (2004) vol. 15 (3) pp. 1397-406 Szilágyi et al. Prediction of physical protein-protein interactions. Phys Biol (2005) vol. 2 (2) pp. S1-16 Tong and Boone. Synthetic genetic array analysis in Saccharomyces cerevisiae. Methods Mol Biol (2006) vol. 313 pp. 171-92 Tong et al. Global mapping of the yeast genetic interaction network. Science (2004) vol. 303 (5659) pp. 808-13 Tong et al. A combined experimental and computational strategy to define protein interaction networks for peptide recognition modules. Science (2002) vol. 295 (5553) pp. 321-4 Toret et al. Multiple pathways regulate endocytic coat disassembly in Saccharomyces cerevisiae for optimal downstream trafficking. Traffic (2008) vol. 9 (5) pp. 848-59 Traub. Tickets to ride: selecting cargo for clathrin-regulated internalization. Nat.Rev.Mol.Cell Biol. (2009) vol. 10 (9) pp. 583-596 Traub. Common principles in clathrin-mediated sorting at the Golgi and the plasma membrane. Biochim.Biophys.Acta (2005) vol. 1744 (3) pp. 415-437 Traub. Sorting it out: AP-2 and alternate clathrin adaptors in endocytic cargo selection. J.Cell Biol. (2003) vol. 163 (2) pp. 203-208 Traub and Apodaca. AP-1B: polarized sorting at the endosome. Nat Cell Biol (2003) vol. 5 (12) pp. 1045-7 Ungar and Hughson. SNARE protein structure and function. Annu.Rev.Cell Dev.Biol. (2003) vol. 19 pp. 493-517 Ungewickell and Hinrichsen. Endocytosis: clathrin-mediated membrane budding. Curr.Opin.Cell Biol. (2007) vol. 19 (4) pp. 417-25 Valdez-Taubas and Pelham. Swf1-dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation. EMBO J (2005) vol. 24 (14) pp. 2524-32 Valdez-Taubas and Pelham. Slow diffusion of proteins in the yeast plasma membrane allows polarity to be maintained by endocytic cycling. Curr.Biol. (2003) vol. 13 (18) pp. 1636-40 Valdivia et al. The yeast clathrin adaptor protein complex 1 is required for the efficient retention of a subset of late Golgi membrane proteins. Dev Cell (2002) vol. 2 (3) pp. 283-94 van Vliet et al. Intracellular sorting and transport of proteins. Prog.Biophys.Mol.Biol. (2003) vol. 83 (1) pp. 1-45  127  Veit et al. Multiple palmitoylation of synaptotagmin and the t-SNARE SNAP-25. FEBS Lett (1996) vol. 385 (1-2) pp. 119-23 von Mering et al. Comparative assessment of large-scale data sets of protein-protein interactions. Nature (2002) vol. 417 (6887) pp. 399-403 von Mollard et al. The yeast v-SNARE Vti1p mediates two vesicle transport pathways through interactions with the t-SNAREs Sed5p and Pep12p. J.Cell Biol. (1997) vol. 137 (7) pp. 1511-24 Voronov et al. Synaptojanin 1-linked phosphoinositide dyshomeostasis and cognitive deficits in mouse models of Down's syndrome. Proc.Natl.Acad.Sci.U.S.A. (2008) vol. 105 (27) pp. 9415-20 Wahler et al. Enzyme fingerprints of activity, and stereo- and enantioselectivity from fluorogenic and chromogenic substrate arrays. Chemistry (2002) vol. 8 (14) pp. 3211-28 Wang et al. Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi. Cell (2003) vol. 114 (3) pp. 299-310 Wasiak et al. Enthoprotin: a novel clathrin-associated protein identified through subcellular proteomics. J.Cell Biol. (2002) vol. 158 (5) pp. 855-62 Wei. Hermansky-Pudlak syndrome: a disease of protein trafficking and organelle function. Pigment Cell Res (2006) vol. 19 (1) pp. 19-42 Wendland et al. Yeast epsins contain an essential N-terminal ENTH domain, bind clathrin and are required for endocytosis. EMBO J (1999) vol. 18 (16) pp. 4383-93 Wendland and Emr. Pan1p, yeast eps15, functions as a multivalent adaptor that coordinates protein-protein interactions essential for endocytosis. J.Cell Biol. (1998) vol. 141 (1) pp. 7184 Whitney et al. Cytoplasmic coat proteins involved in endosome function. Cell (1995) vol. 83 (5) pp. 703-13 Yu et al. Structural analysis of the interaction between Dishevelled2 and clathrin AP-2 adaptor, a critical step in noncanonical Wnt signaling. Structure (2010) vol. 18 (10) pp. 1311-20 Yu et al. Association of Dishevelled with the clathrin AP-2 adaptor is required for Frizzled endocytosis and planar cell polarity signaling. Dev Cell (2007) vol. 12 (1) pp. 129-41 Yuen et al. Systematic genome instability screens in yeast and their potential relevance to cancer. Proc.Natl.Acad.Sci.U.S.A. (2007) vol. 104 (10) pp. 3925-30 Zeidman et al. Protein acyl thioesterases (Review). Mol Membr Biol (2009) vol. 26 (1) pp. 32-41  128  Ziman et al. Chs6p-dependent anterograde transport of Chs3p from the chitosome to the plasma membrane in Saccharomyces cerevisiae. Mol.Biol.Cell (1998) vol. 9 (6) pp. 1565-76  129  APPENDIX A: Supplemental material for chapter 2 Table A1. Plasmids and yeast strains used in this study Type Plasmid Plasmid  Name pCS7 pCS30  Description/Genotype SNC1-GFP in pRS316 GFP-SNC1-SUC2 in pRS306 (GSS; TPI1 promoter)  Plasmid  pMD9  GFP-SNC1(V40A M43A)SUC2 in pRS306 (GSS en-; TPI1 promoter)  Plasmid  pHB4  SUC2 5'UTR-ADHprNATR-GFP-SNC1-SUC2 (GSS; ADH promoter)  Source Notes (Schluter et al., 2008)a This Sequences from pGS study (Lewis et al., 2000)b were PCR-amplified to introduce a SmaI site before the Snc1 stop codon, and subcloning this into XhoI/SmaIdigested pRS306. The resulting plasmid was digested with XbaI/SmaI, end-filled, and ligated to SUC2 sequences on a SmaIHpaI fragment from pSEYC306 (Darsow et al., 2000)c This V40A and M43A study mutations were introduced into the SNC1 coding region of pCS30 by site-directed mutagenesis This pHB4 was created in a study multi-step process. pHB1 was first created by co-transformation of SacI/Kpn1-cut pRS416 with the following PCR products: (1) the NATR selection marker, ADH promoter and yeGFP coding sequence amplified from pYMN9 (Janke et al., 2004)h and (2) a 2378 bp fragment amplified from yeast genomic DNA after integration of pCS30, encoding the SNC1-SUC2 fusion and including 458 bp of SUC2 3’UTR. A PCR fragment encoding the  130  Type  Name  Description/Genotype  Source  Plasmid  pMD65  SUC2 5'UTR-ADHprNATR-GFP-NPFxD-SSO1 (NPF-Sso1)  This study  Notes Nyv1 cytosolic domain (aa 1-229), amplified from yeast genomic DNA, was used to replace the Snc1 cytosolic domain (aa 190) by cotransformation with EcoR1-digested pHB1, creating pHB2. To facilitate integration into the SUC2 locus, 252bp of SUC2 5’UTR were amplified by PCR and incorporated upstream of the NATR cassette in BglIIdigested pHB2, creating pHB3. This introduced a unique NotI restriction site upstream of the SUC2 5’UTR, such that NotI/SnaB1 digestion of pHB3 (or its derivatives) releases the chimeric reporter and the NATR marker on a cassette that is targeted to the SUC2 locus. pHB4 was then created by cotransforming ClaI/SalIdigested pHB3 with a PCR fragment containing the SNC1 sequence from pCS30. pMD51 was created by co-transforming ClaI/SalI-digested pHB3 with a PCR fragment containing the SNC1(V40A M43A) sequence from pMD9. The NPFxD endocytosis signal was appended to the N-terminus of the SNC1(V40A M43A) sequence in pMD51 by  131  Type  Name  Description/Genotype  Source  Plasmid  pMD69  SUC2 5'UTR-ADHprNATR-GFP-3xNPFxDSSO1-SUC2 in pRS416 (NGSS)  This study  Notes co-transforming SpeIdigested pMD51 with a PCR product encoding MVLTNANPFSD, to generate pMD52. The SNC1(V40A M43A) coding sequence was then replaced with that of SSO1 by cotransforming ClaI/SalI digested pMD52 with a PCR fragment containing the SSO1 sequence amplified from yeast genomic DNA to create pMD60. pMD65 was made by co-transforming ClaI/SalI-digested pMD52 with a PCR fragment that directed replacement of SNC1SUC2 sequences with the SSO1 coding sequence. pMD67 was created by insertion of two additional NPFxD motifs N-terminal in tandem with the existing NPFxD motif in pMD52 by synthesizing a PCR product containing 2 VLTNANPFSD motifs by primer extension, using overlapping oligonucleotides with 5’ homology to the ADH promoter and 3’ homology to the NPFxD motif in pMD52. The SNC1(V40A M43A) sequence of pMD67 was then replaced with SSO1 by cotransforming ClaI/SalI digested pMD67 with PCR-amplified SSO1,  132  Type  Name  Description/Genotype  Source  Plasmid  pSM1493  STE6-GFP in pRS426  Plasmid  pBW1427  LDB17-4XGFP tagging construct  Huyer et al., 2004d This study  Notes resulting in pMD69. gift from Susan Michaelis A plasmid for Cterminal tagging of LDB17 with 4 tandem copies of GFP+ was created in a multistep process. Site directed mutagenesis was first used to convert the ApaLI site in pGFP+(NATR) (Scholz et al., 2000)e to an HpaI site, and the SacI site to a PmlI site, generating pBW1395. The 1014bp PmlI/HpaI fragment from pBW1395 was cloned into PmlIlinearized pBW1395 to generate pBW1396, which has two copies of GFP+ flanked by unique HpaI and PmlI sites. The 2028bp PmlI/HpaI 2xGFP+ fragment from pBW1396 was then cloned into PmlIlinearized pBW1396, generating pBW1397, which has four copies of GFP+ flanked by unique HpaI and PmlI sites. PCR amplification and TA cloning was used to clone 349 bp of the LDB17 3'UTR immediately following the LDB17 ORF while introducing flanking BglII/XbaI sites. The 353bp BglII/XbaI fragment was verified by DNA sequencing and ligated into BglII/XbaIcut pBW1397,  133  Type  Name  Description/Genotype  Source  Plasmid  pOAD  pOAD  Plasmid  pOBD2  pOBD2  Plasmid  p3384  Yfr024c(291-373) in pOBD2  Plasmid  p3388  Abp1(351-592) in pOBD2  Plasmid  p3390  Bzz1(476-564) in pOBD2  Plasmid  p3391  Bzz1(562-633) in pOBD2  Plasmid  p3392  Bzz1(476-633) in pOBD2  Plasmid  p3497  Myo3(1054-1271) in pOBD2  Plasmid  p3519  Bem1(51-240) in pOBD2  Uetz et al., 2000f Uetz et al., 2000f Tong et al., 2002g Tong et al., 2002g Tong et al., 2002g Tong et al., 2002g Tong et al., 2002g Tong et al., 2002g Tong et al.,  Notes generating pBW1426. The LDB17 ORF from nt 10 to 1473 was amplified by PCR, introducing an inframe PvuII site immediately preceding the stop codon, subjected to TA cloning, and sequenced. The 1352bp PvuII/HpaI fragment was then ligated into HpaI-cut pBW1398, generating the pBW1427, which contains a 6970bp 4xGFP+-NATR cassette flanked by LDB17 sequences that can be released by HpaI/XbaI digestion. gift from Stan Fields  gift from Stan Fields  gift from Charlie Boone.  gift from Charlie Boone.  gift from Charlie Boone.  gift from Charlie Boone.  gift from Charlie Boone.  gift from Charlie Boone.  gift from Charlie Boone.  134  Type  Name  Description/Genotype  Plasmid  p3520  Bem1(1-551) in pOBD2  Plasmid  p3697  Rvs167(401-482) in pOBD2  Plasmid  p3699  Sla1(336-435) in pOBD2  Plasmid  p3700  Sla1(1-150) in pOBD2  Plasmid  p3735  Sla1(1-435) in pOBD2  Plasmid  p3737  Sla1(61-150) in pOBD2  Plasmid  p3755  Ysc84(391-468) in pOBD2  Plasmid  p3771  Bbc1(1-90) in pOBD2  Plasmid  p3842  Hof1(576-669) in pOBD2  Yeast Strain Yeast Strain Yeast Strain  BY4741  MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 MATa his3Δ1 leu2Δ0 lys2∆0 ura3Δ0 BY4741 suc2::GFP-SNC1SUC2::URA3  Yeast Strain  HBY222  BY4741 rvs167∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  CSY448  BY4741 ldb17∆::KAN suc2::GFP-SNC1-  This study  BY4742 HBY280  Source Notes 2002g Tong et gift from Charlie Boone. al., 2002g Tong et gift from Charlie Boone. al., 2002g Tong et gift from Charlie Boone. al., 2002g Tong et gift from Charlie Boone. al., 2002g Tong et gift from Charlie Boone. al., 2002g Tong et gift from Charlie Boone. al., 2002g Tong et gift from Charlie Boone. al., 2002g Tong et gift from Charlie Boone. al., 2002g Tong et gift from Charlie Boone. al., 2002g Open Biosystems Open Biosystems This study  The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was  135  Type  Name  Description/Genotype SUC2::URA3  Source  Yeast Strain  CSY461  BY4741 bzz1∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  CSY520  BY4741 lsb3∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  CSY453  BY4741 inp52∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  CSY451  BY4741 abp1∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  HBY277  BY4741 myo5∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  HBY282  BY4741 fus3∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast  HBY278  BY4741 ede1∆::KAN  This  Notes linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter  136  Type Strain  Name  Description/Genotype suc2::GFP-SNC1SUC2::URA3  Source study  Yeast Strain  HBY279  BY4741 sla1∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  HBY281  BY4741 vrp1∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  HBY284  BY4741 yol098c∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  CSY452  BY4741 crn1∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  CSY455  BY4741 twf1∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  CSY449  BY4741 rvs161∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Notes plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives.  137  Type Yeast Strain  Name CSY521  Description/Genotype BY4741 bbc1∆::KAN suc2::GFP-SNC1SUC2::URA3  Source This study  Yeast Strain  CSY435  BY4741 yap1802∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  CSY522  BY4741 syp1∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  CSY523  BY4741 cap1∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  CSY525  BY4741 cap2∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  CSY444  BY4741 yap1801∆::KAN suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  CSY462  BY4741 suc2::GFPsnc1(V40A,M43A)SUC2::URA3  This study  Yeast  MDY581  BY4741 yap1801∆::KAN  This  Notes The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in BY4741 and its knockout derivatives. The GSS EN- reporter plasmid pMD9 was linearized with XbaI and integrated at the SUC2 locus in BY4741 The YAP1802 ORF was  138  Type Strain  Name  Description/Genotype yap1802∆::HPH  Source study  Yeast Strain  HBY323  BY4741 yap1801∆::KAN yap1802∆::HPH suc2::GFPSNC1-SUC2::URA3  This study  Yeast Strain  MDY582  BY4741 yap1801∆::KAN yap1802∆CBM-3HA::HIS3  This study  Yeast Strain Yeast Strain  MDY583  BY4741 yap1801∆::KAN YAP1802-3HA::HIS3 BY4741 suc2::GFPSNC1(V40A,M43A)SUC2::URA3  This study This study  Yeast Strain  HBY333  BY4741 suc2∆::NAT-GFP3xNPFxD-SSO1-SUC2  This study  Yeast Strain  HBY334  BY4741 yap1801∆::KAN This suc2∆::NAT-GFP-3xNPFxD- study SSO1-SUC2  CSY462  Notes replaced with the hygromycin resistance marker in BY4741 yap1801::KAN by transformation of a PCR fragment amplified from pFA6hphNT1 (Janke et al., 2004)h, generating MDY581. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in MDY581 The final 4 C-terminal residues of Yap1802 (564-568) were replaced with a 3xHA tag in BY4741 yap1801::KAN to make MDY582  The GSS EN- reporter plasmid pMD9 was linearized with XbaI and integrated at the SUC2 locus in BY4741 to create CSY462 The 3xNGSS reporter was integrated at the SUC2 locus in BY4741 and derivatives by transformation with a NotI/SnaBI fragment released from pMD69 to create strains HBY333-HBY338. Integration of reporters was confirmed by colony PCR. The 3xNGSS reporter was integrated at the SUC2 locus in BY4741 and derivatives by transformation with a  139  Type  Name  Description/Genotype  Source  Yeast Strain  HBY335  BY4741 yap1802∆::KAN This suc2∆::NAT-GFP-3xNPFxD- study SSO1-SUC2  Yeast Strain  HBY336  BY4741 yap1801∆::KAN This yap1802∆::HPH study suc2∆::NAT-GFP-3xNPFxDSSO1-SUC2  Yeast Strain  HBY337  BY4741 ldb17∆::KAN This suc2∆::NAT-GFP-3xNPFxD- study SSO1-SUC2  Yeast  HBY338  BY4741 sla1∆::KAN  This  Notes NotI/SnaBI fragment released from pMD69 to create strains HBY333-HBY338. Integration of reporters was confirmed by colony PCR. The 3xNGSS reporter was integrated at the SUC2 locus in BY4741 and derivatives by transformation with a NotI/SnaBI fragment released from pMD69 to create strains HBY333-HBY338. Integration of reporters was confirmed by colony PCR. The 3xNGSS reporter was integrated at the SUC2 locus in BY4741 and derivatives by transformation with a NotI/SnaBI fragment released from pMD69 to create strains HBY333-HBY338. Integration of reporters was confirmed by colony PCR. The 3xNGSS reporter was integrated at the SUC2 locus in BY4741 and derivatives by transformation with a NotI/SnaBI fragment released from pMD69 to create strains HBY333-HBY338. Integration of reporters was confirmed by colony PCR. The 3xNGSS reporter  140  Type Strain  Name  Description/Genotype Source suc2∆::NAT-GFP-3xNPFxD- study SSO1-SUC2  Yeast Strain  LCY1785  BY4741 LDB1713MYC::KAN  This study  Yeast Strain  LCY1786  BY4741 ldb17∆PRD13MYC::KAN  This study  Yeast Strain  LCY1797  BY4741 LDB1713MYC::KAN BZZ13HA::HIS3  This study  Yeast Strain  LCY1798  BY4741 ldb17∆PRD13MYC::KAN BZZ13HA::HIS3  This study  Yeast Strain  CSY76  BY4741 LDB17-GFP::HIS3  This study  Yeast Strain  CSY80  BY4741 ldb17∆PRDGFP::HIS3  This study  Notes was integrated at the SUC2 locus in BY4741 and derivatives by transformation with a NotI/SnaBI fragment released from pMD69 to create strains HBY333-HBY338. Integration of reporters was confirmed by colony PCR. C-terminal tag encoding a 13xmyc epitope was integrated at the LDB17 locus as previously described (Longtine et al., 1998)i C-terminal tag encoding a 13xmyc epitope was integrated at the LDB17 locus before Pro475, truncating Ldb17 and removing the prolinerich domain (Longtine et al., 1998)i C-terminal tag encoding a 3xHA epitope (Longtine et al., 1998)i was integrated at the BZZ1 locus in LCY1786 C-terminal tag encoding a 3xHA epitope (Longtine et al., 1998)i was integrated at the BZZ1 locus in LCY1786 To create the control strains (CSY76 and CSY78), the GFP-HIS3 cassette was integrated before the LDB17 stop codon. Expression of Ldb17PRD∆ in CSY80 and CSY79 was  141  Type  Name  Description/Genotype  Source  Yeast Strain  CSY78  BY4741 bzz1∆::KAN LDB17-GFP::HIS3  This study  Yeast Strain  CSY79  BY4741 bzz1∆::KAN ldb17∆PRD-GFP::HIS3  This study  Yeast Strain  HBY377  CSY76 suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  HBY378  CSY80 suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  HBY379  CSY78 suc2::GFP-SNC1SUC2::URA3  This study  Yeast Strain  HBY380  CSY79 suc2::GFP-SNC1SUC2::URA3  This study  Notes accomplished by integration of a GFPHIS3 cassette pGFP+; (Scholz et al., 2000)e before Pro475, truncating Ldb17 and removing the prolinerich domain. To create the control strains (CSY76 and CSY78), the GFP-HIS3 cassette was integrated before the LDB17 stop codon. Expression of Ldb17PRD∆ in CSY80 and CSY79 was accomplished by integration of a GFPHIS3 cassette pGFP+; (Scholz et al., 2000)e before Pro475, truncating Ldb17 and removing the prolinerich domain. The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in the indicated strain The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in the indicated strain The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in the indicated strain The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus in the  142  Type  Name  Description/Genotype  Source  Yeast Strain  MDY620  BY4741 LDB174XGFP::NAT  This study  Yeast Strain  MDY628  BY4741 sla1∆::KAN LDB17-4XGFP::NAT  This study  Yeast Strain  CSY84  BY4741 LDB17-GFP::NAT SLA1-RFP::HIS3  This study  Yeast Strain  CSY85  BY4741 LDB17-GFP::NAT MYO5-RFP::HIS3  This study  Yeast Strain  CSY86  BY4741 LDB17-GFP::NAT ABP1-RFP::HIS3  This study  Yeast Strain  SEY6210  S. Emr  Yeast Strain  BWY2563  MATa leu2-3,112 ura3-52 his3-∆200 trp1-∆901 lys2-801 suc2-∆9 MetSEY6210 yap1801∆::HIS3  Yeast Strain  BWY2565  SEY6210 yap1802∆::LEU2  Wendla nd and Emr, 1998k  Yeast Strain  BWY2567  SEY6210 yap1801∆::HIS3 yap1802∆::LEU2  Wendla nd and  Wendla nd and Emr, 1998k  Notes indicated strain MDY620 and MDY628 were constructed by transforming BY4741 and BY4741 sla1∆ with HpaI/XbaI digested pBW1427. MDY620 and MDY628 were constructed by transforming BY4741 and BY4741 sla1∆ with HpaI/XbaI digested pBW1427. C-terminal tag encoding mRFP1.5 (Campbell et al., 2002)j was integrated as previously described (Longtine et al., 1998)i C-terminal tag encoding mRFP1.5 (Campbell et al., 2002)j was integrated as previously described (Longtine et al., 1998)i C-terminal tag encoding mRFP1.5 (Campbell et al., 2002)j was integrated as previously described (Longtine et al., 1998)i  BY2563, BY2565 and BY2567 were backcrossed from strains described in (Wendland and Emr, 1998)k BY2563, BY2565 and BY2567 were backcrossed from strains described in (Wendland and Emr, 1998)k BY2563, BY2565 and BY2567 were  143  Type  Name  Description/Genotype  Source Emr, 1998k  Yeast Strain  BWY2552  SEY6210 ldb17∆::NAT  This study  Yeast Strain  BWY2068  SEY6210 end3∆::KAN  This study  Yeast Strain  HBY551  SEY6210 ABP1-GFP::HIS3  This study  Yeast Strain  HBY552  BWY2552 ABP1GFP::HIS3  This study  Yeast Strain  MDY562  SEY6210 SLA1-RFP::HIS3  This study  Yeast Strain  MDY564  SEY6210 ldb17∆::NAT SLA1-RFP::HIS3  This study  Yeast Strain  PJ694a  James et al., 1996m  Yeast Strain  PJ694a  MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4∆ gal80∆ LYS::GAL1-HIS3 GAL2-ADE2 met2::GAL7lacZ MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4∆  Notes backcrossed from strains described in (Wendland and Emr, 1998)k LDB17 was replaced by NATR MX4 amplified from p4339 (Tong and Boone, 2006)l in SEY6210 to create BWY2552 END3 was replaced by KANR MX6 amplified from pFA6a-KanMX6 (Longtine et al., 1998)i to create BY2068 C-terminal tag encoding an improved version of GFP (GFP+) (Scholz et al., 2000)e was integrated in SEY6210 C-terminal tag encoding an improved version of GFP (GFP+) (Scholz et al., 2000)e was integrated in WY2552 C-terminal tag encoding mRFP1.5 (Campbell et al., 2002)j was integrated as previously described (Longtine et al., 1998) in SEY6210 C-terminal tag encoding mRFP1.5 (Campbell et al., 2002)j was integrated in BWY2552 as previously described (Longtine et al., 1998)i  James et al., 1996m  144  Type  Name  Description/Genotype gal80∆ LYS::GAL1-HIS3 GAL2-ADE2 met2::GAL7lacZ PJ694a pOAD-LDB17  Source  Notes  Yeast Strain  Y2HLDB17  S. Fields  BMY102  PJ694a pOAD-ldb17∆PRD  This study  Yeast Strain  Y6613  C. Boone  Yeast Strain  MDY220  MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 lyp1∆::STE3pr-LEU2 cyh Y6613 suc2::GFP-SNC1SUC2::URA3  PJ69-4a containing pOAD-LDB17 was obtained from the genome wide Y2H activation domain collection (gift from Stan Fields, U of Washington). PJ69-4a containing pOAD-ldb17 ∆PRD was generated by cotransformation of PJ694-a with an LDB17 PCR fragment lacking the C-term PRD domain (Pro475-Lys491) and PvuII/NcoI-linearized pOAD, and was subsequently confirmed by sequencing and western blotting. gift from Charlie Boone.  Yeast Strain  Yeast Strain  Y7043  C. Boone  Yeast Strain  MDY519  MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 can1::STE2pr-LEU2 lyp1 cyh2 Y7043 suc2::GFP-SNC1SUC2::URA3  This study  This study  Synthetic genetic analysis (SGA) starting strains Y6613 (Tong and Boone, 2006)g and Y7043 (gift of C. Boone) were transformed with Xba1-linearized pCS30, yielding MDY220 and MDY519. gift from Charlie Boone.  Synthetic genetic analysis (SGA) starting strains Y6613 (Tong and Boone, 2006)g and Y7043 (gift of C. Boone) were  145  Type  Name  Description/Genotype  Source  Yeast Strain  MDY525  Y7043 suc2::GFP-SNC1SUC2::NAT  This study  Yeast Strain  MDY59  Y7043 suc2::GFPSNC1(S59A)-SUC2::NAT  This study  Yeast Strain  MDY79  Y7043 suc2::GFPSNC1(S59D)-SUC2::NAT  This study  Yeast Strain  MDY324  Y7043 suc2::GFPSNC1(V40A,M43A)SUC2::URA3 pNAT  This study  Yeast Strain  MDY48  MDY519 apm1∆::NAT  This study  Yeast Strain  MDY46  MDY519 apm2∆::NAT  This study  Yeast Strain  MDY47  MDY519 apl4∆::NAT  This study  Notes transformed with Xba1-linearized pCS30, yielding MDY220 and MDY519. Y7043 was transformed with pHB4 was digested with NotI and SnaB1 pHB4 was mutated to introduce the SNC1 S59A mutation, digested with NotI/SnaBI and transformed into Y7043 pHB4 was mutated to introduce the SNC1 S59D mutation, digested with NotI/SnaBI and transformed into Y7044 The GSS EN- reporter plasmid pMD9 was linearized with XbaI and integrated at the SUC2 locus in the indicated strain Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker  146  Type  Name  Description/Genotype  Source  Yeast Strain  MDY35  MDY519 yap1801∆::NAT  This study  Yeast Strain  MDY47  MDY519 apl4∆::NAT  This study  Yeast Strain  MDY48  MDY519 apm1∆::NAT  This study  Yeast Strain  MDY46  MDY519 apm2∆::NAT  This study  Yeast Strain  MDY34  MDY519 ent1∆::NAT  This study  Notes was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with  147  Type  Name  Description/Genotype  Source  Yeast Strain  MDY35  MDY519 ent2∆::NAT  This study  Yeast Strain  MDY36  MDY519 ent4∆::NAT  This study  Yeast Strain  MDY53  MDY519 glc8∆::NAT  This study  Yeast Strain  MDY43  MDY519 inp52∆::NAT  This study  Yeast Strain  MDY37  MDY519 prk1∆::NAT  This study  Notes appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and  148  Type  Name  Description/Genotype  Source  Yeast Strain  MDY44  MDY519 sec28∆::NAT  This study  Yeast Strain  MDY41  MDY519 syp1∆::NAT  This study  Yeast Strain  MDY45  MDY519 ubi4∆::NAT  This study  Yeast Strain  MDY52  MDY519 vps13∆::NAT  This study  Yeast Strain  MDY33  MDY519 yap1801∆::NAT  This study  Yeast Strain  MDY40  MDY519 yap1802∆::NAT  This study  Notes Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker  149  Type  Name  Description/Genotype  Source  Yeast Strain  MDY38  MDY519 ydr348c∆::NAT  This study  Yeast Strain  MDY54  MDY519 yvh1∆::NAT  This study  Yeast Strain  MDY88  MDY519 apl6∆::NAT  This study  Yeast Strain  MDY14  MDY519 atg20∆::NAT  This study  Yeast Strain  MDY87  MDY519 ent5∆::NAT  This study  Notes was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with  150  Type  Name  Description/Genotype  Source  Yeast Strain  MDY129  MDY519 gga2∆::NAT  This study  Yeast Strain  MDY131  MDY519 inp53∆::NAT  This study  Yeast Strain  MDY127  MDY519 nhx1∆::NAT  This study  Yeast Strain  MDY10  MDY519 snx4∆::NAT  This study  Yeast Strain  MDY169  MDY519 snx41∆::NAT  This study  Notes appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and  151  Type  Name  Description/Genotype  Source  Yeast Strain  MDY271  MDY519 vam3∆::NAT  This study  Yeast Strain  MDY92  MDY519 vps4∆::NAT  This study  Yeast Strain  MDY39  MDY519 vps5∆::NAT  This study  Yeast Strain  MDY270  MDY519 vps9∆::NAT  This study  Yeast Strain  MDY95  MDY519 vps54∆::NAT  This study  Yeast Strain  CSY56  MDY519 vps55∆::NAT  This study  Notes Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker  152  Type  Name  Description/Genotype  Source  Yeast Strain  CSY57  MDY519 vps68∆::NAT  This study  Yeast Strain  MDY125  MDY519 yfr043c∆::NAT  This study  Yeast Strain  MDY170  MDY519 ypt31∆::NAT  This study  Yeast Strain  MDY16  MDY519 ypt35∆::NAT  This study  Yeast Strain  MDY292  MDY519 trp1∆::NAT  This study  Notes was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with  153  Type  Name  Description/Genotype  Source  Yeast Strain  MDY282  MDY519 age2∆::NAT  This study  Yeast Strain  MDY171  MDY519 cka1∆::NAT  This study  Yeast Strain  MDY280  MDY519 ckb1∆::NAT  This study  Yeast Strain  MDY13  MDY519 ent3∆::NAT  This study  Yeast Strain  MDY85  MDY519 erg4∆::NAT  This study  Notes appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and  154  Type  Name  Description/Genotype  Source  Yeast Strain  MDY133  MDY519 fks1∆::NAT  This study  Yeast Strain  MDY279  MDY519 gcs1∆::NAT  This study  Yeast Strain  MDY277  MDY519 gef1∆::NAT  This study  Yeast Strain  MDY42  MDY519 hul5∆::NAT  This study  Yeast Strain  MDY83  MDY519 las21∆::NAT  This study  Yeast Strain  MDY82  MDY519 lcb4∆::NAT  This study  Notes Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker  155  Type  Name  Description/Genotype  Source  Yeast Strain  MDY265  MDY519 sak1∆::NAT  This study  Yeast Strain  MDY268  MDY519 scy1∆::NAT  This study  Yeast Strain  MDY284  MDY519 vam7∆::NAT  This study  Yeast Strain  MDY283  MDY519 vps8∆::NAT  This study  Yeast Strain  MDY286  MDY519 vps17∆::NAT  This study  Notes was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with  156  Type  Name  Description/Genotype  Source  Yeast Strain  MDY134  MDY519 vps21∆::NAT  This study  Yeast Strain  MDY278  MDY519 vps38∆::NAT  This study  Yeast Strain  MDY281  MDY519 vps51∆::NAT  This study  Yeast Strain  MDY287  MDY519 ykr078w∆::NAT  This study  Yeast Strain  MDY285  MDY519 ypr097w∆::NAT  This study  Notes appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and  157  Type  Name  Description/Genotype  Source  Yeast Strain  MDY80  MDY519 ypt6∆::NAT  This study  Yeast Strain  MDY360  MDY519 apm3∆::NAT  This study  Yeast Strain  MDY389  MDY519 arf1∆::NAT  This study  Yeast Strain  MDY332  MDY519 arr4∆::NAT  This study  Yeast Strain  MDY333  MDY519 bsd2∆::NAT  This study  Yeast Strain  MDY415  MDY519 hap4∆::NAT  This study  Notes Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker  158  Type  Name  Description/Genotype  Source  Yeast Strain  MDY371  MDY519 lsp1∆::NAT  This study  Yeast Strain  MDY413  MDY519 mon2∆::NAT  This study  Yeast Strain  MDY357  MDY519 mvp1∆::NAT  This study  Yeast Strain  MDY372  MDY519 pil1∆::NAT  This study  Yeast Strain  MDY383  MDY519 ptc1∆::NAT  This study  Notes was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with  159  Type  Name  Description/Genotype  Source  Yeast Strain  MDY391  MDY519 rav1∆::NAT  This study  Yeast Strain  MDY366  MDY519 rts1∆::NAT  This study  Yeast Strain  MDY421  MDY519 sft2∆::NAT  This study  Yeast Strain  MDY400  MDY519 vrp1∆::NAT  This study  Yeast Strain  MDY51  MDY519 akr1∆::NAT  This study  Notes appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and  160  Type  Name  Description/Genotype  Source  Yeast Strain  MDY332  MDY519 get3∆::NAT  This study  Yeast Strain  MDY393  MDY519 stv1∆::NAT  This study  Yeast Strain  MDY398  MDY519 ubp3∆::NAT  This study  Yeast Strain  MDY385  MDY519 vps1∆::NAT  This study  Yeast Strain  MDY417  MDY519 vps27∆::NAT  This study  Notes Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l. Gene replacement with the NATR MX4 marker was accomplished by transformation of MDY519 with appropriate PCR products amplified from p4339 (Tong and Boone, 2006)l.  161  Table A2. Full list of mutants with cell surface GSS levels greater to or equal to the median value for all strains. Note that a low densitometry value indicates a high level of cell surface reporter. Densitometry values were subtracted from the median value for all strains to generate "SCORE". Rank  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37  ORF  Gene  YCR009C YDR207C YOR035C YLR337C YAL013W YPL045W YNL069C YDR388W YMR198W YDL146W YKR094C YBL007C YCR044C YIL049W YKL002W YIL154C YDR276C YLR370C YLR056W YIL128W YHR026W YER056CA YLR338W YLR399C YMR311C YGR240C YDR442W YOL004W YHR161C YEL031W YBL047C YJL080C YNL215W YMR091C YIL090W YDR364C YPL031C  RVS161 UME6 SHE4 VRP1 DEP1 VPS16 RPL16B RVS167 CIK1 LDB17 RPL40B SLA1 PER1 DFG10 DID4 IMP2' PMP3 ARC18 ERG3 MET18 PPA1 RPL34A  BDF1 GLC8 PFK1 SIN3 YAP1801 SPF1 EDE1 SCP160 IES2 NPL6 ICE2 CDC40 PHO85  Normalized densitometry values MATa MATalpha 0.11 0.04 0.05 0.10 0.14 0.08 0.14 0.12 0.13 0.13 0.03 0.24 0.13 0.14 0.14 0.06 0.22 0.14 0.15 0.10 0.20 0.16 0.16 0.18 0.07 0.35 0.18 0.24 0.21 0.22 0.32 0.13 0.23 0.23 0.24 0.28 0.10 0.14 0.26 0.26 0.06 0.15 0.21 0.15 0.29 0.29 0.29 0.32  0.19 0.40 0.36 0.26  0.45 0.38 0.33 0.27 0.42  0.29  SCORE average 0.07 0.07 0.11 0.13 0.13 0.13 0.13 0.13 0.14 0.14 0.14 0.15 0.15 0.16 0.16 0.18 0.21 0.21 0.21 0.23 0.23 0.23  0.93 0.93 0.90 0.87 0.87 0.87 0.87 0.87 0.87 0.86 0.86 0.86 0.86 0.85 0.85 0.82 0.80 0.80 0.79 0.78 0.77 0.77  0.24 0.25 0.25 0.26 0.26 0.26 0.26 0.27 0.27 0.27 0.28 0.29 0.29 0.29 0.30  0.77 0.75 0.75 0.75 0.75 0.75 0.75 0.74 0.74 0.73 0.72 0.71 0.71 0.71 0.70  162  Rank  38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81  ORF  Gene  YLR119W YDL081C YNR006W YIL076W YGR241C YLR025W YPR029C YKL007W YIL093C YGL058W YJL176C YER074W YBR127C YAL047C YNL079C YJR121W YCR088W YKL119C YGR242W YPL178W YHL002W YHL025W YMR116C YMR109W YLR425W YNL105W YBL027W YDL002C YNR010W YIL084C YNL106C YFL025C YNR023W YNR052C YGL168W YJL062W YIR034C YMR263W YGL084C YEL013W YIR003W YKR035C YLR171W YPL002C  SRN2 RPP1A VPS27 SEC28 YAP1802 SNF7 APL4 CAP1 RSM25 RAD6 SWI3 RPS24A VMA2 SPC72 TPM1 ATP2 ABP1 VPH2 CBC2 HSE1 SNF6 ASC1 MYO5 TUS1 RPL19B NHP10 CSE2 SDS3 INP52 BST1 SNF12 POP2 HUR1 LAS21 LYS1 SAP30 GUP1 VAC8  SNF8  Normalized densitometry values MATa MATalpha 0.31 0.30 0.33 0.43 0.21 0.32 0.35 0.29 0.33 0.23 0.44 0.26 0.42 0.34 0.25 0.46 0.71 0.00 0.39 0.33 0.36 0.37 0.50 0.24 0.37 0.32 0.42 0.38 0.43 0.33 0.38 0.30 0.47 0.39 0.17 0.61 0.35 0.43 0.40 0.39 0.25 0.54 0.42 0.38 0.26 0.56 0.26 0.57 0.43 0.29 0.56 0.35 0.51 0.65 0.21 0.86 0.00 0.36 0.51 0.50 0.38 0.57 0.31 0.56 0.32 0.38 0.51 0.38 0.51 0.51 0.39 0.50 0.40 0.26 0.64 0.45  SCORE average 0.31 0.32 0.32 0.32 0.32 0.33 0.33 0.34 0.34 0.35 0.35 0.36 0.36 0.37 0.37 0.37 0.37 0.38 0.38 0.38 0.39 0.39 0.39 0.39 0.39 0.40 0.40 0.41 0.42 0.43 0.43 0.43 0.43 0.43 0.43 0.44 0.44 0.44 0.44 0.45 0.45 0.45 0.45 0.45  0.70 0.69 0.69 0.68 0.68 0.68 0.67 0.67 0.66 0.65 0.65 0.65 0.64 0.64 0.63 0.63 0.63 0.62 0.62 0.62 0.62 0.62 0.62 0.61 0.61 0.61 0.61 0.59 0.59 0.58 0.58 0.58 0.57 0.57 0.57 0.57 0.56 0.56 0.56 0.56 0.55 0.55 0.55 0.55  163  Rank  ORF  Gene  82 YCR028CA 83 YGR101W 84 YLR200W 85 YGL027C 86 YDL243C 87 YLR170C 88 YDL160C 89 YOR209C 90 YER092W 91 YAL068C 92 YGL219C 93 YGR204W 94 YLR169W 95 YGL066W 96 YKR001C 97 YOR275C 98 YPL100W 99 YPR067W 100 YKL204W 101 YNL219C 102 YBL083C 103 YPL042C 104 YJL175W 105 YNL197C 106 YDR226W 107 YOR126C 108 YBL058W 109 YOL129W 110 YGR206W 111 YML019W 112 YDL194W 113 YNR005C 114 YBL006C 115 YKL041W 116 YNL111C 117 YFR031CA 118 YBR095C 119 YBR156C 120 YGR100W 121 YBL082C 122 YLL030C 123 YLR417W  RIM1 PCP1 YKE2 CWH41 AAD4 APS1 DHH1 NPT1 IES5 MDM34 ADE3 SGF73 VPS1 RIM20 ATG21 ISA2 EAP1 ALG9 SSN3 WHI3 ADK1 IAH1 SHP1 VPS68 OST6 SNF3 LDB7 VPS24 CYB5 RPL2A  SLI15 MDR1 ALG3 VPS36  Normalized densitometry values MATa MATalpha 0.45 0.24 0.46 0.32 0.27 0.34 0.36 0.35 0.53 0.32 0.33 0.43 0.42 0.39 0.37 0.50 0.33 0.55 0.50 0.50 0.84 0.97 0.19 0.39 0.51 0.44 0.54 0.66 0.53 0.53 0.48 0.27 0.44 0.84 0.52 0.52 0.06  SCORE average 0.45  0.55  0.58 0.80  0.46 0.46 0.46 0.47 0.47 0.47 0.48 0.48 0.48 0.48 0.48 0.48 0.48 0.48 0.49 0.49 0.50 0.50 0.50 0.50 0.50 0.50 0.51 0.51 0.51 0.51 0.52 0.52 0.52 0.52 0.52 0.53 0.53 0.53 0.53  0.54 0.54 0.54 0.53 0.53 0.53 0.53 0.53 0.52 0.52 0.52 0.52 0.52 0.52 0.51 0.51 0.51 0.51 0.51 0.51 0.51 0.50 0.50 0.50 0.50 0.49 0.49 0.48 0.48 0.48 0.48 0.48 0.48 0.48 0.47  0.64 0.23 0.56 0.56 1.03 0.55  0.54 0.54 0.54 0.54 0.55 0.55  0.47 0.47 0.47 0.46 0.46 0.46  0.67 0.60 0.47 0.67 0.47 0.61 0.60 0.61 0.43 0.64 0.63 0.54 0.55 0.59 0.62 0.66 0.45  0.16 0.04 0.51 0.83 0.51 0.64 0.53 0.60 0.51 0.39  164  Rank  124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167  ORF  Gene  YIL020C YNR030W YKR100C YIL034C YLR139C YIL163C YPL256C YPR030W YHL019C YML014W YJL153C YHR030C YPR164W YPL179W YDR006C YJR073C YML008C YER120W YKL073W YGL076C YPR070W YNL248C YPR024W YKR099W YCL005W YDR074W YLR428C YNL016W YIR023W YNL252C YML048W YGR080W YNL269W YPL158C YNL238W YER130C YKL074C YBR289W YMR272C YCR063W YHL027W YPL057C YLR403W YPL118W  HIS6 ALG12 SKG1 CAP2 SLS1 CLN2 CSR2 APM2 TRM9 INO1 SLT2 MMS1 PPQ1 SOK1 OPI3 ERG6 SCS2 LHS1 RPL7A MED1 RPA49 YME1 BAS1 LDB16 TPS2 PUB1 DAL81 MRPL17 GSF2 TWF1 BSC4 KEX2 MUD2 SNF5 SCS7 BUD31 RIM101 SUR1 SFP1 MRP51  Normalized densitometry values MATa MATalpha 0.20 0.90 0.53 0.57 0.58 0.52 0.46 0.64 0.55 0.12 1.00 0.62 0.50 0.39 0.73 0.59 0.53 0.56 0.24 0.88 0.64 0.48 0.36 0.76 0.63 0.50 0.53 0.60 0.56 1.04 0.09 0.49 0.64 0.67 0.47 0.52 0.62 0.73 0.41 0.57 0.80 0.35 0.53 0.62 0.13 1.02 0.64 0.52 0.64 0.52 0.50 0.66 0.14 1.02 0.58 0.49 0.68 0.43 0.74 0.13 1.04 0.59 0.59 0.59 0.60 0.57 0.47 0.71 0.59 0.37 0.82 0.57 0.62 0.45 0.74 0.46 0.73 0.39 0.80 0.60  SCORE average 0.55 0.55 0.55 0.55 0.55 0.56 0.56 0.56 0.56 0.56 0.56 0.56 0.56 0.56 0.56 0.56 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.59 0.59 0.59 0.59 0.59 0.59 0.59 0.59 0.59 0.59 0.60 0.60 0.60  0.46 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.43 0.43 0.43 0.43 0.43 0.43 0.43 0.43 0.42 0.42 0.42 0.42 0.42 0.42 0.42 0.42 0.42 0.41 0.41 0.41 0.41 0.41 0.41 0.41 0.41  165  Rank  168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211  ORF  Gene  YGR256W YLL033W YNL097C YGR061C YCL007C YJL183W YHR081W YDR448W YIL027C YGR157W YOL108C YOR030W YOR008C YPR101W YGR056W YCR002C YDL176W YEL036C YFR001W YJL060W YBR229C YLL038C YLL039C YOR026W YLR087C YOL036W YGR122W YBR097W YOR274W YJR075W YNL284C YPL027W YNL220W YHR082C YJR034W YNL280C YDL075W YCR028C YLR069C YDL073W YLR358C YDR129C YPR052C YIR026C  GND2 PHO23 ADE6 MNN11 LRP1 ADA2 KRE27 CHO2 INO4 DFG16 SLG1 SNT309 RSC1 CDC10 ANP1 LOC1 BNA3 ROT2 ENT4 UBI4 BUB3 CSF1  VPS15 MOD5 HOC1 MRPL10 SMA1 ADE12 KSP1 PET191 ERG24 RPL31A FEN2 MEF1  SAC6 NHP6A YVH1  Normalized densitometry values MATa MATalpha 0.47 0.72 0.69 0.50 0.61 0.59 0.63 0.58 0.60 0.88 0.33 0.29 0.92 0.57 0.64 0.70 0.51 0.65 0.57 0.61 0.77 0.45 0.73 0.49 0.47 0.75 0.56 0.67 0.61 0.01 1.22 0.62 0.62 0.81 0.43 0.62 0.66 0.58 0.56 0.68 0.70 0.54 0.66 0.58 0.14 1.11 0.42 0.82 0.62 0.65 0.61 0.66 0.59 0.63 0.54 0.71 0.52 0.74 0.59 0.67 0.63 1.09 0.18 0.61 0.66 0.63 0.64 0.22 1.05 0.64 1.11 0.17 0.73 0.55 0.91 0.38  SCORE average 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.61 0.61 0.61 0.61 0.61 0.61 0.61 0.61 0.61 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.62 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.64 0.64 0.64 0.64 0.64 0.64  0.41 0.41 0.41 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.36 0.36  166  Rank  ORF  Gene  YMR023C YGL127C YLR382C YGL167C YCR030C YML017W YMR063W YBR255W YOR141C YLR150W YGL200C YGL014W YGL072C YEL063C YAL055W YDR532C YNL153C YJL124C YDR346C YJL020C YEL060C YJL128C YEL067C YBR150C YKL081W YPL034W YDL005C YPR006C YGL016W YIR009W YJR116W YLR412W YPL129W YPL066W YNR031C YJL067W YBR144C YJL068C YFL013WA 251 YMR031W -A 252 YLR149C 253 YIL116W  MSS1 SOH1 NAM2 PMR1 SYP1 PSP2 RIM9  212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250  ARP8 STM1 EMP24 PUF4 CAN1 PEX22 GIM3 LSM1 SVF1 BBC1 PRB1 PBS2 TBS1 TEF4 MED2 ICL2 KAP122 MSL1  TAF14 SSK2  HIS5  Normalized densitometry values MATa MATalpha 0.26 1.02 0.41 0.88 0.64 0.56 0.73 0.51 0.78 0.69 0.60 0.34 0.96 0.67 0.63 1.03 0.28 0.63 0.67 0.65 0.65 0.66 0.34 0.97 0.33 0.98 0.72 0.58 1.22 0.09 0.66 0.68 0.64 0.61 0.70 0.75 0.57 0.64 0.68 0.59 0.73 0.53 0.80 0.54 0.79 0.43 0.90 0.66 0.68 0.67 0.75 0.58 0.50 0.83 0.43 0.90 0.80 0.54 0.70 0.64 0.67 0.93 0.41 0.66 0.69 0.47 0.88 0.76 0.58 0.38 0.97 0.57 0.78  SCORE average 0.64 0.64 0.64 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.67 0.67 0.67 0.67 0.67 0.67 0.67 0.67 0.67 0.67 0.67 0.67 0.67 0.67  0.36 0.36 0.36 0.36 0.36 0.36 0.36 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.33 0.33 0.33 0.33 0.33 0.33 0.33  0.56  0.79  0.67  0.33  0.56 0.25  0.79 1.10  0.67 0.68  0.33 0.33  167  Rank  254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296  ORF  Gene  YLR113W YLR422W YMR024W YNL199C YHR092C YFL034W YKL065C YMR038C YGR160W YOR067C YJL056C YKL063C YGR126W YDR225W YBR106W YGR259C YPL212C YDR233C YGL045W YIL130W YOL009C YKR035W -A YLR120C YJR055W YCR048W YGL149W YJL120W YDL161W YFL013C YPR173C YML012W YIR005W YHR079C YCL046W YLR136C YPL036W YDR072C YCL008C YDR335W YNL264C YOR189W YPL227C YDR455C  HOG1 MRPL3 GCR2 HXT4  CCS1 ALG8 ZAP1  HTA1 PHO88 PUS1 RTN1 RIM8 MDM12 DID2 YPS1 HIT1 ARE1  ENT1 IES1 VPS4 ERV25 IST3 IRE1 TIS11 PMA2 IPT1 STP22 MSN5 PDR17 IES4 ALG5  Normalized densitometry values MATa MATalpha 0.75 0.60 0.65 0.70 0.34 1.01 0.66 0.69 0.85 0.51 0.70 0.66 0.72 0.64 0.68 0.40 0.97 0.65 0.72 0.51 0.85 0.68 0.69 0.92 0.45 0.81 0.56 0.75 0.62 0.58 0.80 0.55 0.83 0.54 0.84 0.55 0.83 0.66 0.72 0.53 0.85 0.66 0.73 0.58 0.42 0.63 0.52 0.66 0.66 0.79 0.72 0.73 0.71 0.79 0.66 0.83 0.57 0.78 1.03 0.51 0.52 0.73 0.44  0.80 0.96 0.75 0.86 0.73 0.73 0.59 0.67 0.66 0.68 0.60 0.73 0.56 0.83 0.62 0.37 0.70 0.89 0.88 0.68 0.97  SCORE average 0.68 0.68 0.68 0.68 0.68 0.68 0.68 0.68 0.68 0.68 0.68 0.68 0.68 0.69 0.69 0.69 0.69 0.69 0.69 0.69 0.69 0.69  0.33 0.33 0.33 0.33 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.31 0.31 0.31 0.31  0.69 0.69 0.69 0.69 0.69 0.69 0.69 0.69 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70  0.31 0.31 0.31 0.31 0.31 0.31 0.31 0.31 0.31 0.31 0.31 0.31 0.31 0.31 0.31 0.31 0.31 0.30 0.30 0.30 0.30  168  Rank  ORF  Gene  297 YMR032W 298 YNL266W 299 YMR316CA 300 YPL049C 301 YLR262C 302 YMR139W 303 YCR060W 304 YOR255W 305 YDR414C 306 YPL145C 307 YOR211C 308 YOL007C 309 YAL010C 310 YOR127W 311 YGR078C 312 YPL079W 313 YBL016W 314 YER155C 315 YNL084C 316 YDL023C 317 YMR126C 318 YGR036C 319 YPL101W 320 YOR036W 321 YGL012W 322 YDR368W 323 YOR016C 324 YNL004W 325 YPL239W 326 YER154W 327 YJR088C 328 YDR221W 329 YMR307W 330 YNR050C 331 YDR424C 332 YDL231C 333 YAL053W 334 YDL096C 335 YBR266C 336 YKL001C 337 YJR106W 338 YFR024CA  HOF1  DIG1 YPT6 RIM11 TAH1 OSW1 ERD1 KES1 MGM1 MDM10 RGA1 PAC10 RPL21B FUS3 BEM2 END3  CAX4 ELP4 PEP12 ERG4 YPR1 ERP4 HRB1 YAR1 OXA1  GAS1 LYS9 DYN2 BRE4  SLM6 MET14 ECM27 LSB3  Normalized densitometry values MATa MATalpha 0.70 0.70 0.47 0.94 0.46 0.54 0.67 0.47 0.21 0.75 0.73 0.44 0.51 0.71 0.52 0.61 0.14 0.63 0.95 1.20 0.70 0.71 0.95 0.71 0.72 0.46 0.69 0.62 0.64 0.75 0.64 0.79 0.74 0.72 0.31 0.86 0.56 0.68 0.48 0.62 0.58 0.73  0.95 0.87 0.74 0.94 1.20 0.67 0.69 0.99 0.91 0.91 0.82 1.28 0.79 0.48 0.23 0.73 0.72 0.48 0.72 0.72 0.97 0.74 0.81 0.79 0.69 0.80 0.65 0.71 1.14 0.58 0.89 0.76 0.97 0.83 0.86 0.72  SCORE average 0.70 0.70 0.70  0.30 0.30 0.30  0.70 0.70 0.70 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72  0.30 0.30 0.30 0.30 0.30 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.29 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28  169  Rank  339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381  ORF  Gene  YGL198W YBR248C YNL294C YGR143W YKL072W YEL045C YKL071W YOL072W YLR006C YJR082C YBR019C YLR371W YJR118C YPL193W YDR234W YER117W YER087W YJR010C-A YPR131C YFL016C YGR058W YKL139W YNL198C YLR262CA YKL032C YOL006C YLR429W YNR021W YML094W YMR092C YFL031W YGL109W YJL095W YIR021W YER187W YLR320W YNL050C YPL226W YMR289W YGR254W YGR138C YKL160W YMR074C  YIP4 HIS7 RIM21 SKN1 STB6  THP1 SSK1 EAF6 GAL10 ROM2 ILM1 RSA1 LYS4 RPL23B SPC1 NAT3 MDJ1 CTK1  IXR1 TOP1 CRN1 GIM5 AIP1 HAC1 BCK1 MRS1 MMS22 NEW1 ENO1 TPO2 ELF1  Normalized densitometry values MATa MATalpha 0.66 0.79 0.41 1.04 0.62 0.83 0.64 0.82 0.73 0.73 0.73 0.72 0.73 1.01 0.45 0.80 0.66 0.71 0.75 0.64 0.82 0.73 0.24 1.23 0.26 1.21 0.73 1.04 0.43 0.73 0.53 0.94 0.89 0.58 0.74 0.69 0.78 0.74 0.73 0.74 0.58 0.89 0.75 0.45 0.64 0.74 0.69 0.72 0.75 0.57 0.71 1.01 0.52 0.46 0.92 0.52 0.97 0.91 0.89 0.75 0.62  0.72 1.03 0.83 0.73 0.79 0.75 0.73 0.91 0.77 0.47 0.96 1.03 0.57 0.96 0.52 0.57 0.59 0.74 0.86  SCORE average 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.74 0.74 0.74 0.74 0.74  0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27  0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74  0.27 0.27 0.27 0.27 0.27 0.27 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26  170  Rank  ORF  382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425  YBR235W YHR114W YGR252W YIL085C YNL196C YJR044C YNL123W YER069W YJL121C YDL047W YJL215C YJL038C YCR017C YDR050C YOL100W YJL065C YGL256W YLL014W YKL136W YJL185C YNL043C YLR455W YNL165W YBR036C YBR114W YER175C YDR028C YMR242C YMR145C YGR135W YDL184C YBR220C YLL054C YPL064C YDR329C YNL070W YPL061W YOL098C YJL182C YBR294W YOR128C YKR021W YNL324W YKR096W  Gene  BZZ1 GCN5 KTR7 VPS55 ARG5,6 RPE1 SIT4  CWH43 TPI1 PKH2 DLS1 ADH4  CSG2 RAD16 TMT1 REG1 RPL20A NDE1 PRE9 RPL41A  CWC27 PEX3 TOM7 ALD6  SUL1 ADE2  Normalized densitometry values MATa MATalpha 0.85 0.63 0.79 0.70 0.93 0.56 0.85 0.64 0.51 0.98 0.85 0.64 0.91 0.58 0.37 1.12 0.75 0.74 0.70 0.79 0.54 0.95 0.75 0.76 0.74 0.75 0.75 0.72 0.78 0.61 0.89 0.66 0.84 0.66 0.84 0.94 0.56 1.03 0.47 0.64 0.86 0.75 0.91 0.59 1.02 0.48 0.56 0.94 0.82 0.69 0.60 0.90 0.80 0.70 0.89 0.62 0.53 0.97 0.79 0.71 0.69 0.81 0.63 0.88 0.98 0.53 1.34 0.17 0.65 0.86 0.66 0.85 0.67 0.84 0.41 1.10 0.64 0.87 1.00 0.52 0.71 0.81 0.75 0.77  SCORE average 0.74 0.74 0.74 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76  0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25  171  Rank  426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469  ORF  Gene  YOL028C YFL003C YGL231C YPL165C YAL035W YIL011W YKL213C YPR074C YJR010W YNL148C YDR293C YBR059C YCL058C YGR023W YOR002W YML055W YKL064W YBL025W YPL240C YCR011C YOL076W YBR301W YOR345C YMR317W YLR099C YIL025C YGR155W YCL045C YOL059W YIL132C YMR275C YOL013C YDR011W YMR148W YDR265W YKL066W YDL076C YMR179W YJR105W YDR071C YMR027W YMR241W YLR386W YHR087W  YAP7 MSH4 SET6 FUN12 TIR3 DOA1 TKL1 MET3 ALF1 SSD1 AKL1 FYV5 MTL1 ALG6 SPC2 MNR2 RRN10 HSP82 ADP1 MDM20 DAN3  ICT1 CYS4 GPD2 CSM2 BUL1 HRD1 SNQ2 PEX10 RXT3 SPT21 ADO1 PAA1 YHM2 VAC14  Normalized densitometry values MATa MATalpha 0.81 0.71 0.97 0.54 0.75 0.77 0.82 0.70 0.60 0.92 0.88 0.64 0.77 0.75 0.80 0.73 0.48 1.05 0.76 0.76 0.81 0.72 0.72 0.81 0.64 0.89 0.76 0.77 0.74 0.79 0.75 0.78 0.81 0.72 0.86 0.67 0.49 1.04 0.73 0.80 0.77 0.52 1.01 0.56 0.97 0.77 0.56 0.97 0.85 0.68 0.77 0.76 0.81 0.73 0.80 0.74 0.86 0.68 0.82 0.72 0.76 0.78 0.97 0.57 0.76 0.78 0.66 0.88 0.77 0.77 0.43 1.11 0.83 0.71 0.77 0.60 0.94 0.67 0.88 0.35 1.19 0.73 0.81 0.50 1.04  SCORE average 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77  0.25 0.25 0.25 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23  172  Rank  470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513  ORF  Gene  YOR130C YLL047W YFL032W YML084W YPL041C YOR378W YGL015C YNL089C YCL074W YPL017C YNL255C YGR215W YEL054C YER161C YPR053C YDR010C YMR031C YPL077C YGR130C YPR065W YGL024W YOL152W YMR252C YNL042W YML029W YDR209C YOL001W YML083C YNL192W YNL299W YPL139C YKR097W YER068W YFL004W YGR069W YJL127C YPL004C YLR152C YPL102C YNL316C YER122C YJL130C YBR141C YPL262W  ORT1  GIS2 RSM27 RPL12A SPT2  ROX1 FRE7 BOP3 USA1 PHO80 CHS1 TRF5 UME1 PCK1 MOT2 VTC2 SPT10 LSP1  PHA2 GLO3 URA2 FUM1  Normalized densitometry values MATa MATalpha 0.56 0.99 0.95 0.60 0.80 0.75 0.62 0.93 0.49 1.06 0.73 0.82 0.49 1.07 0.69 0.86 1.00 0.55 0.55 1.00 0.93 0.62 0.72 0.84 0.43 1.13 0.72 0.83 0.62 0.93 0.86 0.69 0.71 0.84 0.77 0.78 0.78 0.77 0.81 0.74 0.92 0.63 0.60 0.96 0.56 1.00 1.20 0.36 0.76 0.80 0.96 0.60 0.48 1.08 0.81 0.75 0.78 0.54 1.02 0.78 0.79 0.77 0.79 0.58 0.98 0.89 0.68 0.94 0.63 0.76 0.80 0.65 0.92 0.86 0.71 0.70 0.87 0.54 1.03 0.52 1.05 0.78 0.84 0.73 0.84 0.73  SCORE average 0.77 0.77 0.77 0.77 0.77 0.77 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78 0.78  0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22  173  Rank  514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557  ORF  Gene  YML099C YBR139W YPL062W YJR102C YPL056C YJR122W YKL098W YGL160W YMR315W YDR477W YOR331C YGR020C YGR188C YJR052W YGL205W YHR178W YIL155C YDR192C YKL075C YOR366W YGR184C YPR069C YDR005C YHR110W YLR027C YMR251W YGR208W YBL044W YCR053W YNL246W YKL029C YPR063C YDR241W YJL139C YKL174C YPR153W YKL207W YOR182C YGR260W YHR003C YJL071W YGR154C YGL246C YMR154C  ARG81  VPS25 CAF17  SNF1 VMA7 BUB1 RAD7 POX1 STB5 GUT2 NUP42  UBR1 SPE3 MAF1 ERP5 AAT2 SER2 THR4 VPS75 MAE1 BUD26 YUR1 TPO5  RPS30B TNA1 ARG2 RAI1 RIM13  Normalized densitometry values MATa MATalpha 0.65 0.92 0.82 0.75 0.82 0.75 1.24 0.33 0.61 0.97 0.79 0.67 0.90 0.78 0.80 0.59 0.99 0.79 0.79 0.79 0.90 0.68 0.67 0.91 0.79 0.68 0.91 0.83 0.75 0.93 0.66 0.87 0.72 0.72 0.86 0.96 0.63 0.77 0.82 0.83 0.76 0.88 0.71 0.72 0.87 0.77 0.82 0.80 0.79 0.94 0.65 0.61 0.98 0.84 0.75 0.69 0.90 0.85 0.74 0.85 0.75 0.76 0.84 0.81 0.78 0.84 0.76 0.83 0.76 0.73 0.87 0.78 0.82 0.74 0.86 0.76 0.84 0.73 0.87 0.84 0.76 0.72 0.88  SCORE average 0.78 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80  0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.20 0.20 0.20  174  Rank  558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600  ORF  Gene  YDR525W YDR440W YPL234C YCR076C YOL060C YER184C YGR269W YHR050W YPR091C YER176W YNL250W YLR226W YBR023C YPR166C YLR354C YJL027C YMR123W YBR201W YGL161C YMR269W YDR372C YOL104C YNL325C YOR288C YOR285W YGL174W YNL010W YFR034C YMR316CB YLL002W YIL056W YNR002C YOR239W YLR192C YGR153W YDR466W YDR181C YGL105W YHR100C YLR434C YMR077C YJL212C YOR312C  API2 DOT1 TFP3 MAM3  SMF2 ECM32 RAD50 BUR2 CHS3 MRP2 TAL1 PKR1 DER1 YIP5 VPS74 NDJ1 FIG4 MPD1 BUD13 PHO4  RTT109 ATO2 ABP140 HCR1 PKH3 SAS4 ARC1  VPS20 OPT1 RPL20B  Normalized densitometry values MATa MATalpha 0.67 0.93 0.80 0.80 0.86 0.74 1.02 0.58 0.89 0.71 0.90 0.70 0.81 0.79 0.79 0.81 0.72 0.89 0.83 0.77 0.80 0.66 0.94 0.59 1.02 0.74 0.87 1.02 0.59 1.10 0.51 0.80 0.81 0.87 0.74 0.96 0.65 0.64 0.97 0.72 0.89 0.79 0.83 0.54 1.07 0.84 0.77 0.76 0.85 0.73 0.88 0.90 0.72 0.54 1.08 0.67 0.79 1.01 0.68 0.70 0.69 0.79 0.79 1.07 0.81 0.61 0.81 0.67  0.94 0.83 0.60 0.94 0.92 0.92 0.83 0.83 0.55 1.01 0.95 0.81  SCORE average 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81  0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20  0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81  0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.19 0.19 0.19 0.19 0.19 0.19 0.19  175  Rank  ORF  601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644  YFR011C YNR022C YDR360W YOL016C YDR289C YJR131W YOR267C YLR325C YJR107W YEL042W YNL009W YOL093W YDR315C YKL025C YPL070W YFR043C YIL038C YLR207W YIL139C YLR164W YDR159W YPR009W YDR171W YOL126C YKL031W YBR082C YNL268W YGR227W YOR043W YJL211C YJL055W YPR179C YJR108W YLR052W YDR382W YLR138W YGR228W YKL003C YPL092W YGR263C YLR133W YNL047C YBR125C YDR461W  Gene  MRPL50 CMK2 RTT103 MNS1 HRK1 RPL38 GDA1 IDP3 TRM10 IPK1 PAN3 MUK1 NOT3 HRD3 REV7 SAC3 SUT2 HSP42 MDH2 UBC4 LYP1 DIE2 WHI2  HDA3 ABM1 IES3 RPP2B NHA1 MRP17 SSU1 CKI1 SLM2 PTC4 MFA1  Normalized densitometry values MATa MATalpha 0.97 0.65 0.92 0.70 0.85 0.77 0.75 0.87 0.83 0.79 0.92 0.71 0.82 0.81 0.82 0.80 0.74 0.88 0.75 0.88 0.80 0.83 0.68 0.95 0.70 0.93 0.81 0.82 0.82 0.81 0.70 0.92 0.95 0.68 0.82 0.81 1.05 0.58 0.90 0.73 0.85 0.78 1.09 0.54 1.04 0.59 0.68 0.95 0.84 0.79 0.90 0.73 0.76 0.88 0.82 0.81 0.74 0.90 0.74 0.90 0.67 0.97 0.78 0.85 0.78 0.85 0.89 0.75 0.62 1.02 0.95 0.69 0.94 0.70 0.82 0.59 1.05 0.88 0.76 0.34 1.30 0.81 0.83 1.00 0.64 0.82  SCORE average 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82  0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19  176  Rank  645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688  ORF  Gene  YKR030W YLR432W YGL141W YDL162C YJR109C YFR019W YGL146C YMR312W YBR014C YNL270C YEL007W YOR273C YKL008C YLR356W YDR444W YDL197C YKL069W YHR163W YDR057W YPL078C YMR044W YGL129C YBL033C YML128C YPL154C YBR286W YNL121C YJL088W YJL216C YDR063W YIL005W YLR082C YHR151C YOR270C YNL091W YDL128W YIL133C YNL164C YCL051W YGL108C YOR271C YOR124C YLR143W YCL025C  GMH1 IMD3 HUL5 CPA2 FAB1 ELP6 ALP1 TPO4 LAC1  ASF2 SOL3 YOS9 ATP4 IOC4 RSM23 RIB1 MSC1 PEP4 APE3 TOM70 ARG3  EPS1 SRL2 VPH1 NST1 VCX1 RPL16A IBD2 LRE1  UBP2 AGP1  Normalized densitometry values MATa MATalpha 0.97 0.67 0.71 0.93 0.88 0.76 0.87 0.77 0.85 0.79 0.95 0.69 0.68 0.96 0.77 0.87 0.68 0.96 0.51 1.13 0.82 1.08 0.56 0.73 0.92 0.70 0.94 0.98 0.66 1.01 0.64 0.86 0.79 0.50 1.15 0.78 0.87 0.78 0.87 1.02 0.63 0.96 0.69 0.49 1.16 0.78 0.87 1.03 0.62 0.90 0.75 0.94 0.71 0.55 1.10 0.63 1.02 0.75 0.90 0.88 0.78 0.91 0.74 0.75 0.90 0.93 0.73 0.83 0.83 0.88 0.78 0.97 0.68 0.83 0.72 0.94 0.97 0.69 1.11 0.55 0.79 0.87 0.84 0.82 0.65 1.01  SCORE average 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83  0.19 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.17 0.17 0.17 0.17 0.17  177  Rank  689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731  ORF  Gene  YJL178C YLL053C YOR333C YNL214W YGR127W YDL136W YJL030W YJR087W YEL015W YPR068C YAL016W YAL062W YDR330W YDR042C YJL016W YNL169C YGL107C YJR100C YNL146W YBL101C YBR128C YER177W YOR325W YLR031W YBR228W YEL056W YER179W YGL126W YNL040W YLR384C YMR026C YHL011C YKR028W YGR261C YER167W YHR015W YER119CA YNL008C YER085C YML082W YIL112W YNR074C YPL246C  ATG27  PEX17 RPL35B MAD2 EDC3 HOS1 TPD3 GDH3 UBX5  PSD1 RMD9  ECM21 ATG14 BMH1  SLX1 HAT2 DMC1 SCS3 IKI3 PEX12 PRS3 SAP190 APL6 BCK2 MIP6  ASI3  HOS4 AIF1 RBD2  Normalized densitometry values MATa MATalpha 0.79 0.87 0.81 0.85 0.89 0.77 0.89 0.77 1.03 0.63 0.59 1.07 0.74 0.92 0.79 0.87 1.00 0.67 0.91 0.76 0.83 0.85 0.82 0.94 0.73 0.83 0.84 0.84 0.83 0.83 0.93 0.74 0.74 0.92 0.95 0.72 1.01 0.66 0.88 0.79 0.84 0.83 0.86 0.81 0.87 0.80 1.00 0.67 0.69 0.99 0.79 0.88 0.87 0.81 0.93 0.74 0.86 0.81 0.73 0.94 0.84 0.85 0.82 0.77 0.90 1.06 0.62 0.95 0.73 0.82 0.86 0.77 0.98 0.69 0.74 0.65 0.92  0.91 0.70 0.99 0.94 1.03 0.77  SCORE average 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84  0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17  0.84 0.84 0.84 0.84 0.84 0.84  0.17 0.17 0.16 0.16 0.16 0.16  178  Rank  ORF  Gene  732 YCL026CA 733 YDR247W 734 YOL105C 735 YLR151C 736 YLR402W 737 YCL036W 738 YNL045W 739 YBR233W 740 YHR035W 741 YCR068W 742 YLR450W 743 YIL008W 744 YOR024W 745 YPR152C 746 YAL005C 747 YNL259C 748 YLL052C 749 YGL210W 750 YLR333C 751 YBR022W 752 YMR099C 753 YMR068W 754 YMR067C 755 YPL018W 756 YPL059W 757 YLR444C 758 YHR029C 759 YDL189W 760 YDR507C 761 YAL061W 762 YPL230W 763 YOL058W 764 YBR238C 765 YBR001C 766 YER059W 767 YNL099C 768 YGR037C 769 YML058W 770 YFL049W 771 YGL017W 772 YDL072C 773 YMR172W 774 YOL052C  FRM2 VHS1 WSC3 PCD1 GFD2 PBP2 ATG15 HMG2 URM1  SSA1 ATX1 AQY2 YPT32 RPS25B POA1 AVO2 UBX4 CTF19 GRX5 YHI9 RBS1 GIN4  ARG1 NTH2 PCL6 OCA1 ACB1 SML1 ATE1 HOT1 SPE2  Normalized densitometry values MATa MATalpha 0.64 1.04 0.84 0.75 0.94 0.86 0.77 1.02 1.01 1.21 0.87 0.75 0.97 1.01 0.85 0.76 0.70 0.84 1.07 0.70 0.66 0.86 0.79 0.96 0.86 0.84 1.10 0.99 0.65 0.85 0.91 0.88 0.77 0.93 0.75 0.59 0.93 0.75 0.84 0.88 0.89 0.68 0.72  0.84 0.93 0.74 0.82 0.91 0.66 0.68 0.48 0.82 0.93 0.72 0.68 0.83 0.92 0.99 0.85 0.62 0.98 1.03 0.83 0.90 0.73 0.83 0.59 0.70 1.04 0.85 0.85 0.78 0.81 0.92 0.76 0.94 1.10 0.77 0.94 0.86 0.82 0.81 1.02 0.98  SCORE average 0.84  0.16  0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85  0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16  179  Rank  775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818  ORF  Gene  YKL110C YNL241C YJL172W YOR129C YDR069C YDR273W YBR224W YNL273W YBL056W YDR127W YER080W YDR017C YDR245W YGR200C YDR379W YBR219C YEL018W YLR332W YDL018C YJL122W YLR263W YMR302C YPL222W YNL296W YLR199C YDR290W YNR008W YPR026W YKL070W YOR137C YKL168C YDR313C YOR363C YOL116W YDL239C YGR125W YDR015C YOL064C YBL107C YGR212W YJR110W YDR043C YEL023C YPR188C  KTI12 ZWF1 CPS1 DOA4 DON1 TOF1 PTC3 ARO1 KCS1 MNN10 ELP2 RGA2 EAF5 MID2 ERP3 RED1 PRP12  LRO1 ATH1 SIA1 KKQ8 PIB1 PI(4,5)P2 MSN1 ADY3  MET22 SLI1 YMR1 NRG1 MLC2  Normalized densitometry values MATa MATalpha 0.84 0.85 0.86 0.83 0.92 0.78 0.80 0.89 0.85 0.68 1.01 0.98 0.71 0.85 1.00 0.70 0.76 0.93 1.02 0.68 1.04 0.65 0.75 0.95 1.00 0.70 0.79 0.91 0.85 0.85 0.85 0.97 0.73 0.91 0.79 0.88 0.82 0.86 0.84 1.02 0.68 0.69 1.01 0.57 1.13 0.88 0.82 0.88 0.82 0.97 0.73 0.92 0.78 0.86 0.84 0.89 0.81 0.49 1.21 0.83 0.87 0.95 0.75 0.89 0.81 0.90 0.80 1.09 0.61 1.01 0.69 1.03 0.68 0.78 0.92 0.85 0.86 0.93 0.78 0.86 0.84 0.96 0.75 0.67 1.04  SCORE average 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85  0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15  180  Rank  819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862  ORF  Gene  YHR041C YKL047W YFR010W YPR060C YGR229C YDR323C YLR242C YDL238C YNL155W YLR146C YJL210W YDR470C YGR123C YLR191W YGL079W YJL082W YMR025W YPL054W YCR045C YOL044W YDR048C YGR163W YMR297W YOL099C YKL027W YGL133W YLR046C YNL044W YMR316W YMR284W YEL062W YMR195W YDR099W YEL068C YPR132W YLL007C YDR024W YJL161W YER182W YNL265C YNL275W YGL144C YDR257C YGL244W  SRB2 UBP6 ARO7 SMI1 PEP7 ARV1 GUD1 SPE4 PEX2 UGO1 PPT1 PEX13 IML2 CSI1 LEE1 PEX15 GTR2 PRC1  ITC1 YIP3 DIA1 YKU70 NPR2 ICY1 BMH2 RPS23B FYV1  IST1 ROG1 SET7 RTF1  Normalized densitometry values MATa MATalpha 0.27 1.44 0.52 1.19 0.99 0.72 0.87 0.84 1.00 0.71 0.73 0.98 0.79 0.92 0.85 0.86 0.95 0.76 1.04 0.67 0.79 0.93 0.71 1.00 0.98 0.73 0.90 0.81 0.89 0.83 0.84 0.88 0.74 0.98 0.68 1.03 0.97 0.74 0.93 0.78 0.59 1.13 0.84 0.87 1.01 0.70 0.77 0.95 0.85 0.87 0.81 0.91 0.92 0.80 1.02 0.70 0.74 0.97 0.97 0.75 0.84 0.88 1.00 0.72 0.89 0.83 0.67 1.05 0.91 0.81 1.07 0.65 0.47 1.25 0.89 0.83 0.98 0.74 0.86 0.72 1.01 0.82 0.90 0.80 0.93 0.86  SCORE average 0.85 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86  0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14  181  Rank  863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904  ORF  Gene  YER111C YJR084W YGL242C YEL071W YHL017W YCR024CA YPR046W YEL009C YNL100W YIL105C YLR351C YGR137W YJR015W YGR063C YFR032C YOL057W YDR264C YGR088W YER144C YGR170W YBR262C YJR094WA YNL225C YEL025C YLR296W YJR031C YPL208W YOR078W YNL315C YIL140W YBR226C YOR032C YDR184C YOL103W YGR223C YDR249C YMR022W YJR119C YOL125W YCR089W YMR081C YFR040W  SWI4 CSN12 DLD3 PMP1 MCM16 GCN4 SLM1 NIT3  SPT4  AKR1 CTT1 UBP5 PSD2 RPL43B CNM67  GEA1 RKM1 BUD21 ATP11 AXL2 HMS1 ATC1 ITR2 HSV2 QRI8  FIG2 ISF1 SAP155  Normalized densitometry values MATa MATalpha 0.82 0.91 0.69 1.03 0.99 0.73 0.65 1.08 0.92 0.80 0.78 0.95 1.02 0.72 0.58 0.87 0.95 1.10 0.91 0.99 0.87 1.08 0.87 0.83 0.85 0.84 0.88 1.16 0.87 1.00 0.86 0.86 0.74 0.72 0.45 0.97 0.91 0.86 0.98 0.78 0.90 0.73 0.84 0.72 0.87 0.80 0.98 0.88  0.70 1.00 1.15 0.86 0.78 0.63 0.82 0.74 0.86 0.65 0.91 0.88 0.90 0.85 0.57  0.73 0.87 0.88 0.99 1.01 1.29 0.77 0.82 0.88 0.76 0.96 0.83 1.00 0.90 1.02 0.94 0.75 0.85  SCORE average 0.86 0.86 0.86 0.86 0.86 0.86  0.14 0.14 0.14 0.14 0.14 0.14  0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.87 0.87 0.87 0.87 0.87 0.87  0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14  0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87  0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14  182  Rank  905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948  ORF  Gene  YPL223C YBR065C YLR154C YNR075W YGR235C YDR009W YPR079W YPL112C YKR041W YEL066W YAL058W YNL136W YKL090W YDR479C YPL159C YBR230C YAL067C YLR098C YNL012W YER010C YOR379C YBR176W YKR007W YLR324W YOR028C YDR250C YLL061W YER001W YEL014C YDR443C YDR203W YCL035C YLR353W YGL173C YFR044C YGL059W YDR421W YPL084W YPR156C YLR461W YLL056C YIL162W YHR200W YDR539W  GRE1 ECM2 RNH203 COS10 GAL3 MRL1 PEX25 HPA3 CNE1 EAF7 CUE2 PEX29 PET20 SEO1 CHA4 SPO1  ECM31 MEH1 PEX30 CIN5 MMP1 MNN1 SSN2 GRX1 BUD8 KEM1  ARO80 BRO1 TPO3 PAU4 SUC2 RPN10  Normalized densitometry values MATa MATalpha 0.75 0.99 0.86 0.87 0.97 0.77 0.74 1.00 0.76 0.98 0.94 0.80 0.91 0.83 0.68 1.06 0.79 0.95 0.78 0.96 0.90 0.84 0.67 1.07 0.93 0.81 0.81 0.93 0.96 0.78 1.06 0.68 0.78 0.96 0.83 0.91 0.79 0.96 0.63 1.11 0.85 0.90 0.63 1.11 0.95 0.79 0.94 0.80 0.95 0.79 0.70 1.04 1.09 0.66 0.77 0.98 1.05 0.70 0.52 1.23 1.15 0.60 0.98 0.76 0.68 1.07 0.63 1.12 0.89 0.86 0.95 0.80 0.80 0.95 0.87 0.92 0.83 0.96 0.79 0.95 0.80 0.88 0.82 0.93 0.96 0.79  SCORE average 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.88 0.88 0.88  0.14 0.14 0.14 0.14 0.14 0.14 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13  183  Rank  ORF  949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992  YGR266W YAL020C YOR276W YDR217C YGR164W YPR151C YIL141W YMR189W YKL026C YDL149W YMR062C YML075C YKL005C YMR262W YOR292C YHR184W YDR377W YNL291C YGR248W YAL065C YMR036C YGL025C YGR118W YHR018C YML122C YDL012C YLR032W YBR084W YNL001W YIL002C YKR044W YPL216W YOR132W YJL170C YKL067W YER005W YGL208W YGR015C YGL261C YIR004W YPL021W YDR239C YPL111W YER097W  Gene  ATS1 CAF20 RAD9 SUE1 GCV2 GPX1 ATG9 ECM40 HMG1 BYE1  SSP1 ATP17 MID1 SOL4 MIH1 PGD1 RPS23A ARG4  RAD5 MIS1 DOM34 INP51 UIP5 VPS17 ASG7 YNK1 YND1 SIP2  DJP1 ECM23 CAR1  Normalized densitometry values MATa MATalpha 0.96 0.79 0.75 1.00 0.67 1.08 0.71 1.04 0.81 0.94 0.78 0.97 1.01 0.74 0.88 0.88 0.89 0.87 0.93 0.82 0.88 0.86 0.89 0.85 0.90 0.73 1.02 0.89 0.86 0.76 1.00 1.05 0.70 0.69 1.07 0.92 0.84 0.84 0.91 0.76 1.00 0.95 0.81 0.84 0.92 1.06 0.70 0.88 0.88 0.87 0.89 0.90 0.86 0.76 1.00 0.71 1.05 0.95 0.81 0.93 0.83 0.89 0.88 0.68 1.08 1.07 0.70 1.02 0.74 0.75 1.01 0.88 0.89 0.93 0.83 0.76 1.00 0.88 1.07 0.70 0.96 0.81 0.80 0.96 0.77 1.00  SCORE average 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88  0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12  184  Rank  993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036  ORF  Gene  YPL155C YJL214W YPL156C YLL055W YFR038W YAL060W YGL053W YGL197W YPL058C YDR521W YOL063C YHR137W YDL091C YPR120C YLR453C YJL023C YCR106W YDL167C YLR427W YOL055C YBR168W YLR093C YJL037W YGR159C YKL077W YOR073W YBL091C YJR018W YGR168C YMR182C YDL204W YKL123W YDR193W YNL083W YPR149W YOR279C YJR070C YMR011W YBR025C YJL134W YLR421C YLR258W YJL043W YMR111C  KIP2 HXT8 PRM4  BDH1 PRM8 MDS3 PDR12  ARO9 UBX3 CLB5 RIF2 PET130 RDS1 NRP1 MAG2 THI20 PEX32 NYV1 NSR1 SGO1 MAP2  RGM1 RTN2  SAL1 NCE102 RFM1 LIA1 HXT2 LCB3 RPN13 GSY2  Normalized densitometry values MATa MATalpha 1.12 0.65 0.72 1.04 1.02 0.74 0.93 0.84 1.17 0.59 0.95 0.82 0.86 0.90 0.86 0.91 0.76 1.01 0.90 0.86 1.02 0.75 0.92 0.85 0.87 0.90 0.87 0.90 0.90 0.87 1.06 0.70 0.91 0.86 0.92 0.85 0.79 0.98 1.03 0.74 0.79 0.98 0.87 0.90 0.85 0.92 0.69 1.08 1.09 0.68 0.96 0.81 0.88 0.89 0.89 0.66 1.11 1.06 0.71 0.81 0.96 0.69 1.08 0.95 0.82 0.90 0.88 0.89 0.88 0.85 0.92 0.81 0.96 0.88 0.89 0.93 0.85 0.84 0.94 0.85 0.93 0.99 0.79 0.83 0.95 0.95 0.82  SCORE average 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89  0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12  185  Rank  ORF  1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079  YMR141C YLR219W YGR192C YLR074C YJR050W YJR062C YJL177W YBL095W YER055C YGR087C YOR375C YKR055W YDR344C YMR016C YNL293W YKL184W YFR013W YMR243C YHL013C YDL127W YML121W YJR059W YLR110C YDR262W YIL160C YJL024C YDR073W YLR456W YLR365W YJL007C YKL159C YGL226CA YPL213W YPR150W YOL115W YLR266C YER123W YJR039W YBL043W YBR138C YIL156W YJL137C YOR263C  Gene  MSC3 TDH3 BUD20 ISY1 NTA1 RPL17B HIS1 PDC6 GDH1 RHO4 SOK2 MSB3 SPE1 IOC3 ZRC1 PCL2 GTR1 PTK2 CCW12 POT1 APS3 SNF11  RCN1 OST5 LEA1 PAP2 PDR8 YCK3 ECM13 UBP7 GLG2  Normalized densitometry values MATa MATalpha 1.08 0.70 0.93 0.85 0.96 0.82 0.72 1.06 0.74 1.04 0.85 0.93 0.97 0.81 0.87 0.91 0.86 0.91 0.98 0.80 0.95 0.83 0.81 0.96 0.90 0.88 0.85 0.93 0.87 0.91 1.01 0.77 0.91 0.87 0.74 1.04 0.85 0.93 0.92 0.86 0.89 0.89 0.89 0.90 0.80 0.98 0.88 0.91 1.09 0.69 1.02 0.77 0.93 0.85 0.88 0.90 0.72 1.06 1.04 0.74 0.91 0.87 0.94 0.84 0.89 0.74 1.01 1.12 0.81 0.89 0.80 0.95 0.92 1.05 0.89  1.05 0.78 0.67 0.98 0.90 0.98 0.84 0.87 0.74 0.90  SCORE average 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89  0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11  0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89  0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11  186  Rank  ORF  1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122  YLR280C YDR270W YOR025W YBL063W YHR158C YKL091C YPR039W YBR005W YGL218W YBR245C YER156C YIL024C YJR021C YOR134W YGR027C YBR287W YDR186C YOR229W YJL012C YPL127C YGR124W YCR077C YOL065C YMR121C YOR290C YBR204C YHL003C YMR223W YDR348C YOR286W YDR080W YPR171W YMR070W YFR032CA YHR191C YLR017W YHR146W YOL011W YGR066C YDR269C YJL218W YLR426W YBL100C  Gene  CCC2 HST3 KIP1 KEL1  RCR1 ISW1  REC107 BAG7 RPS25A  WTM2 VTC4 HHO1 ASN2 PAT1 INP54 RPL15B SNF2 LAG1 UBP8  VPS41 BSP1 MOT3 RPL29 CTF8 MEU1 CRP1 PLB3  Normalized densitometry values MATa MATalpha 0.70 1.08 0.81 0.97 0.99 0.80 0.96 0.82 0.93 0.86 1.00 0.79 1.00 0.79 1.05 0.74 0.89 0.94 0.85 0.88 0.91 0.99 0.80 0.80 0.99 0.81 0.98 1.04 0.74 0.88 0.91 0.96 0.83 0.95 0.84 0.99 0.80 0.90 0.89 1.03 0.76 1.01 0.78 1.00 0.79 0.90 0.90 0.90 0.99 0.80 0.66 1.13 0.71 1.08 0.83 0.97 0.68 1.11 0.67 1.12 0.90 0.89 0.87 0.92 0.94 0.86 1.07 1.01 0.97 0.86 1.02 0.84 0.75 0.75  0.73 0.78 0.82 0.94 0.78 0.96 1.04 1.05 0.90  SCORE average 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90  0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11  0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90  0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11  187  Rank  1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165  ORF  Gene  YNL329C YOR029W YNL208W YBR275C YOR170W YMR078C YNR032CA YPR092W YDR369C YPR045C YKL170W YLR346C YOR192C YKL151C YCL047C YBL078C YOR052C YOR111W YDR524C YLR404W YPR109W YMR254C YNL029C YML079W YCL010C YLR142W YOR044W YOR177C YBL010C YHR176W YBR197C YMR319C YNR027W YGL021W YCR105W YIL059C YJR150C YJR115W YOR293W YOR230W YER180C YKL179C YGL131C  PEX6  RIF1 CTF18 HUB1  XRS2  Normalized densitometry values MATa MATalpha 0.94 0.86 1.05 0.74 0.90 0.91 0.88 0.96 0.84 0.95 0.85 1.04 0.76 0.86 0.73 1.09  MRPL38  ATG8  AGE1  KTR5 SGF29 PUT1 MPC54 FMO1 FET4 BUD17 ALK1 ADH7 DAN1 RPS10A WTM1 ISC10 COY1 SNT2  0.98 0.83 0.83 0.81 0.90 0.92 0.78 0.88 0.91 0.98 0.75 0.97 0.68 0.82 0.96 0.83 0.80 0.90 0.81 0.92 0.83 0.92 0.95 1.06 0.96 0.95 1.00 0.71 0.94 1.03 1.04 0.66  0.94 1.07 0.70 0.90 0.81 0.97 0.97 0.99 0.90 0.88 1.02 0.92 0.89 0.82 1.05 0.83 1.12 0.99 0.84 0.97 1.00 0.90 1.00 0.88 0.98 0.88 0.85 0.74 0.85 0.85 0.80 1.09 0.86 0.78 0.77 1.14  SCORE average 0.90 0.90 0.90 0.90 0.90 0.90 0.90  0.11 0.11 0.11 0.11 0.11 0.11 0.11  0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90  0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10  188  Rank  ORF  Gene  1166 YMR251W -A 1167 YJR035W 1168 YGL202W 1169 YGR239C 1170 YGL140C 1171 YKL107W 1172 YGL157W 1173 YMR007W 1174 YOR245C 1175 YOR184W 1176 YGL152C 1177 YGL181W 1178 YGR133W 1179 YDR497C 1180 YOR306C 1181 YDR173C 1182 YDR058C 1183 YEL041W 1184 YPL170W 1185 YAR031W 1186 YIL071C 1187 YLR435W 1188 YNR024W 1189 YDL039C 1190 YMR303C 1191 YLR410W 1192 YML018C 1193 YPR018W 1194 YOL041C 1195 YER039CA 1196 YBL104C 1197 YJL186W 1198 YLL012W 1199 YOR318C 1200 YLR104W 1201 YDL242W 1202 YBR147W 1203 YFR009W 1204 YDR482C 1205 YPL133C 1206 YBR131W 1207 YHR021W  HOR7 RAD26 ARO8 PEX21  DGA1 SER1 GTS1 PEX4 ITR1 MCH5 ARG82 TGL2 DAP1 PRM9 PCI8 TSR2 PRM7 ADH2 VIP1 RLF2 NOP12  MNN5 YEH1  GCN20 CWC21 RDS2 CCZ1 ECM12  Normalized densitometry values MATa MATalpha 0.81 1.00  SCORE average 0.90  0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10  1.00 0.90 0.95 0.89 0.97 0.92 0.92 0.88 0.90 0.88 0.85 1.02 0.96 0.97 0.91 0.96 0.81 1.02 0.85 0.81 0.73 0.96 0.93 1.11 0.85 1.02 0.76 0.81 1.20  0.81 0.91 0.86 0.92 0.84 0.89 0.89 0.93 0.91 0.93 0.96 0.79 0.84 0.84 0.85 1.00 0.79 0.96 1.00 1.08 0.85 0.88 0.70 0.96 0.80 1.06 1.01 0.62  0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91  0.96 1.08 0.93 1.00 0.88 1.05 0.93 0.92 0.91 0.95 0.91 1.00  0.85 0.73 0.88 0.82 0.94 0.77 0.89 0.89 0.91 0.87 0.91 0.82  0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91  189  Rank  ORF  1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250  -A YBR227C YGL170C YOR280C YBR276C YBR222C YBL062W YNL212W YER081W YKL216W YGR250C YDR261C YIL012W YEL008W YLR225C YFL015C YER007W YML022W YJR079W YDR378C YPR022C YIL016W YKR036C YHR096C YDL214C YLR206W YBR105C YDR272W YFR014C YLL021W YHR183W YGR129W YBL009W YDR371W YDL086W YPL207W YMR259C YDL130W YML042W YBR078W YDL186W YDR007W YHL039W YNL289W  Gene  MCX1 SPO74 FSH3 PPS1 PCS60 VID27 SER3 URA1 EXG2  PAC2 APT1 LSM6 SNL1 CAF4 HXT5 PRR2 ENT2 VID24 GLO2 CMK1 SPA2 GND1 SYF2 CTS2  RPP1B CAT2 ECM33 TRP1 PCL1  Normalized densitometry values MATa MATalpha 0.96 0.90 0.98 0.94 0.87 1.01 0.95 0.99 0.91 1.03 0.85 0.99 0.82 0.94 0.80 0.72 0.93 1.25 0.44 0.73 1.04 0.91 0.91 0.98 1.00 0.94 0.79 0.87 0.64 0.76 1.06 0.99 0.87 0.84 0.93 0.83 0.95 1.11 0.95 0.70 0.98 0.96 0.86  0.85 0.92 0.84 0.88 0.95 0.81 0.87 0.83 0.79 0.97 0.83 1.00 0.87 1.02 1.10 0.89 0.58 1.39 1.09 0.79 0.91 0.84 0.82 0.89 1.03 0.96 1.19 1.07 0.77 0.83 0.96 0.99 0.90 0.99 0.88 0.72 0.87 1.13 0.85 0.87 0.97  SCORE average 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91  0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09  190  Rank  ORF  1251 YNL190W 1252 YLR437C 1253 YDR363W -A 1254 YBR046C 1255 YMR034C 1256 YLR299W 1257 YDR251W 1258 YDR003W 1259 YHR086W 1260 YPL152W 1261 YGL071W 1262 YML095C 1263 YDL206W 1264 YDR283C 1265 YLR213C 1266 YJL208C 1267 YNL125C 1268 YGR070W 1269 YEL040W 1270 YDR210W 1271 YBR169C 1272 YPL130W 1273 YKL121W 1274 YGR096W 1275 YCL042W 1276 YLR220W 1277 YFR012W 1278 YDR112W 1279 YNL015W 1280 YDR486C 1281 YLR454W 1282 YFL052W 1283 YDR271C 1284 YDR219C 1285 YJL064W 1286 YLR335W 1287 YNL176C 1288 YNL028W 1289 YEL064C 1290 YIL173W 1291 YNL027W 1292 YLL060C 1293 YHR123W  Gene  SEM1 ZTA1 ECM38 PAM1 RCR2 NAM8 RRD2 RCS1 RAD10 GCN2 CRR1 NUC1 ESBP6 ROM1 UTR2 SSE2 SPO19 TPC1 CCC1  PBI2 VPS60  NUP2  AVT2 VTH1 CRZ1 GTT2 EPT1  Normalized densitometry values MATa MATalpha 0.94 0.89 1.01 0.82 0.90 0.92 1.01 0.77 0.75 0.99 1.02 0.93 0.98 0.89 0.84 0.84 1.03 0.79 0.87 0.86 0.89 1.12 0.80 0.97 0.79 0.73 0.97 0.94 0.70 0.79 0.75 0.78 0.94 0.81 0.71 0.97 1.04 0.80 0.77 1.10 0.87 0.89 1.06 1.08 0.83  0.82 1.06 1.07 0.84 0.81 0.90 0.85 0.94 0.99 0.91 0.99 0.80 1.04 0.96 0.97 0.94 0.71 1.03 0.86 1.04 1.10 0.86 0.89 1.13 1.04 1.09 1.05 0.89 1.03 1.12 0.87 0.80 1.04 1.07 0.74 0.96 0.94 0.78 0.75 1.00  SCORE average 0.91 0.91 0.91  0.09 0.09 0.09  0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92  0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09  191  Rank  1294 1295 1296 1297 1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337  ORF  Gene  YDR152W YJR090C YPL162C YJL217W YDR176W YJL066C YBR054W YPR184W YOR084W YLR072W YAL059W YPL068C YOL107W YIR001C YPR174C YEL003W YOR108W YBR107C YHL040C YDL020C YPR172W YKR105C YKL190W YBR074W YLR372W YML131W YDR408C YLL059C YCR043C YHR033W YJR124C YOR231W YGL243W YDR185C YDR068W YDR231C YJL135W YDL106C YML047C YJL184W YJR025C YGR182C YMR310C YBR263W  GIR2 GRR1  NGG1 MPM1 YRO2 GDB1  ECM1  SGN1 GIM4 LEU9 IML3 ARN1 RPN4  CNB1 SUR4 ADE8  MKK1 TAD1 DOS2 COX20 PHO2 PRM6 GON7 BNA1  SHM1  Normalized densitometry values MATa MATalpha 0.97 0.87 0.92 1.04 0.79 0.75 1.09 0.70 1.14 0.96 0.88 0.93 0.90 0.83 1.01 0.99 0.85 0.98 0.86 0.95 0.89 0.88 0.95 0.73 1.11 1.00 0.84 0.77 1.07 0.93 0.91 1.03 0.81 1.06 0.78 0.92 0.92 0.97 0.87 0.77 1.07 0.83 1.01 1.06 0.78 0.97 0.87 0.84 1.00 1.04 0.80 0.92 0.92 1.02 0.83 0.93 0.91 1.04 0.80 1.04 0.81 0.96 0.88 0.91 0.94 1.09 0.75 0.91 0.94 0.70 1.14 0.99 0.86 0.94 0.90 0.95 0.89 0.92 0.92 0.93 0.91 0.93 0.95 0.90 0.98 0.86  SCORE average 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92  0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08  192  Rank  ORF  1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 1354 1355 1356 1357 1358 1359 1360 1361 1362 1363 1364 1365 1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381  YGR011W YPL115C YDR459C YBR225W YDR198C YMR214W YDR445C YLR118C YLL026W YBR162C YML013W YDR370C YJL169W YHR113W YFL047W YOL092W YEL011W YER110C YMR247C YMR161W YER044C YMR226C YJL106W YLR111W YHR022C YPL026C YER088C YLR049C YDL095W YMR144W YBR035C YIL054W YBR291C YMR205C YOR058C YMR053C YJR153W YGL121C YMR127C YDR179C YGR196C YJL200C YNL234W YBR085W  Gene  BEM3  SCJ1  HSP104 TOS1 SEL1  RGD2 GLC3 KAP123 HLJ1 ERG28 IME2  SKS1 DOT6 PMT1 PDX3 CTP1 PFK2 ASE1 STB2 PGU1 GPG1 SAS2 CSN9 FYV8  AAC3  Normalized densitometry values MATa MATalpha 1.01 0.83 0.93 0.91 0.89 0.95 0.92 0.92 1.05 0.80 0.82 1.02 0.90 0.95 0.98 0.87 0.93 0.92 1.02 0.83 1.04 0.80 0.92 0.92 0.93 0.91 0.90 0.95 0.93 0.91 0.81 1.04 0.83 1.02 0.64 1.20 0.90 0.94 0.90 0.94 0.92 0.88 0.97 1.03 0.82 0.89 0.96 1.06 0.79 0.74 1.11 0.91 0.93 0.94 0.91 0.72 1.13 1.05 0.80 0.92 1.05 0.80 0.89 0.96 0.93 0.98 0.87 0.88 0.97 0.92 0.93 1.10 0.75 0.91 0.94 0.95 0.90 0.96 0.89 0.76 1.09 0.93 0.87 0.98  SCORE average 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93  0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08  193  Rank  1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 1403 1404 1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 1420 1421 1422 1423  ORF  Gene  YMR175W YCL040W YNL326C YLR304C YFR026C YDL168W YJR133W YBR015C YBR200W YDR263C YGR122CA YDR326C YJL168C YMR258C YIL138C YDR034C YNL300W YLR137W YML003W YMR318C YGR202C YPL080C YGR017W YMR244W YEL016C YGL235W YNR004W YDR253C YMR110C YLR228C YKR098C YDR333C YER075C YER087CA YIL013C YJR024C YLR044C YDR535C YKR092C YOL061W YLR265C YBR130C  SIP18 GLK1 PFA3 ACO1 SFA1 XPT1 MNN2 BEM1 DIN7  SET2 TPM2 LYS14  ADH6 PCT1  NPP2  MET32 ECM22 UBP11 PTP3  PDR11 PDC1 SRP40 PRS5 NEJ1 SHE3  Normalized densitometry values MATa MATalpha 0.89 0.96 0.97 0.88 0.91 0.94 1.02 0.84 1.09 0.77 1.00 0.85 1.06 0.80 0.75 1.11 0.93 0.93 0.79 1.07 1.13 0.73  SCORE average 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93  0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07  1.05 0.89 0.69 1.17 0.93 0.74 1.11 0.86 0.85 0.79 0.75 1.03 0.79 1.03 0.93 1.07 0.97 1.00 0.93 0.94 0.98 1.14 1.10  0.81 0.96 1.17 0.69  0.92 0.88 0.72 0.76  0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93  1.10 0.86 1.04 0.90 0.59 0.95 1.06 1.11  0.76 1.00 0.82 0.96 1.27 0.92 0.80 0.75  0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93  1.12 0.74 0.99 1.01 1.07 1.11 0.83 1.06 0.83 0.93 0.79 0.89 0.86  194  Rank  1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466  ORF  Gene  YGL086W YDR096W YKL076C YOR018W YEL017CA YPL257W YLR297W YDL022W YBL068W YIR016W YNL013C YGR268C YAL036C YJR135C YLR224W YBR264C YKR040C YKL146W YAR029W YOR093C YDL190C YLR352W YNL332W YHL035C YGR275W YKR093W YAL004W YDR380W YLL023C YML102W YNL055C YNL195C YOL075C YCR007C YNL304W YKL044W YJL165C YPR084W YLR433C YER040W YMR142C YOL136C YGR053C  MAD1 GIS1 PSY1 ROD1 PMP2  GPD1 PRS4  HUA1 RBG1 MCM22 YPT10 AVT3  UFD2 THI12 RTT102 PTR2 ARO10 CAC2 POR1  YPT11 HAL5 CNA1 GLN3 RPL13B PFK27  Normalized densitometry values MATa MATalpha 0.92 0.94 0.87 0.99 1.06 0.80 0.82 1.04 1.07 0.79 0.85 0.88 0.91 0.91 0.96 0.77 1.09 0.87 0.79 0.97 1.00 0.93 0.98 0.80 0.79 0.71 1.07 1.02 0.98 0.83 0.75 0.86 1.00 0.77 0.84 0.62 0.84 0.87 0.96 0.86 1.02 0.93 0.69 0.82 0.84 0.73 0.94 0.99  1.01 0.98 0.96 0.95 0.90 1.09 0.77 1.00 1.07 0.90 0.86 0.93 0.88 1.06 1.07 1.16 0.79 0.84 0.89 1.03 1.11 1.01 0.87 1.09 1.02 1.25 1.03 1.00 0.90 1.01 0.85 0.94 1.17 1.05 1.03 1.14 0.93 0.88  SCORE average 0.93 0.93 0.93 0.93 0.93  0.07 0.07 0.07 0.07 0.07  0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.94 0.94 0.94  0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07  195  Rank  1467 1468 1469 1470 1471 1472 1473 1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510  ORF  Gene  YLR034C YDL171C YOR296W YBR043C YOL003C YFR035C YNR071C YPL033C YGR019W YBL094C YDL054C YOR059C YJL199C YDR540C YEL047C YLR282C YEL037C YLL058W YDR297W YPL220W YMR238W YJL052W YKR091W YHR207C YJL171C YMR155W YPR005C YLL046C YGL094C YKL034W YIL001W YDR467C YDR422C YML002W YIL015W YLL045C YDR405W YGL199C YNR066C YBL003C YOL106W YBR299W YOL091W YFR055W  SMF3 GLT1 QDR3  UGA1 MCH1 MBB1  RAD23 SUR2 RPL1A DFG5 TDH1 SRL3 SET5  HAL1 RNP1 PAN2 TUL1  SIP1 BAR1 RPL8B MRP20  HTA2 MAL32 SPO21  Normalized densitometry values MATa MATalpha 0.95 0.92 0.85 1.03 0.90 0.97 0.87 1.00 1.02 0.85 1.02 0.85 0.72 1.16 0.94 0.94 0.94 0.94 0.96 0.92 0.94 0.94 0.94 0.93 0.89 0.98 0.95 0.92 0.95 0.92 1.02 0.85 0.98 0.90 0.97 0.90 0.95 0.92 0.89 0.99 0.92 0.95 0.97 0.90 0.80 1.08 0.94 0.93 1.06 0.82 1.09 0.78 1.00 0.88 0.90 0.98 0.91 0.97 0.94 0.94 1.01 0.87 0.83 1.05 0.92 0.96 0.89 0.98 1.06 0.81 0.79 1.08 0.94 0.90 0.98 0.97 0.91 0.88 1.00 0.67 1.21 0.81 1.07 0.80 1.08 0.98 0.90  SCORE average 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94  0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07  196  Rank  ORF  1511 1512 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553  YPR003C YPR141C YLL005C YOL025W YMR294W -A YKR018C YMR255W YAL051W YNL187W YPR044C YPL053C YJL206C YKL143W YNL231C YMR163C YGR057C YNL318C YGR161C YJL198W YOR360C YML038C YGL158W YHR016C YNL120C YJR080C YNL046W YAR003W YHR017W YBR115C YIL073C YDR122W YMR120C YOL151W YBR209W YDR031W YDR252W YDL241W YGR234W YNL140C YER070W YPR054W YJL132W YDR244W  Gene  KAR3 SPO75 LAG2  GFD1 OAF1  KTR6 LTV1 PDR16 LST7 HXT14 RTS3 PHO90 PDE2 YMD8 RCK1 YSC84  SWD1 YSC83 LYS2 SPO22 KIN1 ADE17 GRE2  BTT1 YHB1 RNR1 SMK1 PEX5  Normalized densitometry values MATa MATalpha 0.97 0.91 1.03 0.85 0.85 1.03 0.81 1.07 1.17 0.71 0.79 0.74 0.92 0.89 0.72 0.70 0.83 0.87 1.07 0.80 1.04 1.10 0.91 0.91 0.79 0.99 0.95 0.99 0.95 0.73 1.02 0.76 1.02 0.82 0.91 1.03 0.89 0.94 0.81 1.07 1.00 1.03 0.92 1.03 0.53 0.83 1.00 0.92  1.09 1.14 0.96 0.99 1.16 1.18 1.05 1.02 0.81 1.08 0.84 0.78 0.97 0.97 1.09 0.89 0.93 0.89 0.93 1.15 0.86 1.13 0.87 1.06 0.97 0.85 0.99 0.94 1.07 0.82 0.88 0.85 0.97 0.85 1.35 1.06 0.89 0.97  SCORE average 0.94 0.94 0.94 0.94 0.94  0.07 0.06 0.06 0.06 0.06  0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94  0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06  197  Rank  ORF  1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 1564 1565 1566 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591 1592 1593 1594 1595 1596  YLR414C YNL173C YHR025W YOR380W YLR234W YIL037C YNL271C YIL030C YBR295W YOR021C YAL056W YAL021C YOR264W YML053C YBR296C YBR137W YGR136W YPL253C YOR076C YLL057C YIL060W YJR137C YJL029C YBR145W YOL013WA YJL192C YPL221W YAL066W YPL167C YDR295C YKL197C YNL279W YML057W YOR381W YOL081W YGL214W YEL057C YLR443W YLR131C YNL292W YPL267W YDL117W YHR189W  Gene  MDG1 THR1 RDR1 TOP3 PRM2 BNI1 SSM4 PCA1 GPB2 CCR4 DSE3 PHO89 LSB1 VIK1 SKI7 JLP1 ECM17 VPS53 ADH5  SOP4  REV3 HDA2 PEX1 PRM1 CMP2 FRE3 IRA2  ECM7 ACE2 PUS4 CYK3 PTH1  Normalized densitometry values MATa MATalpha 0.95 0.94 0.77 1.11 1.00 0.89 0.96 0.92 0.90 0.99 0.91 0.97 0.94 0.97 0.92 0.89 1.00 1.12 0.76 1.02 0.87 0.94 0.88 1.01 0.97 0.91 0.86 1.03 1.00 0.89 0.97 0.92 1.00 0.89 0.88 1.01 1.17 0.72 1.15 0.73 0.74 1.15 0.94 1.03 0.86 0.82 1.07 0.90 0.86 0.80 1.06 0.84 1.04 0.77 1.15 0.92 1.00 1.13 0.87 1.05 0.86 0.93 0.91 0.89 0.73  0.99 1.03 1.09 0.83 1.05 0.85 1.12 0.74 0.97 0.89 0.76 1.02 0.84 1.03 0.96 0.98 1.00 1.16  SCORE average 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94  0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06  0.94 0.94 0.94 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95  0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06  198  Rank  1597 1598 1599 1600 1601 1602 1603 1604 1605 1606 1607 1608 1609 1610 1611 1612 1613 1614 1615 1616 1617 1618 1619 1620 1621 1622 1623 1624 1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 1637 1638 1639 1640  ORF  Gene  YLR438W YOR365C YER181C YNL230C YOR223W YER071C YKL101W YDL085W YLR187W YLR364W YDL144C YKL086W YDR392W YOL124C YGR221C YFL053W YOL056W YNL335W YMR129W YLR108C YGR279C YGR194C YPL067C YMR105C YCL064C YDR518W YEL017W YJL152W YKL030W YNL249C YPL149W YDR026C YNL011C YNL057W YNL078W YOR383C YOL082W YGR051C YBL065W YMR073C YKL120W YDL135C YGR003W YGR093W  CAR2  ELA1  HSL1 NDE2 SKG3  SRX1 SPT3 TRM11 TOS2 DAK2 GPM3 POM152 SCW4 XKS1 PGM2 CHA1 EUG1 GTT3  MPA43 ATG5  NIS1 FIT3 ATG19  OAC1 RDI1 CUL3  Normalized densitometry values MATa MATalpha 1.02 0.87 0.92 0.97 1.08 0.81 0.90 0.99 0.96 0.94 0.92 0.97 0.95 0.94 0.98 0.92 0.97 0.93 0.79 1.10 0.95 0.94 1.09 0.81 0.63 1.26 0.94 0.96 1.00 0.89 0.93 0.97 1.04 0.86 0.92 0.97 1.04 0.86 0.93 0.97 0.80 1.09 0.95 0.94 0.79 1.10 0.94 0.95 0.84 1.05 0.90 1.00 1.04 0.86 0.95 0.94 0.96 0.94 0.93 0.97 0.74 1.15 0.90 0.99 1.05 0.85 0.85 1.05 0.79 1.10 1.03 0.87 1.03 0.86 0.94 0.96 1.04 0.86 0.93 0.97 0.92 0.97 0.91 0.98 1.01 0.89 0.92 0.98  SCORE average 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95  0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06  199  Rank  ORF  1641 1642 1643 1644 1645 1646 1647 1648 1649 1650 1651 1652 1653 1654 1655 1656 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667 1668 1669 1670 1671 1672 1673 1674 1675 1676 1677 1678 1679 1680 1681 1682 1683 1684  YFR006W YGL262W YMR231W YLR181C YJL213W YJL149W YGL033W YOL122C YFL030W YDR484W YGL175C YHR181W YBR260C YNL237W YPL269W YPL125W YDR439W YAL007C YGR004W YIR039C YPL071C YNL122C YKR051W YGL110C YKL147C YNR013C YCL014W YGR139W YDR143C YPL180W YKL124W YPR021C YNL142W YOL153C YPL119C YDL114W YOR283W YKR043C YKR034W YMR295C YNL129W YOR096W YGR025W YKR078W  Gene  PEP5 VTA1  HOP2 SMF1 AGX1 VPS52 SAE2 SVP26 RGD1 YTP1 KAR9 KAP120 LRS4 ERP2 PEX31 YPS6  CUE3 PHO91 BUD3 SAN1 TCO89 SSH4 AGC1 MEP2 DBP1  DAL80 NRK1 RPS7A  Normalized densitometry values MATa MATalpha 0.93 0.97 1.07 0.83 0.95 0.96 0.94 0.90 1.00 0.94 0.96 0.82 1.08 0.78 1.12 0.86 1.04 0.91 0.99 0.84 1.06 0.91 0.99 1.00 0.90 0.99 0.91 0.93 0.97 1.00 0.90 0.94 0.96 0.92 0.98 0.99 0.91 1.02 0.88 0.96 0.94 1.07 0.83 0.89 1.01 0.95 0.95 0.92 0.98 1.10 0.81 0.68 1.23 0.96 0.94 0.84 1.07 1.03 0.87 0.77 1.13 1.23 0.68 0.78 1.12 0.97 0.93 0.95 0.99 0.92 1.05 0.86 0.94 0.97 1.15 0.75 1.04 0.86 0.19 1.71 0.95 0.96 0.89 1.01  SCORE average 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95  0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05  200  Rank  ORF  1685 1686 1687 1688 1689 1690 1691 1692 1693 1694 1695 1696 1697 1698 1699 1700 1701 1702 1703 1704 1705 1706 1707 1708 1709 1710 1711 1712 1713 1714 1715 1716 1717 1718 1719 1720 1721 1722 1723 1724 1725 1726  YNL157W YDR120C YDR399W YPL055C YOR304W YOR083W YDR458C YHR028C YAL017W YIL094C YHR142W YOR080W YJL103C YOR191W YNL168C YBR278W YGL028C YPL090C YNL020C YCR036W YPR167C YNL086W YDR183W YLR095C YIR019C YOR008CA YBL019W YGL036W YGL196W YDR312W YGL090W YLR400W YBR108W YKL164C YGL248W YFL010C YOR131C YKL171W YOL131W YNL025C YDL130W -A YLR134W  Gene  TRM1 HPT1 LGE1 ISW2 WHI5 DAP2 PSK1 LYS12 CHS7 DIA2 RIS1 DPB3 SCW11 RPS6A ARK1 RBK1 MET16 PLP1 IOC2 MUC1  APN2  Normalized densitometry values MATa MATalpha 1.09 0.82 1.01 0.89 1.04 0.87 0.67 1.24 1.01 0.90 1.01 0.90 0.89 1.02 0.96 0.95 0.89 1.02 0.95 0.92 0.98 0.92 0.99 1.02 0.89 0.86 1.04 0.82 1.09 0.93 0.98 0.86 1.05 0.63 1.28 0.95 0.96 0.89 1.02 0.79 1.12 1.05 0.86 1.05 0.86 0.98 0.93 1.07 0.84 0.84 1.07  SCORE average 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.96 0.96 0.96  0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05  0.97 0.94 1.03 0.88 0.82 0.97 1.06 0.96 0.97 0.83 1.01 0.94 0.97  SSN8 STF1  0.94 0.97 0.88 1.03 1.09 0.94 0.85 0.95 0.94 1.08 0.90 0.98 0.94 0.96 0.94  0.97  0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96  PDC5  0.70  1.21  0.96  SSF2 LIF1  PIR1 PDE1 WWM1  201  Rank  ORF  1727 1728 1729 1730 1731 1732 1733 1734 1735 1736 1737 1738 1739 1740 1741 1742 1743 1744 1745 1746 1747 1748 1749 1750 1751 1752 1753 1754 1755 1756 1757 1758 1759 1760 1761 1762 1763 1764 1765 1766 1767 1768 1769 1770  YDR340W YDR014W YLR449W YMR185W YLR179C YGR217W YNL183C YKL115C YPL247C YJL048C YIL007C YLR368W YMR010W YOL150C YIL006W YDR453C YOR042W YOR348C YBR157C YFL044C YKL140W YOR101W YCL048W YHR031C YJL157C YDR435C YKR104W YGR219W YDR537C YOR356W YMR294W YMR305C YKL218C YDR321W YGR039W YGR052W YFR036W YGL007W YBL061C YKL129C YBR047W YOR173W YER185W YPL225W  Gene  RAD61 FPR4  CCH1 NPR1  UBX6 NAS2 MDM30  YIA6 TSA2 CUE5 PUT4 ICS2 TGL1 RAS1 SPS22 RRM3 FAR1 PPM1  JNM1 SCW10 SRY1 ASP1  CDC26 SKT5 MYO3 DCS2  Normalized densitometry values MATa MATalpha 1.00 0.92 1.25 0.66 0.77 1.14 0.96 1.16 0.75 0.98 0.93 0.93 0.99 1.07 0.85 1.05 0.87 1.20 0.72 1.02 0.89 0.69 1.22 0.62 1.29 0.81 1.11 0.87 1.05 0.94 0.98 1.00 0.91 1.06 0.85 0.92 1.00 0.94 0.97 0.95 0.96 0.93 0.98 0.95 0.96 0.95 0.97 1.05 0.87 0.87 1.04 0.91 1.01 0.89 1.02 1.13 0.78 1.11 0.81 1.16 0.76 0.86 1.06 0.90 1.01 0.89 1.03 0.92 1.00 1.00 0.92 1.10 0.82 0.96 1.05 0.87 0.97 0.95 0.99 0.93 0.94 0.98 0.89 1.03 0.86 1.06  SCORE average 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96  0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.04  202  Rank  1771 1772 1773 1774 1775 1776 1777 1778 1779 1780 1781 1782 1783 1784 1785 1786 1787 1788 1789 1790 1791 1792 1793 1794 1795 1796 1797 1798 1799 1800 1801 1802 1803 1804 1805 1806 1807 1808 1809 1810 1811 1812 1813  ORF  Gene  YLR401C YJL078C YMR173W YMR114C YBL064C YBR241C YBL096C YGR067C YDR480W YNR061C YPL206C YNL145W YKR009C YEL059W YOL014W YOR175C YPL186C YNL030W YDL210W YNL319W YOR308C YGR199W YER011W YER044CA YMR119W YCR083W YBR071W YDR310C YNL276C YOR010C YJL126W YBL046W YIR025W YPL166W YLR390W YBR261C YPL171C YDR035W YLR024C YML096W YNL253W YLR042C YMR215W  DUS3 PRY3 DDR48 PRX1  DIG2  MFA2 FOX2  UIP4 HHF2 UGA4 SNU66 PMT6 TIR1 MEI4 ASI1 TRX3 SUM1 TIR2 NIT2 MND2 ECM19 OYE3 ARO3 UBR2 TEX1 GAS3  Normalized densitometry values MATa MATalpha 0.94 0.98 0.90 1.02 1.00 0.92 0.99 0.93 1.03 0.89 0.98 0.94 1.04 0.88 0.97 0.95 0.93 0.99 0.94 0.98 0.94 0.98 1.09 0.83 0.96 0.96 0.93 1.00 0.91 1.01 0.90 1.02 0.91 1.02 1.09 0.83 1.02 0.91 0.95 0.98 0.97 0.96 0.99 0.93 0.78 1.14 1.09 0.84 0.97 0.96 1.03 1.01 0.71 0.96 0.97 0.97 1.07 0.94 0.95 0.96 1.06 1.00 0.85 1.00 0.94 0.95 1.07  0.96 0.97 0.89 0.91 1.22 0.97 0.95 0.96 0.86 0.98 0.98 0.96 0.87 0.92 1.07 0.93 0.99 0.97 0.85  SCORE average 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96  0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04  0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96  0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04  203  Rank  1814 1815 1816 1817 1818 1819 1820 1821 1822 1823 1824 1825 1826 1827 1828 1829 1830 1831 1832 1833 1834 1835 1836 1837 1838 1839 1840 1841 1842 1843 1844 1845 1846 1847 1848 1849 1850 1851 1852 1853 1854 1855 1856  ORF  Gene  YMR264W YGL263W YNL305C YFL063W YDL232W YOR328W YJR049C YPR062W YKR039W YNR062C YJR152W YLR097C YDR469W YDL131W YNL191W YLR331C YMR153CA YKL175W YOR197W YCR079W YKR033C YDR307W YOR166C YGL096W YML021C YOR147W YER032W YNL032W YPL019C YOR228C YBR018C YGL228W YGR008C YJR030C YOR302W YLR125W YKL092C YGL077C YNL180C YNL322C YHR014W YER086W YLL025W  CUE1 COS12  OST4 PDR10 UTR1 FCY1 GAP1 DAL5 HRT3 SDC1 LYS21  ZRT3 MCA1  TOS8 UNG1 MDM32 FIR1 SIW14 VTC3 GAL7 SHE10 STF2  BUD2 HNM1 RHO5 KRE1 SPO13 ILV1  Normalized densitometry values MATa MATalpha 0.93 1.00 0.99 0.94 0.81 1.11 0.88 1.05 0.96 0.95 0.98 1.02 0.91 0.92 1.01 0.98 0.95 0.93 1.00 1.01 0.91 1.02 0.91 0.90 1.03 1.05 0.88 0.95 0.98 0.96 0.97 1.11 0.82 0.98 1.06 0.97 0.97 0.96 1.04 0.90 0.86 1.06 0.97 1.05 0.92 0.87 0.94 0.89 1.00 0.94 1.10 1.16 0.97 1.08 0.97 1.18 1.02 0.95  0.95 0.87 0.96 0.96 0.98 0.89 1.03 1.07 0.97 0.87 0.96 0.88 1.01 1.06 0.99 1.05 0.93 1.00 0.84 0.77 0.97 0.86 0.76 0.91 0.98  SCORE average 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96 0.96  0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04  0.96 0.96 0.96 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97  0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04  204  Rank  1857 1858 1859 1860 1861 1862 1863 1864 1865 1866 1867 1868 1869 1870 1871 1872 1873 1874 1875 1876 1877 1878 1879 1880 1881 1882 1883 1884 1885 1886 1887 1888 1889 1890 1891 1892 1893 1894 1895 1896 1897 1898  ORF  Gene  YPL268W YIL158W YMR204C YER007CA YNR065C YDR083W YLR084C YHL047C YGR173W YOR311C YHL033C YPL113C YOL039W YDR533C YIR007W YGR092W YCR020C YHL032C YML100W -A YPL047W YNL128W YNL227C YPL163C YBR244W YOL128C YLR294C YOL045W YMR069W YMR234W YMR193W YHR136C YBR203W YHR156C YOR301W YGR181W YML072C YOL158C YOR190W YNL072W YKL105C YDR490C YNL323W  PLC1 INP1  RRP8 RAX2 ARN2 RBG2 HSD1 RPL8A RPP2A HSP31 DBF2 PET18 GUT1  SGF11 TEP1 JJJ1 SVS1 GPX2 YGK3 PSK2 NAT4 RNH1 MRPL24 SPL2 COS111 LIN1 RAX1 TIM13 TCB3 ENB1 SPR1 RNH201 PKH1 LEM3  Normalized densitometry values MATa MATalpha 1.06 0.87 0.94 1.00 0.97 0.97 0.75 1.19  SCORE average 0.97 0.97 0.97 0.97  0.04 0.04 0.04 0.04  1.04 0.95 0.98 0.84 1.14 1.02 1.06 0.97 0.93 1.07 1.01 1.08 0.91 0.98 0.94  0.90 0.98 0.95 1.10 0.80 0.92 0.87 0.96 1.01 0.87 0.93 0.86 1.03 0.95 0.99  0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97  0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04  0.97 1.02 0.99 1.12 0.97 0.92 0.98 0.94 0.93 1.08 1.02 0.99 0.91 1.16 0.92 1.02 0.86 0.90 0.94 1.03 0.89 1.02 0.97  0.97 0.92 0.94 0.82 0.97 1.01 0.96 1.00 1.01 0.86 0.92 0.95 1.03 0.78 1.02 0.92 1.08 1.04 1.00 0.91 1.05 0.92 0.97  0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97  0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03  205  Rank  ORF  1899 1900 1901 1902 1903 1904 1905 1906 1907  YOR183W YOL132W YER079W YDR084C YPR128C YFR015C YNL211C YOR247W YHR001W -A 1908 YCL055W 1909 YDL021W 1910 YGL089C 1911 1912 1913 1914 1915 1916 1917 1918 1919 1920 1921 1922 1923 1924 1925 1926 1927 1928 1929 1930 1931 1932 1933 1934 1935 1936 1937 1938 1939  YPL181W YDL083C YBR052C YBR024W YFR056C YPL096W YML056C YBR280C YLR246W YMR194CA YIL055C YIL009W YKR069W YKL037W YCL034W YAL028W YIL096C YHL006C YIL167W YDR406W YIL166C YKL202W YDR509W YNR067C YBL051C YOL147C YIL014W YIL102C YDR151C  Gene  FYV12 GAS4 TVP23 ANT1 GSY1 SRL1 QCR10 KAR4 GPM2 MF(ALPH A)2 CTI6 RPS16B SCO2 PNG1 IMD4 ERF2  FAA3 MET1 LSB5 FRT2 SHU1 SDL1 PDR15  DSE4 PIN4 PEX11 MNT3 CTH1  Normalized densitometry values MATa MATalpha 0.84 1.10 0.95 0.99 1.00 0.95 0.99 0.95 0.95 0.99 0.96 0.98 0.94 1.01 1.08 0.87 0.92 1.02  SCORE average 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97  0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03  1.03 0.79 1.06  0.91 1.15 0.88  0.97 0.97 0.97  0.03 0.03 0.03  1.01 0.93 0.99 0.81 1.02 0.90 0.92 0.98 1.04 0.93  0.94 1.02 0.95 1.14 0.92 1.05 1.02 0.97 0.90 1.01  0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97  0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03  1.07 0.98 0.89 0.93 0.94 1.04 0.98 0.99 0.96 1.02 0.93 1.10 0.95 1.01 0.95 0.96 0.97 0.98 0.97  0.87 0.97 1.06 1.02 1.00 0.90 0.97 0.95 0.98 0.93 1.02 0.85 1.00 0.94 1.00 0.99  0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.98 0.98  0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03  0.98  206  Rank  ORF  1940 1941 1942 1943 1944 1945 1946 1947 1948 1949 1950 1951 1952 1953 1954 1955 1956 1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983  YMR178W YBR077C YOR031W YBR039W YNL134C YLR065C YKR058W YMR174C YBR300C YHR209W YML101C YMR246W YLR311C YPR002W YGR207C YPL188W YGR189C YGR105W YBR250W YFL056C YER047C YAL023C YGL165C YOL163W YNL254C YNL301C YLR068W YPL095C YPL014W YAL018C YDL088C YPR160W YDR374C YGR097W YJR011C YLL051C YLR385C YMR017W YDL066W YBL060W YKL183W YER164W YDL188C YNL074C  Gene  SLM4 CRS5 ATP3  GLG1 PAI3  CUE4 FAA4 PDH1 POS5 CRH1 VMA21 AAD6 SAP1 PMT2  RPL18B FYV7  ASM4 GPH1 ASK10 FRE6 SWC7 SPO20 IDP1 LOT5 CHD1 PPH22 MLF3  Normalized densitometry values MATa MATalpha 1.01 0.94 1.10 0.86 0.95 1.00 0.98 0.99 0.97 0.92 1.03 1.01 0.94 0.98 0.97 0.86 1.09 1.05 0.90 1.06 0.89 0.90 1.05 1.02 0.94 0.95 1.01 1.02 0.93 0.98 1.01 0.94 0.98 0.98 0.97 0.98 0.97 0.96 1.00 0.98 0.97 0.94 1.02 0.98 0.97 1.00 0.95 0.99 0.96 0.93 1.03 0.93 1.03 1.01 0.95 1.10 0.85 0.94 1.02 1.01 0.95 0.95 1.01 0.99 0.97 0.83 1.12 0.97 0.99 1.02 0.93 0.99 0.97 1.06 0.90 1.12 0.83 1.02 0.93 1.14 0.82 0.99 0.97 0.92 1.03  SCORE average 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98  0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03  207  Rank  ORF  1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025  YBL054W YKR026C YBR073W YDR206W YJL079C YOR342C YOR202W YDR475C YDR536W YDR248C YDR538W YGR213C YMR086CA YPL009C YPL008W YDR483W YOR051C YOR019W YOR359W YJL163C YIR013C YCR025C YLR227C YER046W -A YDR500C YBR188C YDL057W YBR298C YGL258W YBR076W YDR033W YHR076W YNL321W YDR142C YKL211C YML059C YMR169C YCL016C YJR148W YDR102C YLR313C YLR357W  Gene  GCN3 RDH54 EBS1 PRY1 HIS3 STL1 PAD1 RTA1  CHL1 KRE2  VTS1 GAT4 ADY4  RPL37B NTC20 MAL31 ECM8 MRH1 PTC7 PEX7 TRP3 NTE1 ALD3 DCC1 BAT2 SPH1 RSC2  Normalized densitometry values MATa MATalpha 1.03 0.93 1.12 0.84 1.00 0.96 1.10 0.86 0.98 0.97 1.07 0.89 0.95 1.01 0.99 0.97 0.98 0.94 1.01 1.01 0.94 0.80 1.16 0.90 1.06  SCORE average 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98  0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03  0.99 1.09 0.99 0.95 1.21 1.13 1.05 0.96 0.91 0.91 0.90  0.97 0.86 0.96 1.00 0.75 0.83 0.91 1.00 1.05 1.05 1.06  0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98  0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03  0.98 1.01 1.05 0.95 0.94 0.99 0.97 1.04 1.04 1.05 0.99 1.00 0.90 0.98 1.03 0.98  0.98 0.97 0.95 0.91 1.01 1.02 0.97 0.99 0.92 0.92 0.91 0.97 0.96 1.06 0.98 0.93 0.98 0.98  0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98  0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02  208  Rank  ORF  2026 2027 2028 2029 2030 2031 2032 2033 2034 2035 2036 2037 2038 2039 2040 2041 2042 2043 2044 2045 2046 2047 2048 2049 2050 2051 2052 2053 2054 2055 2056 2057 2058 2059 2060 2061 2062 2063 2064 2065 2066 2067 2068 2069  YJL181W YNL295W YER170W YDR336W YCL050C YMR300C YDR061W YNL068C YER073W YMR219W YBR270C YMR152W YLL013C YER046W YMR273C YBR242W YPR038W YHR182W YER188W YER096W YNR007C YLR286C YDR218C YDR191W YMR002W YLR396C YER169W YNL143C YPL050C YHR103W YHR150W YHR112C YKL017C YBR030W YIR038C YDR436W YDR134C YER141W YNL286W YMR087W YOR185C YLR016C YJL049W YJL141C  Gene  ADK2 APA1 ADE4 FKH2 ALD5 ESC1 YIM1 PUF3 SPO73 ZDS1  SHC1 ATG3 CTS1 SPR28 HST4 VPS33 RPH1 MNN9 SBE22 PEX28 HCS1 GTT1 PPZ2 COX15 CUS2 GSP2 PML1 YAK1  Normalized densitometry values MATa MATalpha 1.03 0.93 1.01 0.95 1.19 0.77 1.12 0.84 0.97 0.99 1.29 0.67 0.91 1.05 1.04 0.92 0.96 1.00 1.00 0.96 0.94 1.03 0.98 0.98 0.90 1.07 1.00 0.96 0.99 0.97 0.92 1.05 0.98 0.99 0.88 1.09 1.09 0.88 0.96 1.01 1.23 0.73 0.98 0.98 1.01 0.95 1.13 0.83 0.93 1.03 0.86 1.10 1.17 0.80 0.95 1.02 1.01 0.95 1.04 0.93 0.91 1.06 0.96 1.01 0.98 0.99 0.91 1.05 0.99 0.98 0.96 1.01 1.12 0.85 0.88 1.09 1.07 0.90 0.91 1.06 1.00 0.97 0.96 1.01 1.01 0.95 1.01 0.96  SCORE average 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.98  0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02  209  Rank  2070 2071 2072 2073 2074 2075 2076 2077 2078 2079 2080 2081 2082 2083 2084 2085 2086 2087 2088 2089 2090 2091 2092 2093 2094 2095 2096 2097 2098 2099 2100 2101 2102 2103 2104 2105 2106 2107 2108 2109 2110 2111 2112 2113  ORF  Gene  YPR043W YER002W YLR079W YER051W YAL046C YJL075C YOL027C YPR115W YBR173C YJL201W YKR029C YML123C YBR068C YIL135C YDL052C YKL201C YJL191W YPR138C YBR134W YDR345C YOR324C YCR090C YMR156C YOR307C YIL028W YLR330W YNR063W YNL093W YIL153W YEL065W YMR306W YPL147W YHL026C YIL170W YPL199C YMR040W YIL064W YGR209C YGR007W YOR049C YLR036C YLR014C YDR320C YAL034C  RPL43A NOP16 SIC1  MDM38 UMP1 ECM25 SET3 PHO84 BAP2 VHS2 SLC1 MNN4 RPS14B MEP3 HXT3 FRT1 TPP1 SLY41 CHS5 YPT53 RRD1 SIT1 FKS3 PXA1 HXT12  TRX2 MUQ1 RSB1 PPR1 SWA2 FUN19  Normalized densitometry values MATa MATalpha 0.78 1.19 0.88 1.08 0.91 1.06 0.92 1.05 0.90 1.06 1.07 0.90 0.65 1.32 1.06 0.91 0.98 0.99 1.00 0.97 0.99 0.98 0.96 1.01 0.96 1.01 1.13 0.84 0.97 1.00 1.10 0.87 0.91 1.06 1.05 0.92 1.06 0.91 1.06 0.91 0.95 1.02 0.91 1.06 1.09 0.88 0.92 1.06 0.95 1.02 1.03 0.94 1.05 0.92 1.07 0.91 0.78 1.19 0.83 1.14 1.17 0.80 0.96 1.02 0.99 0.99 1.06 0.92 1.03 0.95 1.03 0.95 1.01 0.96 0.96 1.02 1.05 0.92 0.98 0.99 1.06 0.92 0.90 1.08 0.87 1.10 0.95 1.03  SCORE average 0.98 0.98 0.98 0.98 0.98 0.98 0.98 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99  0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02  210  Rank  ORF  2114 YCR085W 2115 YFR030W 2116 YDL133CA 2117 YOL088C 2118 YOR213C 2119 YOR112W 2120 YBL075C 2121 YNR068C 2122 YJL151C 2123 YDR056C 2124 YDL123W 2125 YLR431C 2126 YLR287CA 2127 YPL176C 2128 YHL041W 2129 YMR135C 2130 YPL182C 2131 YPL272C 2132 YPL052W 2133 YPL116W 2134 YNL330C 2135 YOL032W 2136 YER183C 2137 YER089C 2138 YGL114W 2139 YDR258C 2140 YBR177C 2141 YDR077W 2142 YJL051W 2143 YNL141W 2144 YDL203C 2145 YOR225W 2146 YJL013C 2147 YGL085W 2148 YLL042C 2149 YPR071W 2150 YER045C 2151 YGR021W 2152 YLL032C 2153 YOL084W 2154 YOL042W 2155 YHR125W  Gene  MET10 RPL41B MPD2 SAS5 SSA3 SNA3 SNA4 ATG23 RPS30A  GID8  OAZ1 HOS3 RPD3 FAU1 PTC2 HSP78 EHT1 SED1 AAH1  MAD3 ATG10 ACA1  PHM7 NGL1  Normalized densitometry values MATa MATalpha 1.01 0.97 0.95 1.03 0.91 1.06 0.93 1.05 0.93 1.02 0.96 0.99 0.99 0.79  1.09 1.07 1.16 1.08 0.99 0.82 0.93 0.99 0.90 1.06 1.04 0.94 1.03 1.07 1.00 1.06 0.89 0.92 0.91 1.09 0.98 0.86 0.85 0.96 1.05 0.98 0.98 0.97 0.89  SCORE average 0.99 0.99 0.99  0.02 0.02 0.02  1.05 0.93 1.05 0.96 0.99 1.02 0.99 0.99 1.19 0.99  0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99  0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01  0.89 0.91 0.82 0.90 0.99 1.16 1.06  0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99  0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01  1.08 0.92 0.94 1.04 0.95 0.91 0.98 0.92 1.09 1.06 1.07 0.89 1.00 1.12 1.13 1.02 0.94 1.00 1.00 1.02 1.09  211  Rank  2156 2157 2158 2159 2160 2161 2162 2163 2164 2165 2166 2167 2168 2169 2170 2171 2172 2173 2174 2175 2176 2177 2178 2179 2180 2181 2182 2183 2184 2185 2186 2187 2188 2189 2190 2191 2192 2193 2194 2195 2196 2197 2198 2199  ORF  Gene  YIL121W YER057C YOR069W YGR086C YBR165W YGR018C YPL105C YBR269C YCL069W YOR079C YBR239C YDR078C YGL004C YNL303W YPR125W YNR073C YEL046C YHR127W YMR153W YGR001C YCR098C YDL079C YBR133C YER063W YNL193W YML108W YDR420W YOR097C YLR406C YIL058W YIL040W YMR201C YHL007C YKR080W YNL334C YGR134W YKR005C YGR193C YDL049C YDL065C YDR220C YGL125W YDL124W YGR032W  QDR2 HMF1 VPS5 PIL1 UBS1  VBA3 ATX2 SHU2 RPN14  GLY1 NUP53 GIT1 MRK1 HSL7 THO1  HKR1 RPL31B APQ12 RAD14 STE20 MTD1 SNO2 CAF130 PDX1 KNH1 PEX19 MET13 GSC2  Normalized densitometry values MATa MATalpha 0.82 1.17 0.94 1.04 0.91 1.07 1.02 0.96 0.96 1.03 1.17 0.82 0.97 1.02 1.02 0.96 0.90 1.09 0.93 1.06 1.00 0.99 0.97 1.01 1.12 0.87 0.91 1.08 0.95 1.03 1.03 0.96 0.88 1.11 0.99 1.00 1.11 0.88 0.99 1.00 0.87 1.12 1.02 0.97 0.99 0.96 1.03 1.05 0.94 0.91 1.08 0.97 1.02 0.94 1.05 1.02 0.97 1.05 0.94 0.94 1.04 1.00 0.99 0.99 0.97 1.02 1.00 0.99 0.96 1.03 1.05 0.94 1.12 0.87 1.01 0.98 1.08 0.91 1.09 0.90 0.99 1.00 0.99 1.02 0.97  SCORE average 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99  0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01  212  Rank  2200 2201 2202 2203 2204 2205 2206 2207 2208 2209 2210 2211 2212 2213 2214 2215 2216 2217 2218 2219 2220 2221 2222 2223 2224 2225 2226 2227 2228 2229 2230 2231 2232 2233 2234 2235 2236 2237 2238 2239 2240 2241 2242 2243  ORF  Gene  YPL177C YLR020C YBR099C YIR043C YOL089C YLR344W YNL194C YLR376C YGL159W YLR019W YCR107W YBR288C YDL040C YPL098C YDL240W YDR285W YAL024C YIR035C YBL066C YOR015W YBR199W YER049W YML011C YBR218C YGL063W YMR190C YMR279C YDL094C YEL020C YDL125C YIL052C YMR237W YDR502C YDL019C YOR222W YGL050W YBR101C YGL132W YKR003W YJR149W YER090W YKL061W YDL074C YDL089W  CUP9 YEH2  HAL9 RPL26A PSY3 PSR2 AAD3 APM3 NAT1 MGR2 LRG1 ZIP1 LTE1 SEF1 KTR4  PYC2 PUS2 SGS1  HNT1 RPL34B SAM2 OSH2 ODC2 FES1 OSH6 TRP2 BRE1  Normalized densitometry values MATa MATalpha 1.17 0.82 0.99 1.00 1.01 0.98 0.93 1.06 1.02 0.97 1.05 0.94 0.97 1.02 0.89 1.10 0.96 1.03 1.02 0.97 1.03 0.96 0.91 1.09 0.98 1.01 0.93 1.06 1.04 0.95 0.96 1.03 0.98 1.02 0.98 1.01 1.00 1.03 0.96 1.05 0.94 0.99 1.01 1.02 0.97 1.01 0.99 0.96 1.03 1.00 1.07 0.92 0.88 1.11 1.14 0.86 0.99 1.00 0.73 1.26 1.06 0.93 1.15 0.85 1.01 0.99 0.97 1.02 1.06 0.94 1.00 0.90 1.09 1.02 0.97 1.11 0.89 0.98 1.01 1.01 0.98 1.00 0.99 0.93 1.07  SCORE average 0.99 0.99 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00  0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01  213  Rank  ORF  2244 2245 2246 2247 2248 2249 2250 2251 2252 2253 2254 2255 2256 2257 2258 2259 2260 2261 2262 2263 2264 2265 2266 2267 2268 2269 2270 2271 2272 2273 2274 2275 2276 2277 2278 2279 2280 2281 2282 2283 2284 2285 2286 2287  YOL037C YNR064C YHL012W YNL175C YHR061C YGR059W YJL140W YPR118W YOL029C YKL015W YLR185W YKR010C YKL161C YOR214C YDL027C YAR015W YKL068W YKR102W YLR271W YNL139C YBR195C YBR016W YJL162C YLR077W YER135C YHR106W YJL196C YER035W YJR020W YDR403W YOR384W YHR104W YAL014C YPL099C YOR248W YBR041W YEL030W YGL118C YHR057C YLR334C YML051W YIL149C YER030W YOR133W  Gene  NOP13 GIC1 SPR3 RPB4  PUT3 RPL37A TOF2  ADE1 NUP100 FLO10 RLR1 MSI1 JJJ2  TRR2 ELO1 EDC2 DIT1 FRE5 GRE3 SYN8  FAT1 ECM10 CPR2 GAL80 MLP2 EFT1  Normalized densitometry values MATa MATalpha 1.02 0.98 0.99 1.00 0.93 1.06 0.92 1.08 1.03 0.97 1.11 0.89 1.00 0.99 1.01 1.07 0.92 1.00 1.00 0.90 1.10 0.91 1.08 1.01 0.99 1.09 0.91 1.13 0.87 1.04 0.96 0.88 1.12 0.93 1.07 1.01 0.98 1.00 1.02 0.97 1.01 0.99 1.00 0.99 0.96 1.04 1.02 0.97 1.08 0.92 0.98 1.02 0.99 1.01 0.82 1.18 1.05 0.95 1.09 0.91 1.19 0.81 1.06 0.94 0.92 1.08 1.03 0.97 1.01 0.99 0.96 1.04 1.13 0.87 0.96 1.04 1.03 0.97 0.86 1.14 0.98 1.02 1.09 0.91 0.90 1.10  SCORE average 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00  0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00  214  Rank  ORF  2288 YNL278W 2289 YDR055W 2290 YER091CA 2291 YPL123C 2292 YJR130C 2293 YOR355W 2294 YBR021W 2295 YKR060W 2296 YJL084C 2297 YJR038C 2298 YER062C 2299 YOR092W 2300 YHL028W 2301 YPL040C 2302 YLR363C 2303 YEL010W 2304 YBR205W 2305 YBR178W 2306 YKR012C 2307 YBR093C 2308 YLR194C 2309 YBR187W 2310 YOR053W 2311 YGR286C 2312 YNR060W 2313 YDL115C 2314 YBR056W 2315 YPL097W 2316 YDR428C 2317 YML005W 2318 YBR210W 2319 YER132C 2320 YOR322C 2321 YDR488C 2322 YEL028W 2323 YMR133W 2324 YNL031C 2325 YGR012W 2326 YOL090W 2327 YFR046C 2328 YPL086C 2329 YKL142W 2330 YDR066C  Gene  CAF120 PST1  RNY1 STR2 GDS1 FUR4 UTP30  HOR2 ECM3 WSC4 ISM1 NMD4 KTR3  PHO5  BIO2 FRE4 IWR1 MSY1 TRM12 ERV15 PMD1 LDB19 PAC11 REC114 HHT2 MSH2 CNN1 ELP3 MRP8  Normalized densitometry values MATa MATalpha 1.04 0.96 1.10 0.90 1.07 0.93 1.07 0.94 0.96 0.88 1.01 1.06 0.98 0.99 0.94 0.97 1.04 0.82 1.09 1.00 1.15 1.01 1.19 0.94 0.94 1.08 0.97 0.93 1.08 0.99 1.00 1.08 1.13 1.02 1.02 1.07 1.03 1.01 1.13 1.06 1.03 1.00 1.02 1.00 1.09 1.08  0.93 1.06 1.04 1.12 0.99 0.94 1.02 1.02 1.07 1.03 0.96 1.19 0.92 1.01 0.86 0.99 0.81 1.07 1.06 0.93 1.03 1.07 0.92 1.02 0.92 0.88 0.98 0.99 0.94 0.98 1.00 0.87 0.95 0.97 0.99 0.91 0.92  SCORE average 1.00 1.00 1.00  0.00 0.00 0.00  1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00  0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00  215  Rank  ORF  2331 2332 2333 2334 2335 2336 2337 2338 2339 2340 2341 2342 2343 2344 2345  YPR195C YDR135C YBR297W YPR076W YMR299C YIL050W YML007W YHL030W YMR210W YPL144W YGL232W YCR027C YGR236C YMR015C YDR046C  Gene  YCF1 MAL33 DYN3 PCL7 YAP1 ECM29  TAN1 RHB1 SPG1 ERG5 BAP3  Normalized densitometry values MATa MATalpha 1.02 0.99 1.08 0.93 1.02 0.98 0.98 1.03 1.17 0.84 1.02 0.99 0.94 1.06 0.95 1.06 1.01 1.00 1.11 0.90 1.03 0.98 0.94 1.07 1.00 1.01 0.90 1.11 1.02 0.99  SCORE average 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00  0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00  216  217  Figure A1. Full hierarchical clustering of the genetic interaction data. Hierarchical clustering of normalized, median-centered invertase activity values of double mutant strains generated by crossing top-scoring mutants from the primary screen (array genes, y axis) to a diverse set of 81 trafficking mutants (query genes, x axis). Although 374 top-scoring mutants were used for the crosses, the heat map shows only the 307 array genes for which reliable double mutant data were obtained. Yellow indicates lower than expected levels of GSS at the cell surface, whereas blue indicates higher than expected levels. Red bars indicate representative query genes with known roles in endocytosis or recycling.  218  Figure A2. Characterization of LDB17 mutants and identification of Ldb17-interacting proteins. (A–C) Kymograph representations of Ldb17-GFP relative to markers Sla1-RFP (A), Myo5-RFP (B), and Abp1-RFP (C) at cortical patches over 120 s. (D) Localization of Ldb17-GFP and ldb17ΔPRD in wild-type (WT) and bzz1 cells. Bar, 2 μm. (E) Ldb17 interacts with the syndapin homologue Bzz1 through a C-terminal PRD. Proteins were immunoprecipitated from cells expressing Ldb17-myc, ldb17DPRD-myc, and/or Bzz1-HA with a-HA antibodies and detected by immunoblot analysis with either a-HA or a-myc. (F) Loss of the Ldb17 PRD or Bzz1 increases cell surface levels of the GSS reporter, as measured by liquid assay. Invertase activity is expressed as nanomole glucose  219  released per OD600 (mean of at least three experiments ± SD). (G) Summary of yeast twohybrid interactions between Ldb17-GAL4AD and the 16 different SH3 domain–GAL4BD fusions. Interactions between Ldb17 and SH3 domains from Bzz1, Sla1, Ysc84/Lsb4, and Yfr024c/Lsb3 resulted in strong activation of the HIS3 reporter (resistant to 50 mM 3-AT). (H) PRD dependence of the yeast two-hybrid interactions. Diploid strains containing GALAD and GALBD plasmids are shown on the left; activation of the HIS3 reporter is shown on the right.  220  APPENDIX B: Supplemental material (chapter 3) Table B.1. Plasmids and yeast strains used in this study Type yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain  Name LCY858  Source this study  LCY1156  Description/Genotype can1::SRE2pr-LEU2 lyp1∆ cyh2 his3∆1 leu2∆0 ura3∆0 met15∆0 LYS2+ LC858 apm1∆::NAT  LCY1154  LC858 apm2∆::NAT  this study  LCY1155  LC858 ap14∆::NAT  this study  LCY1141  LC858 yap1801∆::NAT  this study  BY4741  MATa his3-1 leu2-0 ura3-0  this study  LC1994  BY4741 Apm2::GFP(HIS)  this study  LCY1979  BY4741 Apm4::GFP(HIS) Clc1::RFP(NAT)  this study  LCY1977  LCY1994 Clc1::RFP(NAT)  this study  HBY155  LC1979 sla2∆  this study  LCY1978  LC1977 sla2∆  this study  LCY1997  this study  LCY1230  BY4741 Apm2::GFP(HIS3) Clc1::RFP(NAT) BY4741 Apm1::RFP(KAN) Apm2::GFP(HIS3) BY4741 Apm1::RFP(KAN) Ima1::GFP(HIS3) BY4741 Ima1::GFP(HIS3) Clc1::RFP(NAT) BY4741 chs6∆::NAT  LCY3168  1230 apl2∆::KAN  this study  LCY3167  1230 apm1∆::KAN  this study  LCY3170  1230 apm2∆::KAN  this study  LCY3202  1230 apm2∆::KAN apm1∆::URA  this study  LCY3169  1230 ima1∆::KAN  this study  CTY708 CTY603 LCY2439  this study  this study this study this study this study  221  Type yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain yeast strain  Name LCY3203  Description/Genotype 1230 ima1∆::KAN apm2∆::URA  Source this study  LCY3204  BY4741 + pNAT  this study  CTY301  Ima1-GFP(HIS3)  this study  CTY564  Ima1-GFP(HIS3) Apl4::3HA(KAN)  this study  CTY574  Ima1::GFP(HIS3) Apl4N::HA(KAN)  this study  CTY265  Ima1::GFP(HIS3) Apm1::3HA(KAN)  this study  CTY661  Ima1::GFP(HIS3) Apm2::3HA(KAN)  this study  pj694a  MATa trp1-901 leu2-3,112 ura3-52 his3200 gal4∆ gal80∆ LYS::GAL1-HIS3 GAL2ADE2 met2::GAL7-lacZ pj694alpha MATalpha trp1-901 leu2-3,112 ura3-52 his3-200 gal4∆ gal80∆ LYS::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ HBY536 pj694alpha pOBD2-Apm2  James et al., 1996a  HBY544  pj694alpha pOBD2-Apm2 (246-605)  this study  HBY625  pj694alpha pOBD2-Apm2 (391-605)  this study  NR  pj694alpha pOBD2-Apm2 (389-582)  this study  CTY188  pj694a pOAD-Apl4  this study  HBY539  pj694a pOAD-Ima1  this study  HBY626  pj694a pOAD-Mut5N (1-262)  this study  HBY463  LCY1994 ima∆::NAT  this study  HBY567  BY4741 Apm2::3HA(KAN)  this study  HBY688  BY4741 Ima1::3HA(KAN)  this study  LCY2210  BY4741 Ima1::GFP(HIS3) erg6∆::NAT  this study  CTY286  BY4741 Apl2::GFP(HIS3) erg6∆::NAT  this study  James et al., 1996a  this study  222  Type yeast strain  Name HB577  Description/Genotype BY4741 + GFP-Snc1-Suc2::SUC2  Source The GSS reporter plasmid pCS30 was linearized with XbaI and integrated at the SUC2 locus.  yeast strain yeast strain plasmid plasmid plasmid plasmid plasmid plasmid  SEY6210  MATa leu2-3,112 ura3-52 his3-∆200 trp1-∆901 lys2-801 suc2-∆9 MetSEY6210 GFP::SNC1(URA)  S. Emr  pGST-Ima1 pBG1805 pNR1 pNR3 pLC1329 pOAD  pRS415-IMA1 pRS415-IMACM pSec7-DsRed(URA) Uetz et al., 2000  plasmid  pOBD2  Uetz et al., 2000  plasmid  pCS7  SNC1-GFP in pRS316  plasmid  pCS30  GFP-SNC1-SUC2 in pRS306 (GSS; TPI1 promoter)  plasmid  pPPL92  pGST-SNC1  HBY596  this study Open Biosystems Open Biosystems this study this study this study gift from Stan Fields gift from Stan Fields Schluter et al., 2008b Sequences from pGS (Lewis et al., 2000)c were PCRamplified to introduce a SmaI site before the Snc1 stop codon, and subcloning this into XhoI/SmaIdigested pRS306. The resulting plasmid was digested with XbaI/SmaI, endfilled, and ligated to SUC2 sequences on a SmaI-HpaI fragment from pSEYC306 (Darsow et al., 2000)d gift from Anne Spang  James, P., Halladay, J. & Craig, E. A. (1996) Genomic libraries and a host strain designed for highly efficient two- hybrid selection in yeast. Genetics 144: 1425-1436 a  223  Schluter et al. Global analysis of yeast endosomal transport identifies the vps55/68 sorting complex. Mol Biol Cell (2008) vol. 19 (4) pp. 1282-94 b  c Lewis  et al. Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes. Mol.Biol.Cell (2000) vol. 11 (1) pp. 23-38 d Darsow  et al. Invertase fusion proteins for analysis of protein trafficking in yeast. Meth Enzymol (2000) vol. 327 pp. 95-106  224  

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