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Uncovering regulators of adaptor protein complex 1 (AP-1) trafficking pathways Whitfield, Shawn Tamajka 2018

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UNCOVERING REGULATORS OF ADAPTOR PROTEIN COMPLEX 1 (AP-1) TRAFFICKING PATHWAYS.  by  Shawn Tamajka Whitfield  B.Sc., The University of Victoria, 2013      A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY   in   THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Biochemistry and Molecular Biology)     THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)      August 2018    © Shawn Tamajka Whitfield, 2018      ii  Committee Page   The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled:  Uncovering regulators of adaptor protein complex 1 (AP-1) trafficking pathways.  submitted by Shawn Tamajka Whitfield  in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology  Examining Committee: Dr. Elizabeth Conibear, Biochemistry and Molecular Biology Supervisor  Dr. Robert Molday, Biochemistry and Molecular Biology Supervisory Committee Member   Supervisory Committee Member Dr. Calvin Yip, Biochemistry and Molecular Biology University Examiner Dr. Christopher Loewen, Cellular and Physiological Sciences University Examiner   Additional Supervisory Committee Members: Phil Hieter, Biochemistry and Molecular Biology Supervisory Committee Member  Supervisory Committee Member iii  Abstract Clathrin-coated vesicles (CCVs) traffic many cargo proteins throughout the cell to their functional locations. At the center of Golgi/endosome CCV transport is the heterotetrameric AP-1 adaptor protein complex, which coordinates cargo selection and vesicle formation. AP-1 is regulated by a suite of accessory proteins but their identity and functions are incompletely characterized. Here, we identified new AP-1 regulators through targeted yeast genome-wide screens performed in the Conibear lab.  Adaptor protein complexes can contain variant subunits but the effect of this subunit exchange is unclear. In yeast, the functional relationship between the “classical” AP-1 complex containing the medium cargo-selective subunit Apm1 and the variant AP-1 complex (AP-1R) containing Apm2 is unclear. Our genome-wide screens indicated that they sort different cargo, and we found they also respond differently to small molecule inhibitors. We identified Mil1 as a novel specific regulator of the Apm2-containing complex, with active-site mutants supporting its role as a lipase. The data are consistent with a model where AP-1 and AP-1R are recruited to distinct membrane areas, facilitating different trafficking pathways. The second screen, for components involved in the trafficking of the AP-1 cargo Chs3, revealed a connection between the previously-identified AP-1 regulator Laa1 and an uncharacterized ORF that we named Laa2. Laa2 bridges AP-1 and Laa1 through an FGxF gamma-adaptin ear binding motif. Our identification of a yeast “Laa complex” consisting of Laa1, Laa2 and the short coiled-coil protein Slo1 led to the discovery of a similar complex in mammalian cells, consisting of HEATR5A, fasciculation and elongation protein zeta 2 (Fez2) and SCOC. We further showed that HEATR5A is distinct from HEATR5B, which works in the aftiphilin/γ-synergin complex previously implicated in AP-1 function. We found a conserved binding site in Laa2, Fez2, aftiphilin and the aftiphilin-related protein CLBA1 for HEATR5-family proteins, providing a new link between various trafficking pathways. This study identifies novel regulatory proteins that may facilitate AP-1 recruitment and function in particular pathways, and illustrates that proteins are not often purely iv  redundant. Apm1 and Apm2 likely sort different cargo as part of distinct AP-1 isoforms, and HEATR5A and HEATR5B participate in distinct complexes. v  Lay Summary Movement of proteins to specific places in the cell is critical for cell function and survival. Disrupting this movement can have catastrophic consequences including disease or cell death. The Adaptor Protein complex 1 (AP-1) is a central component of the cellular machinery that ensures that proteins are correctly trafficked in the cell. AP-1 is tightly controlled, but our knowledge of all of the factors involved in this control is limited. The studies in this thesis contribute to our understanding of the process by identifying new regulators of AP-1 that are broadly important in protein trafficking in both yeast and humans.    vi  Preface I conceived and wrote the Introduction and Discussion sections and the figures presented therein. The work presented in Chapter 2 and Appendix A has been published: Whitfield ST, Burston HE, Bean BD, Raghuram N, Maldonado-Báez L, Davey M, Wendland B, Conibear E. The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting. Molecular Biology of the Cell. 2016 Feb 1;27(3):588-98. I performed all the experiments and analysis that resulted in the figures presented in the work with the exception of Figures 2.1B, 2.2A,B, 2.4A,B and 2.6A. HEB performed initial follow-up experiments including the calcofluor white experiment (Figure 2.1B), subcellular fractionation of Mil1-GFP (Figure 2.6A), and (with NR) the yeast two-hybrid analyses (Figure 2.4D). LM-B performed the microscopy presented in Figure 2.2B with BW’s supervision and input. Otherwise, I performed microscopy and BDB assisted me in automated counting. MD and I constructed mutant yeast strains and plasmids. EC and I wrote the manuscript with input from co-authors.   The work presented in Chapter 3 and Appendix B is a manuscript in preparation: Whitfield ST, Tam YYC, Sridhar V, Schluter C, Davey M, Mast F, Rachubinski RA, Conibear E. Fez proteins, aftiphilin and CLBA1 are all related to a fundamental Laa complex in yeast through binding to HEATR5-family members. Manuscript in preparation. I performed all the experiments and analysis that resulted in the figures presented in the work with the exception of the following: YYCT performed the initial chs6Δ calcofluor white and E-MAP screens, averaging and Z-scores, initial microscopy for Figures 3.2A and 3.2B, the yeast-two-hybrid in Figure 3.3D and co-IP in Figure 3.3E. MD constructed strains and performed the experiments for Supplementary Figures B1 and B2A and (with VS) Figure 3.3F. CS performed the co-IP for figure 3.2G. FM performed the lifetime imaging for figure 3.3A, with RAR’s supervision and input. VS performed replicates of the experiments in Figure 3.6B,C. EC and I conceived and designed the experiments and I wrote the manuscript with input from EC.  vii  Table of Contents Abstract ........................................................................................................................... iii Lay Summary .................................................................................................................. v Preface ............................................................................................................................vi Table of Contents ........................................................................................................... vii List of Tables ...................................................................................................................xi List of Figures ................................................................................................................. xii List of abbreviations ...................................................................................................... xiii Acknowledgements ..................................................................................................... xviii Dedication ..................................................................................................................... xix Chapter 1: Introduction .................................................................................................... 1 1.1 Introduction ............................................................................................................ 1 1.1.1 Overview of vesicular trafficking pathways ...................................................... 1 1.1.2 Endosomal maturation ..................................................................................... 4 1.2 Establishment of cellular compartment identity ...................................................... 4 1.2.1 Membrane charge ............................................................................................ 5 1.2.2 Lipid packing defects ....................................................................................... 5 1.2.3 Curvature ......................................................................................................... 6 1.2.4 Phosphoinositides ............................................................................................ 6 1.2.5 Small GTPases ................................................................................................ 9 1.3 Vesicle transport .................................................................................................... 9 1.3.1 Initiation and assembly .................................................................................. 11 1.3.2 Budding and scission ..................................................................................... 13 1.3.3 Uncoating and movement .............................................................................. 14 1.3.4 Docking and fusion ........................................................................................ 15 1.4 Adaptor protein complexes .................................................................................. 16 1.4.1 γ/α/δ/ε/ζ and β subunits ................................................................................. 17 1.4.2 µ subunits ...................................................................................................... 20 1.4.3 σ subunits ...................................................................................................... 21 1.4.4 AP hemicomplexes hypothesis ...................................................................... 22 viii  1.4.5 AP complex activation ................................................................................... 22 1.4.6 AP-1 dysfunction in disease ........................................................................... 23 1.5 AP complex regulators/accessory proteins .......................................................... 24 1.5.1 AP accessory protein functions ...................................................................... 24 1.5.2 AP accessory protein dysfunction in disease ................................................. 27 1.6 Research objectives ............................................................................................. 28 Chapter 2: The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting ................................................................ 30 2.1 Synopsis .............................................................................................................. 30 2.2 Introduction .......................................................................................................... 31 2.3 Results ................................................................................................................. 33 2.3.1 Apm2 is part of a functionally distinct AP-1 related complex.......................... 33 2.3.2 AP-1R localizes to the late Golgi and early endosomes ................................ 35 2.3.3 Recognition of tyrosine-based signals by Apm1 and Apm2 ........................... 36 2.3.4 Mil1(Yfl034w) specifically binds the Apm2 C-terminal subdomain B .............. 39 2.3.5 Mil1 has a conserved α/β-hydrolase catalytic motif required for its function .. 42 2.3.6 Mil1 is a peripheral membrane protein that promotes Apm2 recruitment ....... 43 2.4 Discussion ............................................................................................................ 45 2.6 Materials and Methods ......................................................................................... 48 2.6.1 Yeast strains and plasmids ............................................................................ 48 2.6.2 Chemical compounds .................................................................................... 49 2.6.3 Invertase assay .............................................................................................. 49 2.6.4 Calcofluor White assay .................................................................................. 50 2.6.5 Small molecule inhibitor assays ..................................................................... 50 2.6.6 Fluorescence microscopy .............................................................................. 50 2.6.7 Co-immunoprecipitation and Western Blotting ............................................... 51 2.6.8 Yeast two-hybrid assay .................................................................................. 52 2.6.9 Bioinformatic analyses ................................................................................... 52 2.6.10 Membrane fractionation ............................................................................... 52 Chapter 3: A yeast genome-wide screen for AP-1 trafficking regulators reveals that .... 53 3.1 Synopsis .............................................................................................................. 53 ix  3.2 Introduction .......................................................................................................... 54 3.3 Results ................................................................................................................. 56 3.3.1 A genome-wide screen identifies candidate AP-1 regulators ......................... 56 3.3.2 Refining top hits using genetic interaction profiles ......................................... 58 3.3.3 Laa1 and Laa2 (YBL010C) co-localize and form a complex .......................... 60 3.3.4 Laa2 bridges the interaction between AP-1 and Laa1 via an FGxF motif ...... 62 3.3.5 Laa2 is the central component of a yeast complex consisting of Laa1-Laa2-Slo1 ........................................................................................................................ 64 3.3.6 A mammalian form of the Laa complex contains Fez2, SCOC and HEATR5A ................................................................................................................................ 67 3.3.7 Laa2, Fez2, Aftiphilin and CLBA1 share a conserved HEATR5-family binding domain .................................................................................................................... 69 3.4 Discussion ............................................................................................................ 71 3.5 Materials and Methods ......................................................................................... 74 3.5.1 Yeast strains and plasmids ............................................................................ 74 3.5.2 Mammalian plasmids ..................................................................................... 75 3.5.3 Mammalian cell culture .................................................................................. 75 3.5.4 Calcofluor-white based genomic screen and E-MAP ..................................... 76 3.5.5 Screen data analysis and clustering .............................................................. 76 3.5.6 Fluorescence microscopy .............................................................................. 77 3.5.7 Gel filtration .................................................................................................... 77 3.5.8 Co-immunoprecipitation ................................................................................. 78 3.5.9 Immunoblotting .............................................................................................. 78 3.5.10 Lifetime fluorescence measurements .......................................................... 79 3.5.10 Yeast two-hybrid .......................................................................................... 79 Chapter 4: Discussion and conclusions ......................................................................... 80 4.1 Summary of key findings ...................................................................................... 80 4.2 AP-1 µ subunits Apm1 and Apm2 are functionally distinct in yeast ...................... 80 4.3 The predicted lipase is Mil1 is a specific regulator of Apm2 ................................. 81 4.4 The yeast Laa complex (Laa1-Laa2-Slo1) is related to a human HEATR5A-Fez1/2-SCOC complex .............................................................................................. 83 x  4.5 Binding to HEATR5 family members is a common feature of Laa2, Fez1/2, aftiphilin and CLBA1 .................................................................................................. 85 4.6 Conclusion ........................................................................................................... 87 References .................................................................................................................... 88 Appendices ................................................................................................................. 120 Appendix A. Supplementary material for Chapter 2 ................................................. 120 Appendix B. Supplementary material for Chapter 3 ................................................. 124   xi  List of Tables Table 1.1 Major coated-vesicle machinery in eukaryotic cells can be divided into cargo-selective and scaffolding components. .......................................................................... 10 Supplementary Table A1 List of yeast strains used in Chapter 2. ............................... 121 Supplementary Table A2 List of plasmids used in Chapter 2. ..................................... 122 Supplementary Table B1 Ranked list of average Z-scores >1 from duplicate screens of the MATa and MATα yeast gene deletion collections. ................................................ 125 Supplementary Table B2 Median values and group assignments of MCL-calculated clusters from Cytoscape analysis. ............................................................................... 131 Supplementary Table B3 List of yeast strains used in Chapter 3. ............................... 133 Supplementary Table B4 List of plasmids used in Chapter 3. ..................................... 134 Supplementary Table B5 List of antibodies used in Chapter 3. ................................... 135  xii  List of Figures Figure 1.1 Major coat-protein-dependent protein trafficking pathways in a generalized eukaryotic cell. ................................................................................................................ 2 Figure 1.2 Major phosphoinositide (PI) species markers for each cellular compartment. 7 Figure 1.3 Simplified overview of vesicle transport steps shown for AP-1-mediated clathrin-coated vesicle trafficking. ................................................................................. 11 Figure 1.4 Subunit composition of the five AP complexes found in mammals. ............. 17 Figure 2.1 APM1 and APM2 deletions have distinct sorting phenotypes. ..................... 34 Figure 2.2 Apm2 localizes to late Golgi/early endosomes. ............................................ 36 Figure 2.3 The predicted YxxΦ-binding pocket is required for some but not all functions of Apm1 and Apm2........................................................................................................ 38 Figure 2.4 Mil1 interacts with Apm2 through its WQEMP motif. .................................... 40 Figure 2.5 Mil1 has a conserved serine hydrolase catalytic triad. ................................. 43 Figure 2.6 Mil1 is a peripheral membrane protein that promotes Apm2 membrane recruitment. ................................................................................................................... 44 Figure 3.1 A genome-wide screen identifies candidate AP-1 regulators. ...................... 57 Figure 3.2 Laa1 and Laa2 co-localize and form a complex. .......................................... 61 Figure 3.3 Laa2 has an FGxF motif and bridges the interaction between AP-1 and Laa1. ...................................................................................................................................... 63 Figure 3.4 Laa2 is the central component of a yeast complex consisting of Laa1-Laa2-Slo1. .............................................................................................................................. 66 Figure 3.5 A mammalian form of the Laa complex contains Fez2, SCOC and HEATR5A. ...................................................................................................................................... 68 Figure 3.6 Laa2, Fez2, Aftiphilin and CLBA1 share a conserved domain for HEATR5-family binding. ............................................................................................................... 70 Figure 3.7 Model summarizing the yeast and mammalian Laa1/HEATR5-containing complexes. .................................................................................................................... 72 Supplementary Figure A1 The tyrosine-binding pockets of Apm1 and Apm2 are highly conserved, and mutation of residues in these pockets does not destabilize the proteins. .................................................................................................................................... 120 Supplementary Figure A2 Deletion of MIL1 does not affect Apm1-GFP recruitment or number of Golgi compartments. .................................................................................. 121 Supplementary Figure B1 Laa1 and Laa2 run in similar fractions by gel filtration. ...... 124 Supplementary Figure B2 Slo1 deletion affects chs6Δ bypass and Laa complex puncta number. ....................................................................................................................... 124 Supplementary Figure B3 Fez1 also interacts with HEATR5A but not HEATR5B. ..... 125 xiii  List of abbreviations µHD: µ homology domain 3-AT: 3-Amino-1,2,4-triazole A.U.: Arbitrary units ALPS: Amphipathic lipid packing sensor ANTH: AP180 N-terminal homology AP: Adaptor protein AP-1R: AP-1 related APL: Yeast clathrin adaptor protein complex large chain APM: Yeast clathrin adaptor protein complex medium chain APS: Yeast clathrin adaptor protein complex small chain ARF: ADP-ribosylation factor ARH: Autosomal recessive hypercholesterolemia ARP: Actin-related protein BAR: BIN/amphiphysin/Rvs167 CAD: Cationic amphiphilic drug CALM: Clathrin assembly lymphoid myeloid leukemia CCV: Clathrin-coated vesicle CHAPSO: 3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate Chs3: Chitin synthase 3 Clc: Clathrin light chain CLINT : Clathrin interactor CME: Clathrin-mediated endocytosis COP: Coat protein complex CORVET: Class C core vacuole/endosome tethering CW: Calcofluor white DAG: Diacylglycerol DHFR: Dihydrofolate reductase DIC: Differential interference contrast Dlg: Discs large DMSO: Dimethyl sulfoxide DTT: Dithiothreitol xiv  DUF: Domain of unknown function EARP: Endosome-associated recycling protein  EE: Early endosome EEA: Early endosome antigen EH: Eps15 homology E-MAP: Epistatic mini-array profile ENTH: Epsin N-terminal homology ER: Endoplasmic reticulum ESCRT: Endosomal sorting complex required for transport FBE: FN3–BRCT of exomer FCHo: F-BAR domain only Fez: Fasciculation and elongation protein zeta FYCO: FYVE and Coiled-Coil Domain Containing FYVE: Fab-1, YGL023, Vps27 and EEA1 GAD: GAL4-activating domain GAE: Gamma adaptin ear GAP: Gtpase-activating protein GARP: Golgi-associated retrograde protein GBD: GAL4 DNA-binding domain GDI: GDP-dissociation inhibitor GEF: Guanine-nucleotide exchange factor GFP: Green fluorescent protein GGA: Golgi-localized, gamma-adaptin ear homology, Arf-binding GSS: Gfp-Snc1-Suc2 GTP: Guanosine triphosphate HA: Hemagglutinin HEAT: Huntingtin, elongation factor 3, PR65/A subunit of protein phosphatase A, TOR (target of rapamycin) HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIS: Histidine HIV: Human immunodeficiency virus HOPS: Homotypic fusion and vacuole protein sorting HPH: Hygromycin resistance gene xv  Hsc: Heat shock cognate Hyg B: Hygromycin B IgG: Immunoglobulin G IP: Immunoprecipitate JIP: C-Jun NH2-terminal kinase interacting protein LatA: Latrunculin-A LDL-R: Low-density lipoprotein receptor LE: Late endosome LEU: Leucine LPAT: Lysophospholipid acyltransferase MCL: Markov cluster algorithm MEDNIK: Mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia MHC-1: Major histocompatibility complex Mil1: Mu-interacting ligand MUSCLE: Multiple Sequence Comparison by Log-Expectation MVB: Multi-vesicular body N.S.: No significance NAT: Nourseothricin NECAP: Adaptin ear-binding coat-associated protein Nef: Negative effector factor NMR: Nuclear magnetic resonance OD600: Optical density at 600nm OE: Over-expressed ORF: Open reading frame PAGE: Polyacrylamide gel electrophoresis PAPLA: Phosphatidic acid phospholipase A PBS: Phosphate-buffered saline PC: Phosphatidylcholine PCR: Polymerase chain reaction PE: Phosphatidylethanolamine PH: Plekstrin homology PI: Phosphoinositide xvi  PI(3)P: Phosphatidylinositol 3-phosphate PI(3,4)P2: Phosphatidylinositol 3,4-bisphosphate PI(3,4,5)P3: Phosphatidylinositol 3,4,5-triphosphate PI(3,5)P2: Phosphatidylinositol 3,5-bisphosphate PI(4)P: Phosphatidylinositol 4-phosphate PI(4,5)P2: Phosphatidylinositol 4,5-bisphosphate PI(5)P: Phosphatidylinositol 5-phosphate PICALM : Phosphatidylinositol binding clathrin assembly protein PIPKIγ: Type Iγ phosphatidylinositol 4-phosphate 5-kinase  PLA1: Phospholipase A1 PM: Plasma membrane PMSF: Phenylmethylsulfonyl fluoride PS: Phosphatidylserine PtdIns: Phosphatidylinositol PX: Phox homology RE: Recycling endosome RFP: Red fluorescent protein S.E.M.: Standard error of the mean SD: Synthetic dextrose SDS: Sodium dodecyl sulfate SGA: Synthetic genomic array SM: Sec1/munc18 SNARE: Soluble NSF attachment protein receptor SNX: Sorting nexin SPG: Spastic paraplegia TCA: Trichloroacetic acid TdT: TdTomato fluorescent protein TGN: Trans-Golgi network TIRF: Total internal reflection fluorescence TMD: Transmembrane domain t-SNARE: Target SNARE URA: Uracil xvii  UV: Ultraviolet VEGF: Vascular endothelial growth factor VPS: Vacuolar protein sorting v-SNARE: Vesicle SNARE WB: Western blot WPB: Weibel-Palade body WT: Wild-type Y2H: Yeast two-hybrid  YPD: Yeast extract, peptone, dextrose  xviii  Acknowledgements Because I am writing this section last, it is difficult to summon the words to properly express my appreciation for all the people who have helped and supported me throughout, and I will doubtless miss many. I would first like to thank my advisor Elizabeth Conibear for her time and effort, and for being an inspiring writer, presenter and scientist. Her enthusiasm for science is infectious and encourages me to do better. I would also like to thank the experienced scientists from whom I have learned a lot about how to approach science. My committee members Phil Hieter and Bob Molday have been invaluable in their patience and input on my project and in writing numerous reference letters. I am grateful for the financial support from BC Children’s Hospital Research and the Canadian Institutes for Health Research that have helped me to pursue my research. Science is never done alone, and I appreciate the many collaborators who have helped my projects progress. Although I cannot mention them all by name I would especially like to thank Jennifer Hirst for her incredible energy and drive as a collaborator and for her understanding when other things have come up. Another special mention is Margaret “Scottie” Robinson for opportunities to collaborate, for discussions and for the review “Forty Years of Clathrin-coated Vesicles” which may be the single most engaging academic read of my past five years. The Conibear lab has been – and will continue to be – a great place to do science. This is largely because of the friendship of fun, outgoing people. I would especially like to thank Bjorn Bean for being a great scientific role-model, and an even better roommate and friend. I also appreciate conversations, discussions and friendship from Lauren Dalton, who slowly came to appreciate puns despite initially feeling nauseated by them. Mike Davey has worked to keep up with me on the pun-front and has been an endless provider of yeast constructs and technical expertise. Junior students Samantha Dziurdzik and Vaishnavi Sridhar have fooled me into feeling like a wise old owl, and Claire Fowler was a fantastic co-op student until she was co-opted into the Vps13 project.  Support from those outside the lab is also greatly appreciated. I would like to thank Nico Petch for maintaining our friendship since kindergarten and for hosting fun social gatherings. Lauren and Lane Dalton have been close friends and gaming buddies and have welcomed me into not only their home but their parents’ homes as well. Dhanya Sridhar has been my rock, and a teammate in handling whatever has come our way. I am particularly grateful to my parents, sister and grandmother for support and for sitting through my long-winded explanations of my topic, and would like to especially thank my parents for getting up at 2:00am to drive five hours and rescue me in Merritt when I hit a deer.   xix  Dedication         To my parents. 1  Chapter 1: Introduction 1.1 Introduction A hallmark of eukaryotic cells is compartmentalization into membrane-bound organelles, each playing a special role. The ability to isolate and control biochemical responses and protein signaling is extremely powerful. However, it necessitates a system for establishing and maintaining compartment identity, and for transferring materials between compartments. Many proteins only work at a particular cellular location, and the consequences of missorting can be disastrous. Transport intermediates known as vesicles, often bearing proteinaceous coats, are the main means of moving transmembrane and non-cytosolic protein cargo to different cellular destinations. Section 1.1.1 outlines the major coat-protein-dependent trafficking pathways in a generalized eukaryotic cell (Figure 1.1).   1.1.1 Overview of vesicular trafficking pathways  Endoplasmic reticulum and Golgi Transmembrane proteins are synthesized in the endoplasmic reticulum (ER), where they undergo initial modifications such as covalent addition of lipid (lipidation; Resh, 2013) or sugar groups (glycosylation; Breitling and Aebi, 2013). Proteins that pass quality control checks (Koenig and Ploegh, 2014; Briant et al., 2017) are sorted into COPII-coated vesicles for delivery to the Golgi (Zanetti et al., 2012). The related COPI complex mediates retrograde sorting between Golgi stacks and from the Golgi to the ER (Dodonova et al., 2015).  At the Golgi, proteins are further modified by glycosylation (Stanley, 2011) and sorted at the Trans-Golgi Network (TGN) to specific cellular locations. In many organisms the Golgi is the classic “flattened stack of pancakes” whereas in yeast the Golgi appears as scattered dots (Glick and Nakano, 2009). The mammalian TGN is an initial sorting station for vesicles destined for the cell surface (secretory pathways), early/recycling/late endosomes, or lysosomes. In the yeast Saccharomyces cerevisiae 2  the TGN is more difficult to define, and has recently been suggested to act as a hybrid TGN/early endosome/recycling endosome (Day et al., 2018).   Figure 1.1 Major coat-protein-dependent protein trafficking pathways in a generalized eukaryotic cell. Adaptor protein complexes, a main focus of this dissertation, are indicated with coloured circles. Abbreviations: ER: endoplasmic reticulum, TGN: trans-Golgi network, EE: Early endosome, LE: Late endosome, RE: recycling endosome, MVB: multi-vesicular body. Secretion Many different secretory pathways, involving vesicular and tubular transport carriers, are directed from the TGN to the plasma membrane (PM) although endosomes can also serve as intermediaries for TGN-PM transport (Futter et al., 1995; Leitinger et 3  al., 1995). Many unconventional secretory pathways have also been identified (reviewed in Rabouille, 2017; Dimou and Nickel, 2018). The secretory machinery is highly conserved from yeast to mammals (Bennett and Scheller, 1993), but one yeast-specific TGN-PM pathway of note involves the exomer complex (Wang et al., 2006), which is required for sorting a subset of specialized cargo.  Endocytosis PM proteins are internalized through a variety of mechanisms. Clathrin-mediated endocytosis (CME) using the AP-2 complex is the most studied (see reviews: Kaksonen and Roux, 2018; Goode et al., 2015; Elkin et al., 2016). Other machinery, including the TSET complex (Hirst et al., 2014), caveolin, endophilin, and flotilin (reviewed in Howes et al., 2010; Mayor et al., 2014; Doherty and McMahon, 2009; Grant and Donaldson, 2009) mediate clathrin-independent endocytic pathways. Many components of the endocytic trafficking machinery are highly conserved from yeast to humans, but their relative importance varies between yeast and mammalian cells (Kaksonen and Roux, 2018). Whereas clathrin and AP-2 are highly important for mammalian endocytosis, yeast AP-2 may not even bind clathrin (Yeung et al., 1999) and its role in yeast endocytosis is unclear (Goode et al., 2015). Instead, actin seems to be a more important driver of membrane deformation in yeast (Aghamohammadzadeh and Ayscough, 2009), perhaps due to high turgor pressure (Dmitrieff and Nédélec, 2015). Other components of the conserved endocytic machinery, including the yeast AP180 proteins (YAP1801/2), are more important clathrin adaptors than AP-2 in yeast (Maldonado-Báez et al., 2008). Trans-Golgi network and endosomes Sorting from TGN to endosomes is largely mediated by AP (Adaptor Protein) complexes and the related GGA (Golgi-localized, gamma-adaptin ear homology, Arf-binding) family. AP-1 and AP-4 sort from TGN to endosomes (Hirst et al., 2013; Nakatsu et al., 2014), GGA1 and GGA2 (there is also GGA3 in humans) to late endosomes (Costaguta et al., 2001; Hoya et al., 2017), and AP-3 to the vacuole/lysosomes (Newell-Litwa et al., 2007). Endosome-to-TGN recycling of proteins is thought to be mediated from early endosomes mainly by AP-1 (reviewed in Hinners and Tooze, 2003), but 4  GGAs have also been implicated in transport from endosomes to the TGN (Wahle et al., 2005) and to recycling endosomes (Herskowitz et al., 2012; Li et al., 2015). The retromer complex is the main mediator of late endosome-to-TGN recycling (Burd and Cullen, 2014), and AP-5 has recently proposed to act as a backup recycling mechanism (Hirst et al., 2018).  1.1.2 Endosomal maturation The compartments of the endosomal system (early endosomes, late endosomes and recycling endosomes in mammalian cells) are highly interconnected by trafficking and fusion processes (Grant and Donaldson, 2009; Goldenring, 2015; Kobayashi and Fukuda, 2013), and there is some debate as to how distinct any of these compartments truly are, especially in yeast (Day et al., 2018).  The endosomal network may perhaps be most conveniently thought of as undergoing a constant maturation process, with early endosomes transitioning to late endosomes, which become multi-vesicular bodies and ultimately fuse with the vacuole (in yeast) or lysosomes (in mammals) (Scott et al., 2014). Maturation is accompanied by changes in the pH of the compartment (Scott and Gruenberg, 2011), in phosphoinositide composition of the membrane (Wallroth and Haucke, 2018) and in recruitment of Rab GTPases (Casanova and Winckler, 2017; Elkin et al., 2016). The main endosomal maturation machinery are the CORVET, HOPS and ESCRT complexes (reviewed in Schöneberg et al., 2016; Christ et al., 2017; Balderhaar and Ungermann, 2013; Chou et al., 2016).   1.2 Establishment of cellular compartment identity  The recognition and manipulation of membrane characteristics plays a key role in protein trafficking. A large variety of protein domains have evolved to recognize biophysical features such as membrane charge, lipid packing defects, or curvature. Additionally, phosphoinositides and members of the Rab family of small GTPases specifically mark particular compartments and act as platforms for recruitment of trafficking proteins. Usually, proteins recognize multiple factors present on a cellular compartment, a process known as coincidence detection. 5  1.2.1 Membrane charge Many phospholipids contribute to the recognizable properties of the membrane by providing a positive or negative charge. The cytoplasmic face of the bilayer has a net negative charge due to the predominance of phosphatidylserine (PS) and phosphatidylinositol (PI). The net neutral phosphatidylethanolamine (PE) is also found primarily in the cytoplasmic leaflet, whereas the net neutral phosphatidylcholine (PC) and glycosphingolipids are more abundant on the exofacial side of the bilayer (Fadeel and Xue, 2009; Yamaji-Hasegawa and Tsujimoto, 2006). Transmembrane domains of proteins can sense membrane potential and charge, which can affect their topology and functions in membranes (Bogdanov et al., 2014). Many peripheral membrane proteins also have positively-charged regions that interact with negatively-charged phospholipids (Papayannopoulos et al., 2005; Yeung et al., 2006). Phospholipid flippases actively generate and maintain phospholipid asymmetry, which is defined as an increased abundance of specific phospholipids on one side of the membrane compared to the other (Sebastian et al., 2012). The action of flippases and scramblases alters local membrane charge and curvature and can promote trafficking reactions (Hua, 2002; Muthusamy et al., 2009; Xu et al., 2013). Phospholipase and lyso-phospholipid acyltransferase (LPAT) enzymes (reviewed in Brown et al., 2003; Ha et al., 2012) can greatly influence charge and other membrane conditions by virtue of their large-scale anabolism or catabolism of lipids in the membrane. 1.2.2 Lipid packing defects Lipid packing defects occur where the hydrophobic core of the bilayer, which is normally shielded from the cytosol, is revealed as a result of mismatch between the actual and preferred curvature of a membrane (Vanni et al., 2013). This can happen because of a high degree of curvature or presence of conical lipids in the membrane, the insertion of a helix or fatty acid group, or the scarcity of sphingolipids and cholesterol. Multiple protein domains, including the amphipathic lipid packing sensor (ALPS) motifs, recognize lipid packing defects (reviewed in Frost, 2011; Vanni et al., 6  2013; Antonny, 2011). The positively-charged face of the ALPS helix binds to negatively-charged phospholipid head groups while the hydrophobic face burrows into the membrane and interacts directly with fatty acid tails (Vanni et al., 2013). Insertion of ALPS motifs can stabilize curvature or even act as a wedge to increase it. 1.2.3 Curvature  Curvature can be a defining characteristic of membrane compartments. For instance, the PM is largely flat, whereas the small size of transport vesicles makes them naturally highly curved. Rather than being a rigid characteristic of a compartment, curvature can be altered through changes in the composition of lipids or sterols in the membrane, or binding to peripheral membrane proteins. Recognition of curvature can go hand-in-hand with remodeling, as is the case for long, banana-shaped BIN/amphiphysin/Rvs167 (BAR) domains such as those of the retromer complex (Seaman, 2012). Some proteins, such as dynamin, prefer to oligomerize on membranes with a particular membrane curvature (Antonny, 2011) and can stabilize or favour a particular membrane state.  Phospholipid flippases influence membrane curvature in two ways. First, some have a preference for lyso-phospholipids (Baldridge et al., 2013), which are phospholipids with one acyl chain removed. Selective flipping will increase the number of such conical lipids in one leaflet, bending the membrane. Second, flippases force more material into one leaflet of the membrane than the other, and according to the bilayer couple hypothesis (Singer and Oster, 1992) this will cause curvature of the membrane towards the side with higher bulk. 1.2.4 Phosphoinositides Phosphoinositides (PI) are phosphorylated forms of phosphatidylinositol (PtdIns) and are critical identifiers of a compartment, despite constituting only around 1% of phospholipids in the cell (Lemmon, 2008). PtdIns can be phosphorylated at any of the 3, 4 or 5-position hydroxyl groups on the inositol ring, resulting in seven possible distinct PI species (Wallroth and Haucke, 2018). Compartments marked by a major PI species (Figure 1.2) also contain smaller amounts of other PIs.  7   Figure 1.2 Major phosphoinositide (PI) species markers for each cellular compartment. Modified from (Schink et al., 2016; Wallroth and Haucke, 2018). Abbreviations: ER: endoplasmic reticulum, TGN: trans-Golgi network, EE: Early endosome, LE: Late endosome, RE: recycling endosome, MVB: multi-vesicular body. PIs mark distinct compartments The Golgi apparatus and secretory granules are marked by PI(4)P. In higher eukaryotes, the PM uses PI(4,5)P2, PI(3,4)P2 and PI(3,4,5)P3 as spatial and temporal cues for different exo- and endocytic duties (Wallroth and Haucke, 2018; Schink et al., 2016). For example, endocytic patches are marked by PI(4,5)P2. Yeast lack the ability to synthesize PI(3,4)P2 and PI(3,4,5)P3 (Liu and Bankaitis, 2010). The endosomal 8  system is largely marked by PI(3)P, and conversion to PI(3,5)P2 is a hallmark of late endosomes and ultimately the degradative pathway (vacuole/lysosomes) (Wallroth and Haucke, 2018). PI(3,4,5)P3 has also been associated with recycling endosomes and seems to be needed for trafficking from this compartment (Fields et al., 2010).  The ER is not itself identified by a particular phosphoinositide species, although it plays considerable roles in mediating PI conversion and levels in other compartments through membrane contact sites (Raiborg et al., 2015b; Elbaz and Schuldiner, 2011; Prinz, 2014). Some localized PI synthesis occurs at the ER: for example, PI(3)P is needed in formation of phagophores as a preliminary step in autophagy (Wallroth and Haucke, 2018).  PI conversion PI species are interconverted by a number of PI phosphatases and kinases, some acting constitutively and some in a very targeted manner such as for nutrient signaling and organelle positioning (Wallroth and Haucke, 2018). Many coat proteins incorporate specific PI-modifying enzymes for localized synthesis or degradation. In turn, progression between the various PIs can recruit different machinery. For example, a wave of PI(4)P synthesis has been proposed to underlie the sequential recruitment of GGA and AP-1 adaptors at the TGN (Daboussi et al., 2012), with direct binding between Gga2 and the PI-4-kinase Pik1 increasing accumulation of PI(4)P and favouring AP-1 recruitment (Daboussi et al., 2017). Recognition of PIs by protein domains Unsurprisingly, a variety of protein domains have evolved to recognize particular PIs. Binding can be broad or highly specific: plekstrin homology (PH) domains are often not highly selective in binding a particular species of PI , while Phox homology (PX) and FYVE domains are largely specific for PI(3)P (Lemmon, 2008). Many of these domains bind with fairly low affinity, necessitating the presence of multiple PI-binding domains within the protein or of coincidence detection mechanisms to recognize other characteristics of the membrane (Vonkova et al., 2015). ENTH (Epsin N-terminal homology) and ANTH (AP180 N-terminal homology) domains differentially bind PI(4,5)P2 or other PIs but the nature of the ENTH amphipathic helix also allows insertion 9  into and deformation of the membrane and recognition of lipid packing defects (McMahon and Gallop, 2005). 1.2.5 Small GTPases Small Rab GTPase proteins decorate cellular compartments and act as platforms for recruitment of many trafficking proteins (Pfeffer, 2017; Wandinger-Ness and Zerial, 2014). There are almost 70 different Rab proteins in mammalian cells (Wandinger-Ness and Zerial, 2014), many of which display overlapping and distinct functions. The most-recognized Rabs in mammalian cells are Rab5, Rab4 and Rab11, which mainly function at early (Nielsen et al., 1999) and recycling endosomes (Kobayashi and Fukuda, 2013; Takahashi et al., 2012) and autophagosomes (Szatmari et al., 2014), and Rab7 and Rab9, which work at late endosomes (Vanlandingham and Ceresa, 2009). Sequential recruitment and activity of Rabs (Rab cascades) underlies many trafficking events. For example, Rab5-to-Rab7 conversion is a hallmark of early to late endosome progression (Elkin et al., 2016), and intermediate concentrations of Rab5 and Rab7 are themselves recognized by the endosomal maturation machinery.  A prenylated Rab protein reversibly associates with membranes. In a GTP-bound state, Rabs are active on the membrane and bind a variety of effector proteins. The intrinsic GTPase activity of the Rab is modulated by the competing action of guanine-nucleotide exchange factors (GEFs) that promote Rab activation and GTPase-activating proteins (GAPs) that promote GTP hydrolysis and Rab inactivation (Wandinger-Ness and Zerial, 2014). GDP-Dissociation Inhibitors (GDIs) can extract the Rab from the membrane and keep it in a soluble, inactive GDP-bound state in the cytosol. Many trafficking factors either bind to Rabs themselves or to their regulators to affect vesicle transport processes. 1.3 Vesicle transport Coated vesicles: a common theme No matter the compartment, vesicle coats help connect cargo to membrane deformation. Conceptually, various coated-vesicle machineries are remarkably similar: a 10  cargo-selective component connects to a membrane-deforming coat/scaffolding component. The AP complexes, GGAs, COPI and TSET are all thought to share a common ancestor (Hirst et al., 2014), and they also bear high-level resemblance to the COPII, exomer and retromer coats. The main cargo-selective and scaffolding components of each coat are summarized in Table 1.1. Since the underlying principles are so fundamental, studies on one complex can often inform our understanding of the others. Table 1.1 Major coated-vesicle machinery in eukaryotic cells can be divided into cargo-selective and scaffolding components. Abbreviations: TGN: Trans-Golgi network, EE: early endosome, RE: recycling endosome, PM: plasma membrane, Vac: vacuole/lysosomes, ER: endoplasmic reticulum. Complex Cargo selection Coat/scaffold Main pathway AP-1 AP-1 heterotetramer Clathrin TGN-EE, EE/RE-PM AP-2 AP-2 heterotetramer Clathrin Endocytosis AP-3 AP-3 heterotetramer Clathrin/Vps41? TGN-Vac AP-4 AP-4 heterotetramer Unknown TGN-EE AP-5 AP-5 heterotetramer SPG11,15 LE-TGN GGAs GGA1 or GGA2 (or GGA3) Clathrin TGN-LE Exomer Any two of Chs6, Bch2, Bch1, Bud7 Chs5 TGN-PM Retromer Vps26,29,35  Vps5,17 (Snx1,2 in humans*) LE-TGN COPI β-COP, δ-COP, γ-COP,  ζ-COP (F subcomplex) ε-COP, α-COP, β’-COP (B subcomplex) Golgi-ER COPII Sec23,24 of COPII Sec13,31 of COPII ER-Golgi TSET TPLATE, TSAUCER, TCUP, TSPOON TTRAY1,2 Endocytosis *These Snx proteins may also directly bind cargo (Kvainickas et al., 2017; Simonetti et al., 2017). Overview of vesicle transport Vesicle transport processes all share the same set of steps required to move cargo from one compartment to another. I will divide the process into four steps (Figure 1.3): 1) initiation and assembly of the trafficking machinery, including cargo binding 2) budding and scission of the vesicle 3) movement to the destination compartment 4) docking and fusion with the target membrane. I will focus on AP-mediated clathrin-coated vesicle (CCV) trafficking since it is the most relevant to this dissertation.  11   Figure 1.3 Simplified overview of vesicle transport steps shown for AP-1-mediated clathrin-coated vesicle trafficking. AP complexes are recruited to the membrane by coincident binding to various recognition factors, concentrate cargo into a patch on the membrane, and assist budding and vesicle formation. The vesicle is moved to the target membrane and then fuses, delivering the cargo. 1.3.1 Initiation and assembly A number of factors “prime” a membrane for assembly of trafficking machinery, and the coincident detection of multiple signals favours a successful reaction. A nuanced model of CCV endocytosis suggests that a “pioneer module” of trafficking proteins is initially recruited by coordinated binding to PIs in the membrane, small GTPases and cargo. This pioneer module performs cargo selection, scaffolding and initial membrane deformation roles. 12  Recruitment by recognition of membrane properties Membrane characteristics are highly important in recruiting initial trafficking components. For instance, AP-2 and AP-1 have binding sites for PI(4,5)P2 and PI(4)P respectively (Rohde et al., 2002; Heldwein et al., 2004), while the AP-1B isoform of AP-1 is recruited to recycling endosomes by binding PI(3,4,5)P3 (Fields et al., 2010). Other initiation proteins at the PM, such as CALM (Clathrin assembly lymphoid myeloid leukemia protein), FCHo1 and FCHo2 (F-BAR domain only proteins, homologous to yeast Syp1), recognize both curvature and charge of membrane phospholipids. The epsins (epsin1-3, epsinR in humans, Ent1-5 in yeast) recognize curvature (Madsen et al., 2010; Capraro et al., 2010) and phosphoinositides (De Camilli et al., 2002) through ENTH domains. There is some debate as to which of the above initiation proteins really arrives “first” (Cocucci et al., 2012; Henne et al., 2010) but they are considered part of the initiation machinery. PI-modifying proteins that bind initiation machinery can evoke a feed-forward mechanism to increase recruitment. AP-2 stimulates formation of PI(4,5)P2 by binding the PI(4)P 5-kinase PIPKI (Kahlfeldt et al., 2010; Krauss et al., 2006; Bairstow et al., 2006), whereas AP-3 binding to PI4KIIα and GGA2 binding to PI4KIIIβ may create localized PI(4)P (Craige et al., 2008; Daboussi et al., 2017). Recruitment by Arf GTPases Small GTPases of the ADP-ribosylation factor (ARF) family are critical regulators of many trafficking reactions in the late endomembrane system (Jackson and Bouvet, 2014; Donaldson and Jackson, 2011), and, similarly to Rabs, are regulated by specific ARF-GEFs and ARF-GAPs. There are five Arf proteins and more than 20 Arf-like proteins in humans, which exhibit redundant and distinct functions (Guo et al., 2014). Arf1 was one of the first identified factors necessary for AP-1 recruitment to membranes (Traub, 1993; Zhu et al., 1998). Activated Arf1-GTP exposes a myristoylated alpha-helix that inserts into membranes (Liu et al., 2009) and allows Arf1 to act as a platform for recruitment. Arf1 binds and recruits such diverse sorting machinery as exomer, GGAs, AP-1, AP-3, AP-4, and COPI (Guo et al., 2014; Cherfils, 2014), suggesting that it is a common initiator of Golgi network sorting. Coincidence detection and binding of other proteins is absolutely necessary for specificity of sorting reactions in this case. 13  Binding to cargo Binding to signals present in cargo proteins also increases the membrane affinity of sorting complexes such as AP-1 (Ghosh and Kornfeld, 2003; Lee et al., 2008). Usually short linear peptide motifs present in the cytoplasmic regions of transmembrane proteins can be recognized by the main cargo-selective proteins in the coat (e.g. AP complexes) or by accessory proteins that bind to the main cargo-selective complex and expand the repertoire of possible sorting signals, such as the autosomal recessive hypercholesterolemia (ARH) protein which is an adaptor for low-density lipoprotein receptor (He et al., 2002). Interactions between cargo and sorting complexes are relatively weak and transient, but binding cargo increases the odds of a successful vesicle trafficking event.  Coincidence detection ensures that a productive vesicle will form, containing cargo and the correct machinery. Initiation proteins often bind each other (Merrifield and Kaksonen, 2014), increasing their concentration in a patch of membrane and promoting the assembly process. Concurrently, the adaptor component (for example, AP complexes, or the γ-COP and ζ-COP components of COPI) of the vesicle initiation machinery recruits the coat proteins (clathrin or α-COP and β-COP for the previous example) that help in scaffolding and membrane deformation. 1.3.2 Budding and scission Budding The recruitment and clustering of many components of the coated-vesicle machinery causes membrane deformation. The coat protein clathrin is a well-recognized promoter of membrane deformation (Hinrichsen et al., 2006; Kaksonen and Roux, 2018), as its structure favours its polymerization into baskets. Clathrin polymerization is also favoured by membrane curvature (Pucadyil and Holkar, 2016), allowing for a self-reinforcing cascade. Crowding effects and insertion of amphipathic helices from many CCV proteins also contribute to membrane deformation and curvature (Snead et al., 2017; Johannes et al., 2014). Active remodeling of the biophysical characteristics discussed in Section 1.2 by flippases and phospholipases 14  also plays a role. The action of phospholipid flippases is necessary for CCV formation (Muthusamy et al., 2009; Liu et al., 2008). It was recently suggested that conversion from PI(4)P – a cylindrical lipid – to diacylglycerol (DAG) – a conical lipid – underlies transition from a flat to curved membrane for AP-1 (Anitei et al., 2017). Scission Coat assembly causes recruitment of actin nucleation factors including ARP2/3, providing a pulling force that assists scission (Anitei and Hoflack, 2011; Kaksonen and Roux, 2018). At the PM dynamin polymerizes at the neck and pinches off the vesicle (Cocucci et al., 2014), although dynamin may not be required for scission of AP-1 vesicles (Kural et al., 2012). In yeast, the contribution of the dynamin homolog Vps1 is less clear, and it may have a regulatory rather than essential role in scission (Kaksonen and Roux, 2018) with actin as the key driver.  1.3.3 Uncoating and movement Uncoating Once the vesicle has formed and left the donor compartment, many components of the coat have been observed to quickly fall off (Merrifield and Kaksonen, 2014; Ehrlich et al., 2004). In the traditional view of clathrin-coated-vesicle uncoating, auxilin accessory proteins recruit the chaperone Hsc70 to rearrange and destabilize clathrin in the late stages of budding and shortly after scission (Eisenberg and Greene, 2007; Cremona, 2001; Massol et al., 2006). Phospholipid-remodeling CCV accessory proteins such as the PI-phosphatase synaptojanin also change the lipid environment to favour dissociation of coat proteins from the membrane (see Kaksonen and Roux, 2018). There is some uncertainty about how many of the coat components remain on the vesicle for purposes of motor adaptation or compartment selection; for example, some coat components have been observed to interact with the tethering proteins that link vesicle and target membranes and are responsible for vesicle fusion (Trahey and Hay, 2010; Schroeter et al., 2016).  15  Movement Short-range movement of vesicles to the destination compartment can occur by diffusion since the distances involved are not exceptionally great. Accordingly, yeast cells can take advantage of diffusion since they are relatively small. Broadly, in mammalian cells actin-based systems, which can employ the myosin motor protein, are used for short-range transport. Reorganization of the actin network depends on phosphoinositides and the ARP2/3 complex (Ueno et al., 2011; Goley and Welch, 2006). For long-range vesicle trafficking cells use microtubule-based systems (Scheffler and Tran, 2012), involving connection to dynein motor proteins walking toward the minus-end of microtubules and kinesin motor proteins walking (usually) towards the plus-end.  Motor proteins either bind directly to cargo or require cargo adaptors to attach to vesicles (Akhmanova and Hammer, 2010). Full activation and movement of the motor protein is highly regulated by binding to multi-protein complexes and by signals such as phosphorylation. For example, kinesin-1 auto-inhibition by its tail region is only fully relieved by coincident binding of the scaffolding protein JIP1 and the cargo adaptor Fez1 (Blasius et al., 2007). The overall movement of vesicles or organelles is determined by the cumulative actions of different motors: multiple dyneins and kinesins can be attached to a single vesicle and a regulated tug-of-war determines the overall direction of its transport (Maday et al., 2014). 1.3.4 Docking and fusion Docking A vesicle docks with its target compartment through a multi-layered recognition process (Gillingham, 2018; Spang, 2016). First, “tethers” sample the area around the compartment, looking for vesicles destined to dock with the compartment and bring them close. Coiled-coil tethers include the Golgins at the Golgi and EEA1 at early endosomes. Multi-protein tethering complexes act at many different compartments, and include the CORVET and HOPS complexes at endosomes/lysosomes (Seals et al., 2000; Wurmser et al., 2000; Peplowska et al., 2007; reviewed in Balderhaar and 16  Ungermann, 2013), the GARP complex at the TGN (Conibear and Stevens, 2000; reviewed in Bonifacino and Hierro, 2011) and the EARP complex at endosomes (Schindler et al., 2015).  Fusion Fusion between two membranes is mediated by transmembrane proteins known as SNAREs (soluble NSF attachment protein receptors). Complementary SNAREs on vesicle (v-SNAREs) and target (t-SNAREs) membranes form a complex to overcome the huge energy barrier of membrane fusion (reviewed in Yoon and Munson, 2018). Three or four SNARE proteins form a bundle and bring the vesicle and target membranes close together, ultimately leading to fusion. The formation of the SNARE complex is regulated by Sec1/Munc18 (SM) and tethering proteins and can be triggered by specific cellular signals.  1.4 Adaptor protein complexes Adaptor protein complexes act as hubs in coated-vesicle trafficking by interacting with the membrane, cargo proteins, clathrin and regulatory/accessory proteins. AP complexes are heterotetramers consisting of two large (γ,α,δ,ε or ζ and β1-5, ~100kDa each), one medium (µ, ~50kDa) and one small (σ, ~20kDa) adaptin subunit, each with a subset of defined functions. The AP-1 complex consists of γ, β1, µ1 and σ1 subunits. AP complex subunit isoforms, such as µ1A and µ1B, may be expressed in a tissue-specific manner (Nakatsu et al., 2014) and differentially incorporated into the complex (Mattera et al., 2011) (Figure 1.4).  The existence of isoforms of AP complex subunits is thought to expand the repertoire of possible functions and allow for more diverse trafficking pathways. The AP-1 complex is the most variable, with two γ isoforms (γ1/γ2), two µ isoforms (µ1A/µ1B) and three σ isoforms (σ1A/σ1B/σ1C). Of the twelve AP-1 variants that are theoretically possible by combinatorial assembly of large, medium and small subunits, eleven have been shown experimentally (Mattera et al., 2011) in vitro. The composition of the suite of AP complexes in mammalian cells is shown in Figure 1.4. Many studies do not note 17  the exact subunit composition of the AP complex they are studying so it is sometimes difficult to ascribe functions to a particular AP subcomplex.  The following sections focus on the structures and main functions of each type of subunit with a special emphasis on AP-1.  Figure 1.4 Subunit composition of the five AP complexes found in mammals.  Alternate subunits (differentially expressed in particular tissues) are given in grey. 1.4.1 γ/α/δ/ε/ζ and β subunits The large AP subunits contain binding sites for phospholipids, Arf1, clathrin and other coat proteins. Each subunit is arranged into two main subdomains separated by a long, flexible linker (a “hinge”): the core region and “ear” (appendage) region.  18  Core subdomain The core provides a large interaction interface, and is often shown as a curved “L” shape, cradling the µ and σ subunits (Owen et al., 2004; Faini et al., 2013; Robinson, 2015). It largely consists of HEAT repeats, which are alpha-solenoid structures present in a variety of scaffolding proteins. HEAT repeats were named after the set of proteins in which they were first found – huntingtin (involved in Huntington’s disease), elongation factor 3, PR65/A subunit of protein phosphatase A, and the TOR (target of rapamycin (Andrade et al., 2001) –  and are often found in proteins with trafficking roles.  Besides helping to determine the shape and intra-complex interactions of AP complexes, large subunits contribute to membrane recognition. Regions near the N-termini of the core domains have been identified as phosphoinositide binding sites in AP-2 α (Collins et al., 2002) and AP-1 γ (Heldwein et al., 2004). An interface between the γ and σ subunits is the binding site for dileucine motifs (DxxLL), one of the two canonical sorting signals recognized by AP complexes (Mattera et al., 2011; Doray et al., 2007). An Arf1-binding site involved in allosteric activation of AP-1 sits about halfway along the γ core structure (Ren et al., 2013). Arf1 can bind to two AP-1 complexes at once through interactions with γ and β subunits, providing a convenient mechanism for concentrating different AP-1 complexes together by essentially “cross-linking” them in a reversible way. Arf6 may similarly regulate AP-2 recruitment (Krauss et al., 2003; Paleotti et al., 2005; Poupart et al., 2007) but this relationship is less clear.  Hinge region  The hinge regions contain binding sites for clathrin and act as spacers between the core and ear domains. AP-1 γ has been reported to contain two copies of a variant clathrin-binding box (Doray and Kornfeld, 2001) but the β subunits are commonly viewed as the clathrin-binding portions of AP complexes. The β1, β2 and β3 hinges all possess clathrin-box motifs (Lundmark and Carlsson, 2002). Despite evidence suggesting that AP-3 can bind clathrin in vitro (Drake et al., 2000; ter Haar et al., 2000; Dell’Angelica et al., 1998) this does not seem to be the case in vivo (Robinson, 2015; Edeling et al., 2006; Zlatic et al., 2013) 19  Appendage (ear) subdomain Adaptin ear domains bind AP accessory proteins bearing a variety of motifs. While β ears can recruit some accessory proteins (Lundmark and Carlsson, 2002) the traditional region for binding is the non-β appendage; α and γ have been better characterized. Mainly bulky/hydrophobic residues, separated by a defined number of amino acids, fit into pockets on the ear domain (Brett et al., 2002; Ritter et al., 2003; Jha et al., 2004). The crystal structures of AP-2 α and β2 reveal a bipartite structure that can be divided into an N-terminal “sandwich” subdomain of β-strands and a C-terminal “platform” subdomain (Praefcke et al., 2004; Brett et al., 2002; Owen et al., 1999; Traub et al., 1999) (domains shown in Figure 1.3).  The AP2 α-appendage binds DP[F/W] and FXDXF motifs through an overlapping site in the platform subdomain, and WXX[F/W]x[D/E] through the sandwich subdomain (Edeling et al., 2006; Praefcke et al., 2004). The β2 ear also recognizes DP[F/W] motifs. The AP-1 γ ear (also called the gamma adaptin ear or GAE) only has the sandwich subdomain (Nogi et al., 2002; Kent et al., 2002), and binds variants of ΨG[P/D/E][Ψ/L/M] (ΨxxΨ) motifs, where Ψ is an aromatic residue. The AP-4 accessory protein tepsin binds the β4 ear (which by NMR only consists of the platform subdomain) through a [G/S]LFXG[M/L]X[L/V] motif and ε ear through S[A/V]F[S/A]FLN (Mattera et al., 2015; Frazier et al., 2016). Interactions between the ear and individual ear-binding motifs are relatively weak, and many accessory proteins have multiple ear-binding motifs to increase the effective affinity of interaction. AP-5 seems to lack appendage domains (Hirst et al., 2011). Many other trafficking proteins contain a variation on the appendage domain structure. GGA proteins were identified and named because of their homology to the GAE domain (Hirst et al., 2000; Boman et al., 2000; Dell’Angelica et al., 2000). Similar structures are found in the FBE domain of the Chs5 component of the exomer complex (Paczkowski et al., 2012) and the appendages of γ-COP (Watson et al., 2004; Hoffman et al., 2003) and β-COP (De Regis et al., 2008) of COPI, suggesting a common strategy for recruiting accessory proteins for coated-vesicle trafficking. 20  Exchange of large subunits to form alternate complexes Exchange of the “typical” γ1 adaptin for the γ2 adaptin variant in mammalian cells imparts distinct functions (Tavares et al., 2017) to the AP complex despite 60% amino acid identity between the two. The γ2 adaptin complex is likely Arf1-independent (Takatsu et al., 1998), interacts with ubiquitin (Rost et al., 2006) and seems to function in the multi-vesicular body (MVB) pathway (Rost et al., 2008). Although this may seem a large departure from known γ subunit roles, it is consistent with the functions of large subunits in membrane recruitment and cargo selection. AP-2 also has variants based on inclusion of the α adaptin subunits, αA and αC, which exhibit differential expression patterns and localization (Ball et al., 1995; Motley et al., 2006). 1.4.2 µ subunits The medium subunits participate in complex activation, membrane recruitment and cargo recognition. Phosphorylation of the µ subunit increases its affinity for cargo (Ricotta et al., 2002), and may support a conformational change resulting in an “activated” AP complex (Jackson et al., 2010). Binding cargo and phosphoinositides helps stabilize AP complexes on membranes (see section 1.3.1). A basic patch presumed to be involved in PI binding was identified in the crystal structure of the µ subunit AP-2 (Collins et al., 2002), and a similar site may exist in other µ subunits as well – µ1B seems to be able to bind PI(3,4,5)P3 for recruitment to recycling endosomes (Fields et al., 2010). In addition to PI-binding capabilities, many µ domains interact with PI-modifying enzymes (Ling et al., 2007; Kahlfeldt et al., 2010; Krauss et al., 2006; Craige et al., 2008; Fields et al., 2010) and may induce a positive feedback loop for recruitment of more AP complexes. Cargo recognition Tyrosine-based motifs (YxxΦ, where Φ is a bulky, hydrophobic amino acid) are the best-characterized signals recognized by µ. The binding region operates similar to binding of accessory proteins to the GAE, with the Y and Φ residues fitting into complementary pockets (Owen, 1998). A number of other cargo-binding sites on the µ subunit have been identified, including sites that recognize the FDNPVY signal of the 21  low-density lipoprotein receptor (Boll et al., 2002), “acidic cluster” motifs (Negredo et al., 2017) such as those found on carboxypeptidase D and the cation-independent mannose 6-phosphate receptor, and basic motifs such as those found on the polymeric immunoglobulin receptor (Orzech et al., 1999), L-selectin (Dib et al., 2017), and the yeast protein Ste13 (Foote and Nothwehr, 2006).  Similar to the case with the large subunit appendage domain, a µ homology domain (µHD) is present in other proteins with a trafficking function: the stonin and muniscin families (Stonin1/2, FCHO1/2, SGIP1 and yeast Syp1). These proteins interact with AP complexes themselves (Walther et al., 2004) and may work as cargo adaptors to expand the repertoire of sorting signals or number of cargo-binding sites in addition to other functions.  Exchange of medium subunits to form alternate complexes Whether exchange of µ subunits generates functionally distinct isoforms is debated. AP-1A and AP-1B, which contain µ1A and µ1B, respectively, are the most studied µ variant complexes. Considerable work suggests µ1B enables sorting at recycling endosomes (Fölsch et al., 2003) and allows binding to PI(3,4,5)P3 (Fields et al., 2010), PI-modifying proteins (Ling et al., 2007), and Rab8 (Ang et al., 2003). However, µ1A and µ1B bind similar regulatory proteins in vitro (Ling et al., 2007; Shteyn et al., 2011) and some studies have shown they co-localize (Guo et al., 2013).  More recent studies have agreed that µ1A and µ1B enable recognition of different cargo (Sugimoto, 2002; Guo et al., 2013) but suggest they largely overlap in function and can compensate for each other (Guo et al., 2013; Gravotta et al., 2012). Data from model organisms are seemingly contradictory, with AP-1 µ subunits suggested to be redundant in Arabidopsis (Park et al., 2013) and budding yeast (Stepp et al., 1995; Liu et al., 2008), and potentially distinct in zebrafish (Gariano et al., 2014) and worms (Shim et al., 2000). Further studies are needed to resolve this question.  1.4.3 σ subunits The small subunits were initially thought to only contribute to stability of the complex but are now also seen as playing a role in dileucine motif ([D/E]xxxL[L/I]) 22  recognition. Dileucine motifs bind to an interface between the γ and σ subunits in AP-1 (collectively termed the γ/σ hemicomplex) (Doray et al., 2007; Janvier et al., 2003), with the two leucine residues binding in hydrophobic pockets on the σ subunit (Kelly et al., 2008) and the variable (x) residues contributing to specificity. Alternative assortments of AP subunits in a complex can result in different affinities for dileucine motifs (Mattera et al., 2011), allowing for a fine-tuning of cargo sorting. It has been suggested that σ subunits also bind to proteins that affect the function of the AP complex or CCV formation (Schmid and McMahon, 2007). Accordingly, σ1A and σ1B have been suggested to competitively bind ArfGAP1 and regulate endosomal maturation (Candiello et al., 2016). The σ1A, σ1B and σ1C subunits display tissue-specific expression (Glyvuk et al., 2010) and distinct phenotypes upon knockdown. 1.4.4 AP hemicomplexes hypothesis  The consensus view of AP complexes is of stable, obligate heterotetramers. Occasionally, however, studies have found similarities between the mutant phenotypes exhibited by the γ/σ subunits and between the β/µ subunits (Ma et al., 2009; Gu et al., 2013; Deneka et al., 2003). One explanation for this discrepancy is that the subunits may sometimes work as dimeric “hemicomplexes”. Indeed, purified γ/σ hemicomplex can still bind dileucine-based signals (Doray et al., 2007) and mutation of one hemicomplex does not abolish all AP complex functions (Gu et al., 2013). Complicating matters, some subunits may be able to substitute for others in vivo: Silencing β2 gene expression was found to result in less severe defects than knockdown of α because β1 was able to incorporate in the AP-2 complex and compensate (Keyel et al., 2008). Studies using AP subunit knockdowns should at minimum acknowledge the possibility of hemicomplex functioning and not assume all AP complex functions are abolished. 1.4.5 AP complex activation The transition from cytosolic, inactivated AP complex to active, membrane-bound sorting adaptor has been worked out in considerable detail. AP complexes naturally adopt a “closed” conformation, with the µ subunit tucked into the core of the complex 23  and both tyrosine-binding and dileucine-binding sites blocked by the β subunit (Collins et al., 2002). In this state the clathrin-binding site on the β subunit is also occluded by an auto-inhibitory mechanism with the β-hinge bound back into the core (Kelly et al., 2014). Binding to membranes (especially for AP-2; Jackson et al., 2010) or to Arf1 (for AP-1; Ren et al., 2013) causes a large-scale conformational change into the “open” conformation. The µ subunit swings outwards, revealing cargo-binding sites and the β-hinge that recruits clathrin. Phosphorylation of the µ subunit (Ghosh and Kornfeld, 2003) and binding to cargo stabilizes the open conformation (Lee et al., 2008; Canagarajah et al., 2013; Ren et al., 2013). Phosphoinositides continue to be important in membrane stabilization of AP complexes past initiation (Kadlecova et al., 2017). A variety of regulatory proteins can act on AP complexes to favour active or inactive states, FCho proteins (Hollopeter et al., 2014) and NECAPs (Beacham et al., 2018), respectively. 1.4.6 AP-1 dysfunction in disease The importance of AP-1-mediated trafficking is underscored by the drastic effects of its dysfunction. AP-1 null mutants in mammals are embryonic lethal (Ohno, 2006), with the exception of single µ null mutants where there is seemingly some ability of µ1A or µ1B to compensate for each other (Eskelinen et al., 2002). Mutations in individual AP-1 subunits often manifest in tissue-specific ways due to differential expression patterns. Reduced expression of the epithelial-specific µ1B has been linked to Crohn’s disease, an inflammatory disease of the intestines, and intestinal tumours (Park and Guo, 2014). Mutations in the AP1S1 gene encoding σ1A cause MEDNIK (mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia) syndrome (Martinelli and Dionisi-Vici, 2014), while mutations in neuronally-restricted AP1S2 (σ1B) cause neurological conditions including Fried’s disease (Saillour et al., 2007) and Pettigrew syndrome (Cacciagli et al., 2014). Mutations in epithelially-expressed AP1S3 (σ1C) cause the autoinflammatory skin disorder pustular psoriasis (Mahil et al., 2016; Setta-Kaffetzi et al., 2014). Viruses including HIV-1 and HIV-2 hijack both AP-2 and AP-1 for entry into the cell and subsequent trafficking (Collins and Collins, 2014). The HIV-1 Nef (negative effector factor) protein disrupts the immune response by stabilizing AP-1 interaction with 24  MHC-1 (Wonderlich et al., 2008), ultimately leading to increased degradation of MHC-1 through the lysosomal pathway (Roeth et al., 2004). Nef carries a canonical dileucine motif that enables its recognition and trafficking by AP-1 (reviewed in Wonderlich et al., 2011) and AP-2 (Chaudhuri et al., 2007), and can induce trimerization and activation of Arf1-AP-1 complexes (Shen et al., 2015).  1.5 AP complex regulators/accessory proteins High-throughput approaches have so far identified over 100 proteins associated with clathrin-coated vesicles (Robinson, 2015; Borner et al., 2006, 2012, Hirst et al., 2012, 2015), although these proteins are not all present on a vesicle at the same time. Many regulators that help in proper formation and budding of CCVs are excluded from the final vesicle (Kirchhausen et al., 2014) and may not have been identified through these approaches. The AP-2 “accessory proteome” is the most well-characterized, with over 20 proteins found to bind the ear domains (Praefcke et al., 2004; Lundmark and Carlsson, 2002; Schmid and McMahon, 2007). Fewer regulators have been found for AP-1, and many of those may have a certain level of promiscuity and also work with AP-2 (e.g. NECAP proteins) (Miller et al., 2003; Mattera et al., 2004; Ritter et al., 2004). There is only one known ear-binding protein (tepsin) for AP-4 (Mattera et al., 2015; Frazier et al., 2016) and none yet for AP-3 or AP-5.  1.5.1 AP accessory protein functions Most accessory proteins are modular and can perform various functions through their different domains, so it is difficult to categorize them according to one specific role. For a rather comprehensive list of accessory proteins involved in endocytosis, their functions and binding domains/motifs, see (McPherson and Ritter, 2005). Some key roles of CCV accessory proteins are given in the examples below: Cargo-selection AP complexes recognize tyrosine-based and dileucine-based motifs on many proteins but cargo adaptors can help to expand the repertoire of sorting signals. For example, the stonins (Stonin-1/2) and muniscins (FCho1, FCho2, and SGIP1 in 25  mammals, Syp1 in yeast) contain a µ-homology domain (µHD) related to the µ subunit of AP complexes involved in cargo recognition. Epsins can also participate in selection of poly-ubiquitinated proteins (Hawryluk et al., 2006), and ENTH and ANTH domain-containing proteins mediate sorting of SNARE proteins (Chidambaram et al., 2004, 2008; Maritzen et al., 2012a). For example, EpsinR acts an adaptor for recycling the v-SNARE Vti1b to the TGN (Hirst et al., 2004). Recruitment/scaffolding AP complexes can function at many different cellular locations, and require coincidence detection and binding to many different factors to get there. Rabs are important markers of compartment identity, and provide an extra layer of coincidence detection and regulation. Rabaptin-5α links AP-1 to Rab4 and may provide membrane targeting to recycling endosomes (Deneka et al., 2003). Similarly, the scaffolding protein Discs large 1 (Dlg1) in Drosophila binds AP-1 and may help to recruit it during formation of the large cigar-shaped secretory granules, Weibel-Palade bodies (WPBs) (Philippe et al., 2013). Many CCV proteins also contain clathrin-binding sites, providing redundancy in recruiting the coat. Membrane deformation A common theme of CCV accessory proteins is their ability to deform membranes in some way. The ENTH (Epsin N-terminal homology) domain was mentioned in section 1.2.2 as having both membrane-deforming and PI-recognizing capabilities (Legendre-Guillemin, 2004). ENTH domains were first identified in the epsins (epsin1-4 in humans, with epsin4 variously called EpsinR, CLINT or enthoprotin; Ent3 and Ent5 in yeast). ANTH (AP180 Nterminal homology) domains are similar but probably carry out different roles (Duncan and Payne, 2003). BAR domains present on the amphiphysins, endophilins and FCho proteins also help to sculpt the membrane to favour budding (Henne et al., 2010). Membrane deformation can go hand-in-hand with vesicle budding: the AP180-related CALM/PICALM uses an ANTH domain to regulate CCV size and maturation (Miller et al., 2015).  26  Coordination of CCV machinery AP complexes are the most important hubs of CCVs, able to make an astonishing variety of interactions at once, but many other accessory proteins have regions for binding to each other as well. EH domains (Eps15 Homology domains) bind variants on the NPF motif (Paoluzi et al., 1998) (class I), but have also been found to bind FW, WW, or SWG motifs (class II). The EH domain of End3 in yeast is the only one that has been found to bind a H[S/T]F motif (class III) (Confalonieri and Di Fiore, 2002). The EH domains allow Eps15 to interact with other accessory proteins at the PM such as epsin and synaptojanin (Haffner et al., 1997) that contain NPF motifs. Eps15 also has a coiled-coil domain for interaction with Eps15, Eps15R and intersectin, so there can be multiple ways of interacting with other accessory factors even within the same protein.  Many accessory proteins contain multiple AP-ear binding domains, which theoretically allows crosslinking between AP complexes and concentration into a patch on the membrane. The AP-1 accessory proteins aftiphilin and γ-synergin (which form a tripartite complex with HEATR5B) both contain multiple AP-binding motifs as well as EH domains, and may provide both cargo-selection and crosslinking functions (Hirst et al., 2005). The µHD of Stoned B competitively binds both AP-2 and synaptojanin and is suggested to stimulate CCV uncoating (Walther et al., 2001) besides its role in selecting cargo. Favouring different interactions among CCV components at different times can promote progression of vesicle trafficking.  Connecting to motor proteins  AP complexes can interact directly or indirectly with components of cytoskeletal motors. AP-1 directly binds kinesin-3 (Nakagawa et al., 2000) and forms a complex with kinesin-1 through the accessory protein gadkin (formerly called γ-BAR) (Schmidt et al., 2009). Gadkin is also linked to the actin cytoskeleton through direct binding to the actin-nucleating ARP2/3 complex (Maritzen et al., 2012b). AP-2 also uses an adaptor protein, Dab2, to connect to myosin VI for progression along actin filaments (Yu et al., 2009), and there are likely many other motor adaptors yet to be discovered. 27  Regulation of AP complexes  Many regulators stimulate or inhibit the activation of AP complexes, either directly through allostery or indirectly through creating favourable membrane environments. Phosphorylation of the µ subunit by AAK1 has already been mentioned as an activation signal of an AP complex. FCho proteins allosterically regulate AP-2 activation, favouring the closed to open transition (Hollopeter et al., 2014; Umasankar et al., 2014), whereas NECAP proteins have recently been proposed to negatively regulate AP-2 by binding to the phosphorylated, open form and favouring a closed or dephosphorylated form (Beacham et al., 2018). Naturally, proteins (such as ARFGAP1) that affect the activity of Arf1, the allosteric regulator of AP-1, can also influence AP complex recruitment and activation. As previously mentioned, PI-kinases also stimulate AP complex recruitment to membranes, and phosphatases such as synaptojanins destabilize the AP complex on the membranes by removing key PI species (Perera et al., 2006). 1.5.2 AP accessory protein dysfunction in disease AP complex accessory proteins often act semi-redundantly to ensure that vesicle trafficking occurs properly. Compensatory mechanisms may ensure that dysfunction of a single accessory factor does not result in an overt disease phenotype; there are multiple copies of many accessory proteins (e.g. NECAPs, epsins, amphiphysins) that may be able to partially compensate for each other in extenuating circumstances. For instance, Gadkin knockdown affects dendritic cell migration in vitro but not in vivo in a mouse model (Schachtner et al., 2015). Aftiphilin and γ-synergin are needed for the proper release of Weibel-Palade Bodies (WPBs) involved in blood clotting (Lui-Roberts et al., 2008) but have not been linked to a major medical condition. Other cases give a clearer link between AP accessory proteins and disease. ARH is called autosomal recessive hypercholesterolemia protein due to its importance as an adaptor for the low-density lipoprotein receptor (LDL-R); ARH interacts with both AP-1 and AP-2 (Mishra et al., 2002; He et al., 2002). Amphiphysins interact with AP-1 (Huser et al., 2013) and AP-2 and have been linked to cancer, heart problems and susceptibility to Alzheimer’s (reviewed in Prokic et al., 2014). Synaptojanins are linked to Parkinson’s and Alzheimer’s diseases (Drouet and Lesage, 2014). NECAP1 loss of 28  function leads to severe infantile epileptic encephalopathy (Alazami et al., 2014). Complicating matters, many of the above proteins may function at multiple places in the cell and with different complexes. To fully understand the pathogenesis of a disease we must determine the exact contribution of a given accessory protein. 1.6 Research objectives Adaptor protein complexes are critical hubs in vesicular transport networks, and are subject to a high degree of spatiotemporal regulation. Developing a holistic understanding of the machinery involved in clathrin-coated vesicle trafficking will allow for more innovative solutions to treat conditions arising from a dysfunction in this machinery.   Yeast genome-wide screens represent an exceptional tool to probe cellular trafficking biology. Here, we followed up on two quantitative genome-wide screens performed in our lab, which examined the sorting of the v-SNARE Snc1 using an invertase-based reporter (Burston et al., 2008) and the sorting of the model polytopic protein Chs3 using calcofluor white fluorescence (Lam et al., 2006). Snc1 sorting has not previously been attributed to AP-1, while Chs3 is sorted by AP-1 but its exact role and the contributions of its associated machinery are incompletely characterized.   The Snc1 sorting screen revealed a differential role for the two known yeast AP-1 µ subunit isoforms, Apm1 and Apm2. This led us to hypothesize that the two subunits, whose relationship to each other is unclear, are distinct and sort different cargo. In Chapter 2 I detail experiments designed to investigate this hypothesis, to understand the molecular effects of exchanging one µ subunit for another in an AP complex and whether accessory factors dictate specific function in this case.  Trafficking of chitin synthase 3 (Chs3) is highly regulated. Deletion of the exomer complex that regulates Chs3 secretion leads to a well-documented AP-1-dependent Chs3 intracellular retention in an endosomal pool (Weiskoff and Fromme, 2014; Copic et al., 2007; Valdivia et al., 2002; Starr et al., 2012). Subsequent deletion of AP-1 causes a bypass phenotype, but there is debate as to whether AP-1 functions in anterograde or retrograde Chs3 sorting from the TGN to endosomes. We hypothesized 29  that crossing a deletion mutant of the exomer subunit Chs6 with the genome-wide deletion collection would give us a clearer picture of the bypass phenotype and would also allow us to identify new regulators of AP-1. In Chapter 3 I present the results of the genome-wide screen and use network analysis and molecular biology to identify a new AP-1-associated complex in yeast. I then describe experiments designed to investigate unexpected relationships between homologs of this complex in mammalian cells, aiming to understand the potential roles of these accessory proteins.  30  Chapter 2: The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting A version of this chapter has been published as: Whitfield ST, Burston HE, Bean BD, Raghuram N, Maldonado-Báez L, Davey M, Wendland B, Conibear E. The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting. Molecular Biology of the Cell. 2016 Feb 1;27(3):588-98. 2.1 Synopsis In this chapter I pursue the observation that Apm2, a variant medium (µ) subunit in the yeast AP-1 adaptor protein complex, is involved in sorting of the v-SNARE Snc1. Apm1, the medium subunit more commonly incorporated into the AP-1 complex, did not have a similar phenotype in a genome-wide Snc1 sorting screen, prompting the hypothesis that Apm1 and Apm2 are functionally distinct. In humans the cell type-specific expression of alternate µ chains µ1A and µ1B creates distinct forms of AP-1, but it is unclear how these subunits confer differential function. There is debate about whether the µ subunits specify localization to different cellular compartments or recognize different cargo. This uncertainty means that additional studies, in a variety of organisms, are needed to properly understand the functional consequences of subunit exchange. We showed that the yeast variant µ chain Apm2 confers distinct cargo-sorting functions. Loss of Apm2, but not Apm1, increased cell surface levels of the v-SNARE Snc1. Apm2 was unable to replace Apm1 in sorting Chs3, which requires a di-leucine motif recognized by the γ/σ subunits common to both complexes. Apm2 and Apm1 co-localized at Golgi/early endosomes, suggesting they do not associate with distinct compartments. We identified a novel, conserved regulatory protein that is required for Apm2-dependent sorting events. Mil1 is a predicted lipase that binds Apm2, but not Apm1, and contributes to its membrane recruitment. The experiments in this chapter demonstrate distinct functions for AP-1 (containing Apm1) and AP-1R (containing Apm2) in yeast, and identify a predicted lipase (Mil1) as a possible specific contributor to the AP-1R pathway. 31  2.2 Introduction  Clathrin-coated vesicles mediate the transfer of membrane proteins between different cellular compartments. Heterotetrameric adaptor protein (AP) complexes bind short linear motifs on cargo proteins and incorporate them into forming vesicles by linking them to the clathrin coat (Edeling et al., 2006). Five AP complexes (AP-1 to AP-5) have been identified which are believed to regulate distinct trafficking pathways at the Golgi, endosome/lysosome or plasma membrane (Hirst et al., 2013).These AP complexes share the same basic plan, being composed of two large subunits (β1-5 and γ/α/δ/ε/ζ, ~100 kDa), one medium subunit (µ1-5, ~50 kDa) and one small subunit (σ1-5, ~20kDa). Use of alternate subunits has the potential to further expand the diversity of AP complexes (Mattera et al., 2011). For example, three AP-1 subunits (γ, µ1, σ1) have isoforms encoded by different genes, and mutations in the three σ1 isoforms AP1S1, AP1S2 and AP1S3 cause different diseases: MEDNIK syndrome, X-linked mental retardation and pustular psoriasis respectively (Tarpey et al., 2006; Montpetit et al., 2008; Setta-Kaffetzi et al., 2014). Different AP subunits are responsible for binding clathrin, cargo sorting motifs, accessory factors, and membrane lipids (Edeling et al., 2006). Sorting motifs that conform to the YXXΦ consensus (where Φ is a bulky, hydrophobic amino acid) bind a conserved pocket in the medium subunit, whereas dileucine sorting motifs (D/E)XXXL(L/I) are recognized by the small and non-β large subunits (Canagarajah et al., 2013; Traub and Bonifacino, 2013). In the case of AP-1, Arf1 cooperates with cargo and phosphoinositides to mediate membrane recruitment. Arf1-GTP bridges two AP-1 complexes by binding the β and γ subunits (Ren et al., 2013), while the γ subunit of AP-1 binds PI4P at a site similar to that used by the AP-2 α subunit to bind PI(4,5)P2 (Heldwein et al., 2004). Finally, clathrin and accessory factors interact with linker and appendage domains of the large subunits, respectively.  The extent to which the use of variant subunit isoforms alters adaptor function is not well understood. The ubiquitous AP-1A and epithelial-cell-specific AP-1B complexes, which differ only in the incorporation of µ1A or µ1B, provide one of the best-studied examples (Bonifacino, 2014; Nakatsu et al., 2014). Several studies have 32  suggested that differential lipid binding by the µ subunits directs AP-1A to the Golgi but causes AP-1B to localize to recycling endosomes, presenting a plausible basis for their functional diversity (Fölsch et al., 2003; Fields et al., 2010). However, subsequent work showed AP-1A and AP-1B largely co-localize, and that μ1A and μ1B recognize cargo proteins with different affinities (Guo et al., 2013). Thus, the basis for the differential functions of these isoforms is currently unclear (Rodriguez-Boulan et al., 2013), and it is not known if subunit exchange alters the function of other adaptor complexes. Of the three AP complexes in the yeast Saccharomyces cerevisiae, only AP-1 uses alternate subunits. The two forms of AP-1 share the same large (Apl2 and Apl4) and small (Aps1) subunits but incorporate different medium subunits (Apm1 or Apm2). While the classical Apm1-containing AP-1 complex transports well-characterized cargo including chitin synthase III (Chs3) and Sna2 (Valdivia et al., 2002; Renard et al., 2010), no function has been identified for the Apm2-containing complex, which we refer to as AP-1R (AP-1 Related).  Early studies suggested these complexes are biochemically distinct (Stepp et al., 1995; Yeung et al., 1999). Whereas Apm1 and Apm2 were proposed to act redundantly in trafficking the lipid flippase Drs2 (Liu et al., 2008), Apm2 is dispensable for many clathrin-dependent sorting events (Stepp et al., 1995).  Here, we show that the Apm2 subunit confers distinct functions to the yeast AP-1 complex. AP-1R and AP-1 sort distinct cargo proteins and exhibit differential sensitivity to cationic lipophilic drugs. These differences are not readily explained by divergent cargo recognition properties, because AP-1R is unable to substitute for AP-1 in the sorting of cargo via a di-leucine-based motif that is recognized by subunits common to both AP-1 and AP-1R. Strikingly, Apm2 but not Apm1 binds a novel regulatory protein that we call Mil1 (mu-interacting ligand). Mil1 is a predicted serine hydrolase that is required for Apm2-dependent sorting processes and has a role in recruiting Apm2 to membranes. This suggests the binding of specific regulatory factors may be an important mechanism by which subunit exchange confers new functions to clathrin adaptors.   33  2.3 Results 2.3.1 Apm2 is part of a functionally distinct AP-1 related complex  Previously, we uncovered genes required for the trafficking of the v-SNARE Snc1 in a genome-wide screen that assessed changes in the surface levels of the GSS (GFP-Snc1-Suc2) reporter protein (Burston et al., 2009). Genetic and physical interaction analysis identified a cluster of 4 genes, representing three AP-1 subunits (Apm2, Apl4, and Aps1) and the uncharacterized Yfl034w, whose deletion increased surface GSS levels. The identification of Apm2, but not the more abundant µ subunit isoform Apm1, was unexpected and suggested the Apm2-containing AP-1 related complex (AP-1R) has a specific role in Snc1 sorting. To confirm these observations, we used a quantitative assay that measures the invertase (Suc2) activity of GSS present at the cell surface (Figure 2.1A) (Dalton et al., 2015). Loss of APM2, APL4, and APS1 led to a slight but significant increase in GSS surface levels, comparable to that of the endocytosis mutant yap1801 (Burston et al., 2009). This increase was not observed in the absence of APM1, showing that GSS sorting is an AP-1R-specific function and does not require the Apm1-containing AP-1 complex, which we will refer to here as AP-1.  Past studies have shown the AP-1 complex is required for the transport of the chitin synthase Chs3 between Golgi and endosomal compartments (Valdivia et al., 2002). Exomer mutants, such as chs6, have low chitin levels because Chs3 is not transported to the surface, but instead is retained in an AP-1-dependent intracellular recycling loop. Deletion of the large AP-1 subunit Apl2 or of Apm1 bypasses this intracellular retention, restoring Chs3 transport to the cell surface and chitin production. Previous studies found no role for Apm2 in Chs3 sorting using a qualitative assay (Valdivia et al., 2002). To detect subtle defects, we assessed the requirement for Apm2 in Chs3 transport using a sensitive quantitative assay based on the fluorescent compound calcofluor white (CW) (Lam et al., 2006; Burston et al., 2008), which binds chitin produced by cell-surface Chs3. Whereas deletion of AP-1 components (APM1, APL2) restored wild type cell surface levels of Chs3 in chs6 cells, deletion of APM2 or 34  YFL034W did not (Figure 2.1B). These results confirm that Apm2 does not play a role in the AP-1-mediated sorting of Chs3.  Figure 2.1 APM1 and APM2 deletions have distinct sorting phenotypes. (A) APM2 but not APM1 is required for Snc1 sorting. Cell-surface levels of the Snc1 reporter GSS were quantified in yeast deletion strains by measuring invertase activity of liquid cultures, normalized to wild type. Unpaired t-test of mutant strains compared to wild-type, ***: p<0.0001; **: p<0.01. Error bars represent standard error of the mean (n=6). (B) Deletion of APM1 but not APM2 bypasses the Chs3 trafficking defect of a chs6 mutant strain. Chs3 surface activity was quantified as the fluorescence intensity of strains plated on 50μg/mL calcofluor white and reported in arbitrary units (A.U.). Unpaired t-test compared to wild-type, ***: p<0.0001;*: p<0.05. Error bars represent standard error of the mean (n=8). (C) To assess drug sensitivity, yeast were spotted in 10x dilution series and grown on YPD with Hyg B plus indicated drugs, or DMSO as control. Because the Snc1 sorting defect in apm2 mutants was relatively mild, we sought alternative phenotypes that distinguish Apm1 and Apm2. Genome-wide chemogenomic studies (Hoepfner et al., 2014; Lee et al., 2014) suggested that Apm1 and Apm2 have differential drug sensitivities. By exposing apm1 and apm2 strains to a subset of these drugs, we found strains lacking APM1, but not APM2, were sensitive to myriocin, an inhibitor of sphingolipid biosynthesis (Figure 2.1C). In contrast, apm2 mutants were sensitive to cationic amphiphilic drugs (CADs) such as sertraline, as previously reported (Rainey et al., 2010), whereas apm1 mutants exhibited sertraline resistance (Figure 35  2.1C). CADs intercalate in phospholipid bilayers, and may interfere with protein function, including lipid binding (Daniel et al., 2015). The differential sensitivities of these adaptor protein mutants may reflect missorting of cargo that are important in responding to stresses imposed by the drug. These differential drug sensitivities, together with the cargo-specific sorting defects, demonstrate that Apm1 and Apm2 have distinct functions. 2.3.2 AP-1R localizes to the late Golgi and early endosomes  Because Snc1 undergoes a rapid cycle of internalization and recycling to the plasma membrane, increased surface levels in cells lacking AP-1R could reflect reduced endocytosis or an enhanced rate of recycling from intracellular compartments. To determine if Apm2 is part of the endocytosis machinery, the localizations of Apm2-GFP and the AP-2 medium subunit Apm4-GFP were compared with that of clathrin light chain Clc1-RFP in sla2 strains, which stall endocytosis of clathrin-coated pits (Kaksonen et al., 2003; Newpher et al., 2005). Whereas Apm4-GFP co-localized with Clc1-RFP at the plasma membrane, Apm2-GFP was largely localized to intracellular clathrin compartments (Figure 2.2A). A small number of Apm2-GFP puncta were observed at or near the plasma membrane. To determine if these represent sites of endocytosis, we carried out time-lapse imaging using total internal reflection fluorescence (TIRF) microscopy of cells treated with the actin inhibitor Latrunculin-A (LatA) to block the budding of cell surface clathrin-coated pits. The AP-2 subunit Apm4-GFP maintained a strong co-localization with Clc1-RFP at immobile patches on the plasma membrane (Figure 2.2B). In contrast, Apm2-GFP-containing structures near the cell surface were highly mobile and did not co-localize with plasma membrane Clc1-RFP puncta over time. Some lack of co-localization between AP-1 and clathrin has been observed before (Daboussi et al., 2012), suggesting adaptors can be present at membranes without significant clathrin binding. The absence of AP-1R at the plasma membrane is inconsistent with a role in endocytosis and suggests instead that it functions in an intracellular pathway.  36   Figure 2.2 Apm2 localizes to late Golgi/early endosomes. (A) Fluorescence microscopy of live endocytosis-defective sla2 yeast shows Apm2 is not present at cell-surface clathrin-coated pits. White arrows indicate puncta that co-localize in GFP and RFP channels. (B) Kymograph analysis of Apm4-GFP or Apm2-GFP relative to cortical clathrin patches marked by Clc1-RFP. Cells were incubated with the actin inhibitor latrunculin A and imaged by TIRF microscopy. (C) Localization of Apm2-GFP relative to that of Apm1-tdTomato (AP-1 complex), Anp1-RFP (early Golgi), Sec7-DsRed (late Golgi/endosomes), Snf7-RFP (late endosomes). Scale bar, 4μm. To define the intracellular distribution of Apm2-GFP we performed co-localization experiments with fluorescently-tagged marker proteins (n=3, >125 cells/experiment) (Figure 2.2C). Consistent with a role in Golgi/endosomal sorting, 63±3% of Apm2-GFP puncta co-localized with the late-Golgi/early endosome marker Sec7-dsRed. Apm2-GFP only partially co-localized with the early Golgi marker Anp1-RFP (28±3%) and showed little co-localization with the late endosome marker Snf7-RFP (8±1%). These results indicate that AP-1R localizes primarily to the late Golgi/early endosomes, similar to previous findings placing AP-1 at these compartments (Liu et al., 2008; Daboussi et al., 2012). Importantly, Apm2-GFP showed extensive co-localization with Apm1-tdTomato (77±2%) suggesting the bulk of AP-1R is present at the same intracellular compartment as AP-1. 2.3.3 Recognition of tyrosine-based signals by Apm1 and Apm2 The different phenotypes of apm1 and apm2 mutants suggest that Apm1 and Apm2 recognize different cargo for incorporation into vesicles. The μ subunits of AP complexes interact with tyrosine-based motifs (YxxΦ) (Canagarajah et al., 2013), via residues conserved in both Apm1 and Apm2 (Supplementary Figure A1A). The vacuolar 37  protein Sna2 has two tyrosine-based sorting motifs at Y65 and Y75 that govern its trafficking by AP-1 and AP-3, respectively (Renard et al., 2010). We tested sorting of a Sna2 mutant (Sna2Y75A) that lacks the AP-3 signal and whose vacuolar transport depends on AP-1 (Figure 2.3A and 2.3B). Loss of the AP-1 components Apl4 and Apm1 missorted Sna2Y75A-GFP to the cell surface, consistent with previous studies (Renard et al., 2010). The Sna2 tyrosine-based signal is expected to bind the conserved pocket in Apm1. Indeed, a version of Apm1 containing mutations at this site (APM1tyr: F179A D181S) was present at the same level as wild type Apm1 (Supplementary Figure A1B), but was unable to rescue the Sna2Y75A-GFP sorting defect of an apm1 mutant. We found that Apm2 is not required for Sna2Y75A-GFP sorting (Figure 2.3A and 2.3B) (Renard et al., 2010).  However, it is much less abundant than Apm1 and could have a minor but redundant role that would not be detected in knockout experiments. To determine if Apm2 can substitute for Apm1 when expressed at similar level, HA-tagged Apm2 was placed under the Apm1 promoter (APM2OE). We confirmed expression of Apm2 was comparable to that of Apm1 (Supplementary Figure A1B), but observed no rescue of Sna2Y75A-GFP sorting in an apm1 strain. In contrast, re-introduction of wild-type Apm1 significantly restored localization of Sna2Y75A-GFP to the cell surface (n=3, p=0.0005; Figure 2.3B). These results suggest that while Apm1 sorts Sna2 through a tyrosine-based motif, Apm2 does not. Apm2 could lack a functional YxxΦ-binding pocket.  However, wild-type Apm2, but not the Apm2 tyrosine motif-binding mutant (Apm2tyr: F273A D275S), rescued growth on sertraline in an apm2 background (Figure 2.3C), suggesting Apm2 may sort a protein required for sertraline resistance via a tyrosine-containing signal. The Snc1 cytosolic domain lacks tyrosine residues, and may bind to Apm2 via a non-canonical sorting motif. Interestingly, the Apm2tyr mutant also failed to sort GSS in the invertase assay (Figure 2.3D). This suggests that Apm2 sorts another cargo necessary for the normal recycling of Snc1, or that a non-canonical signal in Snc1 binds the same site in Apm2.    38   Figure 2.3 The predicted YxxΦ-binding pocket is required for some but not all functions of Apm1 and Apm2. (A) Expression of an Apm1 mutant lacking a functional tyrosine signal-binding pocket (apm1tyr), or over-expression of APM2 from the APM1 promoter (APM2OE), is unable to restore the vacuolar localization of the AP-1-specific cargo Sna2Y75A-GFP in apm1 mutant cells. Scale bar, 2μm. (B) Quantification of surface fluorescence of Sna2Y75A-GFP. Error bars represent standard error of the mean (n=3). Unpaired t-test compared to apm1Δ, ***: p<0.0001, **: p<0.01. (C) An Apm2 mutant lacking a functional tyrosine signal-binding pocket (apm2tyr) and expressed on a plasmid from its native promoter exhibits sertraline sensitivity. Yeast were plated in a 10x dilution series on YPD containing Hyg B and DMSO or 22.5uM sertraline. (D) The apm2tyr mutant is unable to restore sorting of the Snc1 reporter GSS. Cell-surface GSS levels in the indicated strains were determined by quantifying invertase activity and normalizing to levels in wild type cells. Unpaired t-test compared to wild-type, ***: p<0.0001. Error bars represent standard error of the mean (n=6). (E) Wild type APM1 and apm1tyr, but not overexpressed APM2 (APM2OE), restores the calcofluor resistance of chs6 apm1 mutants. Yeast were plated in a 10x dilution series on buffered YPD containing Hyg B and 50μg/mL calcofluor white (CW). (F) Representative fluorescent images of calcofluor white-stained strains.   39  AP-1 binds Chs3 via a dileucine-based motif (Starr et al., 2012).  Such motifs are recognized by the γ/σ subunits. Thus, mutation of the Apm1 tyrosine-binding pocket is not predicted to affect Chs3 sorting. To test this, serial dilutions of deletion strains were grown on media containing toxic amounts of calcofluor white (Figure 2.3E). Exomer mutants such as chs6 that retain Chs3 inside the cell have reduced chitin and are resistant to calcofluor white, while AP-1 mutants that bypass chs6 and allow Chs3 to reach the surface restore calcofluor white sensitivity. Expression of either wild-type APM1 or apm1tyr in the chs6 apm1 background resulted in a chs6 phenotype (Figure 2.3E and 2.3F), confirming that Chs3 sorting does not require the Apm1 YxxΦ-binding pocket.  Because the dileucine-based motif of Chs3 binds the γ/σ subunits present in both AP-1 and AP-1R, Apm2 should substitute for Apm1 if present at the same level. Surprisingly, expression of APM2 from the APM1 promoter did not alter the calcofluor white-sensitive phenotype of chs6 apm1 strains (Figures 2.3E and 2.3F), indicating AP-1R is incapable of sorting Chs3. This result suggests that differential cargo recognition is not the reason for the different sorting specificity of AP-1 and AP-1R.  2.3.4 Mil1(Yfl034w) specifically binds the Apm2 C-terminal subdomain B One explanation for the functional differences between Apm1 and Apm2 is that they bind different regulatory proteins that direct them into specific pathways. The uncharacterized protein Yfl034w clustered with AP-1R subunits in our genome-wide analysis (Burston et al., 2009) and, like Apm2, was required for sorting of the Snc1 reporter but not Chs3 (Figure 2.1A, B), suggesting it shares a closely related function. Based on results described below, we refer to Yfl034w as Mil1 (medium adaptin-interacting ligand). Physical interactions between Mil1 and components of the AP-1 complex were reported in several proteomic studies (Gavin et al., 2006; Krogan et al., 2006; Collins et al., 2007; Babu et al., 2012). We confirmed this interaction, showing that Mil1-GFP co-purifies with Apl4-HA immunoprecipitated from yeast cell lysates (Figure 2.4A). Like all  40   Figure 2.4 Mil1 interacts with Apm2 through its WQEMP motif. (A) Mil1-GFP co-purified with immunoprecipitated Apl4-3HA, and with a truncated version lacking the gamma appendage (Apl4Δear-HA) known to bind several AP regulatory proteins. Loading of lysate relative to IP was 1:9. All proteins were genomically tagged. (B) Pulldown of Mil1-GFP in strains co-expressing either Apm1-3HA or Apm2-3HA shows Mil1 binds specifically to Apm2. All proteins were genomically tagged. (C) Phyre2 homology model of Apm2, coloured to indicate the N-terminal AP-binding domain (green) and a C-terminal region composed of A (yellow) and B (magenta) subdomains. Key residues predicted to be involved in YxxΦ binding (red), or lipid binding (blue) based on alignment with regions of AP-1 and AP-2 μ subunits are indicated. (D) Yeast two-hybrid mapping of Apm2-Mil1 binding domains. Full-length or truncated Apm2 constructs were fused to the GAL4 DNA-binding domain (GBD) and full-length, truncated or mutated Mil1 constructs were fused to the GAL4-activating domain (GAD). Qualitative interaction strengths are indicated. Mil1WQEMP represents the W143QEMP>AAEAA mutant and Mil1FNIY represents the F152NIY>ANAA mutant. (E) Anti-GFP immunoprecipitation of plasmid-expressed wild-type Mil1-GFP or Mil1WQEMP-GFP from strains co-expressing genomically-tagged Apm2-3HA. (F) Sertraline sensitivity was assessed by plating strains in a 10x dilution series on YPD containing 22.5 μM sertraline, or DMSO as a 41  (Figure 2.4 continued) control. (G) The mil1WQEMP mutant is unable to restore sorting of the Snc1 reporter GSS. Cell-surface GSS levels in the indicated strains were determined by quantifying invertase activity and normalizing to levels in wild type cells. Unpaired t-test compared to wild-type, ***: p<0.0001, *: p<0.05. Error bars represent standard error of the mean (n=10). heterotetrameric clathrin adaptors, the AP-1 consists of a core complex with two appendages (“ears”) projecting from the large subunits by flexible linkers (Edeling et al., 2006). Although the Apl4 (γ) appendage recruits many regulatory proteins, Mil1 was able to bind a truncated form of Apl4 lacking this appendage (Apl4Δear) (Figure 2.4A). We reasoned that if Mil1 is an AP-1R regulator, it might associate preferentially with Apm2, which is part of the core complex. Apm2-HA was present at low levels in the cell lysate, yet we observed a clear enrichment of Apm2-HA upon immunoprecipitation of Mil1-GFP, whereas there was little if any pulldown of Apm1-HA, showing that the physical interaction between Apm2 and Mil1 is highly specific (Figure 2.4B).   A yeast two-hybrid assay was used to map interacting regions. Based on structural homology to other µ adaptins, Apm2 has an N-terminal domain required for its incorporation into the AP complex, while the C-terminus consists of two subdomains: an A domain, containing the tyrosine-binding pocket, and a B domain, which may contribute to membrane recruitment but does not contain known cargo-binding sites (Figure 2.4C) (Fields et al., 2010; Canagarajah et al., 2013). The minimal Mil1-binding region was mapped to residues 389-562, which correspond to the B domain (Figure 2.4D).    Additional mutations were used to define the Apm2 binding site on Mil1. First, the interaction was narrowed to a region between residues 125-175. Point mutations that alter conserved residues in this 50-amino acid fragment revealed that a F152NIY>ANAA mutant retained the yeast two-hybrid interaction with Apm2, but a W143QEMP>AAEAA mutant did not. Furthermore, introducing the W143QEMP>AAEAA mutation into the full length Mil1 protein resulted in a Mil1WQEMP mutant that was stably expressed but did not co-precipitate with Apm2 (Figure 2.4E).  Having identified an Apm2 binding-deficient Mil1 mutant, we tested if the Apm2-Mil1 interaction was important for Apm2 function. Sensitivity of MIL1 deletion strains could be rescued by a plasmid expressing Mil1 or the full length F152NIY>ANAA 42  (Mil1FNIY) mutant, whereas the Mil1WQEMP mutant showed only weak complementation (Figure 2.4F). Additionally, only wild-type Mil1 was able to complement GSS sorting (Figure 2.4G). Thus, the Apm2-Mil1 interaction is functionally important, suggesting Mil1 plays a key role in AP-1R-mediated sorting processes. 2.3.5 Mil1 has a conserved α/β-hydrolase catalytic motif required for its function  Mil1, which contains a C-terminal DUF726 domain, is predicted to be a serine hydrolase with an α/β hydrolase fold (Baxter et al., 2004). Serine hydrolases constitute a large protein family that uses the conserved serine nucleophile to hydrolyze amide, ester and thioester bonds in a variety of substrates (Lenfant et al., 2012). Alignment of Mil1 and its homologs revealed conserved residues that match the Ser-Asp-His catalytic triad of many serine hydrolases, consisting of S759 within a GxSxG motif, D817 and H858 (Figure 2.5A). Structural modeling using Phyre2 (Kelley and Sternberg, 2009) suggested the DUF726 domain of Mil1 is most similar to lipases, and that residues S759, D817 and H858 are oriented in the necessary geometry to form a catalytic triad (Figure 2.5B). The active site of the well-characterized lipase CalB from Candida antarctica is shown for comparison; CalB was one of the highest template matches used for 3D homology modeling. To investigate if Mil1 is likely to have an enzymatic activity important for Apm2-dependent sorting pathways, we tested the sertraline resistance of mutants carrying an alanine substitution of each predicted catalytic residue. A plasmid expressing wild-type Mil1 fully complemented the sertraline sensitivity of a mil1 strain, but the S759A (S>A), D817A (D>A) and H858A (H>A) mutants were only partially functional (Figure 2.5C), despite being expressed at similar levels (data not shown). Each catalytic mutant displayed a similar level of sertraline sensitivity, suggesting that the putative catalytic activity of Mil1 contributes to Apm2 function. 43   Figure 2.5 Mil1 has a conserved serine hydrolase catalytic triad. (A) BLAST alignment of the conserved DUF726 domain in Mil1. Intensity of blue-highlighted residues corresponds to percent identity. Conserved residues corresponding to the predicted Ser-Asp-His catalytic triad are highlighted in red. (B) Structural model showing the catalytic triad of the well-characterized lipase B from Candida antarctica (left) (PDB ID: 1TCA) and the Phyre2 homology model showing the predicted catalytic triad of Mil1 (right). (C) Sertraline sensitivity was assessed by plating strains in a 10x dilution series on YPD containing 22.5uM sertraline, or DMSO as a control. 2.3.6 Mil1 is a peripheral membrane protein that promotes Apm2 recruitment  The transmembrane prediction algorithms Phobius (Käll et al., 2004)(Käll et al., 2004) and TMHMM (Krogh et al., 2001) suggest Mil1 contains four transmembrane domains. However, two of these predicted TMDs overlap the DUF726 α/β hydrolase domain, which is expected to be soluble. Subcellular fractionation was used to examine Mil1 membrane association under conditions that can dislodge peripheral, but not integral, membrane proteins (Figure 2.6A). In the presence of high salt concentrations the transmembrane protein Pep12 was found in the pellet (P100) fraction as expected, whereas Mil1 was present exclusively in the soluble (S100) fraction, indicating that Mil1 is peripherally associated with membranes.  44   Figure 2.6 Mil1 is a peripheral membrane protein that promotes Apm2 membrane recruitment. (A) Subcellular fractionation of Mil1 and the transmembrane protein Pep12. WCE: Whole Cell Extract. P13: low speed pellet fraction, P100: high speed pellet fraction, S100: high speed soluble fraction. Samples were resolved by 15% SDS-PAGE and detected by immunoblotting. (B) Representative maximum intensity Z-projection images from 9 slices at 0.3μm increments, scale bar, 2μm. (C) Localization of Mil1-GFP in wild type or apm2 mutant strains quantified in a single slice. Unpaired t-test *: p<0.05. Error bars represent standard error of the mean (n=3). (D) Representative maximum intensity z-projection images from 9 slices at 0.3μm increments, scale bar, 2μm. (E) Localization of Apm2-GFP expressed in wild type or mil1 mutant strains and quantified in a single slice. Unpaired t-test, ***: p<0.0001, **: p<0.01, *: p<0.05. Error bars represent standard error of the mean (n=4). (F) Proposed model: Mil1 helps to recruit Apm2 to a distinct membrane region. We hypothesized that binding of Mil1 to the B domain of Apm2 could be responsible for its membrane recruitment. Accordingly, we examined the localization of Mil1-GFP in wild-type and apm2 mutants (Figure 2.6B). We observed a slight but significant decrease in the number of Mil1-GFP puncta in apm2 cells compared to wild-type, which could be complemented by introduction of APM2 on a plasmid (Figure 2.6C). Thus, Mil1 localization seems to be largely Apm2-independent. 45  We also considered the hypothesis that Mil1 promotes Apm2 membrane recruitment. We saw a significant decrease in the number of Apm2-GFP puncta in mil1 cells compared to wild-type strains (Figure 2.6D). Loss of Mil1 reduced the number of Apm2 puncta by approximately 50% (Figure 2.6E) but did not significantly change the number of Apm1-GFP or Sec7-dsRed puncta (Supplementary Figures A2A and A2B). Notably, the Mil1S>A catalytic site mutant complemented this defect almost as well as wild type Mil1, but the Mil1WQEMP deficient in Apm2 binding failed to complement, suggesting that direct physical interaction between Mil1 and Apm2, but not Mil1 catalytic activity, is important for recruitment of Apm2, but not Apm1, to membranes. 2.4 Discussion Subunit exchange may represent a general mechanism for creating functionally distinct adaptor complexes. How the incorporation of distinct but highly homologous µ subunits alters the function of the two AP-1 complexes of mammalian epithelial cells, AP-1A and AP-1B, is the subject of current debate (Rodriguez-Boulan et al., 2013). Although AP-1A and AP-1B were shown to localize to different intracellular compartments in early studies, recent work suggested these adaptor complexes are present in the same vesicles, but recognize different cargo (Guo et al., 2013). Our work provides an example of how subunit exchange can create functionally distinct AP-1 adaptor complexes in yeast and highlights the importance of unique interactions with regulatory partners in this process.  We find that inclusion of either Apm1 or Apm2 changes the cargo specificity of AP-1 complexes: whereas the classical Apm1-containing AP-1 complex is involved in sorting Chs3 and Sna2, consistent with previous studies (Valdivia et al., 2002; Renard et al., 2010), the Apm2-containing AP-1R complex works in pathways that sort the v-SNARE Snc1 and grant resistance to sertraline. The lipid flippase Drs2 is the only cargo reported to require both Apm1 and Apm2 for its localization (Liu et al., 2008). Because Drs2 regulates the formation of AP-1/clathrin-coated vesicles by generating membrane curvature, it may constitute an essential component of vesicles in both AP-1 and AP-1R-dependent pathways. 46  We showed Apm2 could not substitute for Apm1 in Sna2 or Chs3 sorting even when expressed at similar levels, suggesting these µ adaptins share little functional overlap. Some of these differences could result from differential cargo binding. The µ adaptins of AP-1, AP-2 and AP-3 bind YxxΦ motifs at a conserved site (Owen, 1998; Jia et al., 2012; Mardones et al., 2013). We found that mutations in Apm1 that disrupt this site block the YxxΦ-dependent AP-1 sorting of Sna2, whereas corresponding mutations in Apm2 cause sertraline sensitivity, suggesting resistance to this drug requires correct sorting of a YxxΦ-containing cargo by Apm2. Thus, while both Apm1 and Apm2 are likely to recognize YxxΦ motifs, small variations at this site could alter the affinity for different cargo, as reported for AP-1 and AP-3 (Mardones et al., 2013).  Apm1 and Apm2 may have additional sites for binding non-canonical signals, as demonstrated for AP-4 (Boll et al., 2002; Burgos et al., 2010). Apm1 binds the Ste13 cytoplasmic domain via an 11aa peptide that lacks recognized motifs (Foote and Nothwehr, 2006). Snc1 also lacks consensus YxxΦ or di-leucine sorting signals, suggesting Apm2 recognizes a divergent sorting signal in the Snc1 cytoplasmic domain or acts indirectly to sort a protein that is itself needed for Snc1 transport. Binding between Apm2 and Snc1 was shown by GST pulldown (H. Burston, doctoral dissertation), but whether the interaction is direct or indirect was not determined. Further work will be needed to define the relative affinities of Apm1 and Apm2 for different sorting signals, and determine if the signals are bound at distinct sites on the µ chain. Although differential recognition of sorting signals might explain some apm1 and apm2 phenotypes, Chs3 sorting – which depends solely on Apm1 – requires a di-leucine motif (Starr et al., 2012). Such motifs are typically recognized by the γ/σ subunits present in both AP-1 and AP-1R (Doray et al., 2007; Kelly et al., 2008; Mattera et al., 2011). Indeed, we found the Apm1 tyrosine-binding pocket required for Sna2 transport was dispensable for Chs3 sorting. The fact that Apm2 is unable to substitute for Apm1 in this pathway, even at comparable levels of expression, suggests that something other than cargo binding affinity distinguishes the AP-1 and AP-1R complexes. It is possible that the Chs3 dileucine signal binds non-canonically to the AP-1 µ subunit, or that Apm2 alters the conformation or accessibility of a dileucine-binding site in the γ/σ subunits. However, we favour an alternative model: that AP-1R is located 47  in a separate compartment or microdomain where it does not encounter Chs3 (Figure 2.6F). Although we found Apm2 and Apm1 are largely present at the same Golgi/endosomal structures, our data are consistent with the model that AP-1 and AP-1R sort cargo into separate vesicle populations.  We identified an Apm2-interacting protein, Mil1, which was required specifically for Apm2-mediated protein trafficking. We hypothesize that Mil1 plays a key role in recruiting Apm2 into a distinct pathway in which Apm1 does not participate. Mil1 is predicted to be structurally similar to fungal lipases and phospholipases, and contains conserved residues that match a Ser-Asp-His catalytic triad and GxSxG motif (Ha et al., 2012). Phospholipases A1 and A2 (PLA1 and PLA2) have been implicated in vesicle formation at the Golgi, either by producing cone-shaped lipids that promote membrane curvature, or by creating particular phospholipid species that recruit trafficking components (Bechler et al., 2012; Ha et al., 2012). The cytosolic phosphatidic acid phospholipase A1 (PAPLA1) is important in the COPII-dependent transport of rhodopsin 1 in flies, but this function does not require its active site GxSxG residues (Kunduri et al., 2014). Similarly, the lipid-binding activity of the phospholipase A1 family member p125A, but not its enzymatic activity, is suggested to maintain COPII residency at ER exit sites (Klinkenberg et al., 2014). We found that Mil1 mediates the efficient recruitment of Apm2 to membranes, and this requires Mil1-Apm2 interaction but not Mil1’s presumed enzymatic activity. Because Mil1 is largely targeted to membranes in the absence of Apm2, it could bind a particular class of lipid and reinforce the recruitment of Apm2 to a specific microdomain, similar to the role proposed for p125A in COPII recruitment (Figure 2.6F).   We found a conserved sequence in a predicted flexible, extended N-terminal region of Mil1 interacted with the “B” lobe of the Apm2 medium chain, distal to known cargo-recognition sites. Thus, Mil1 binding is unlikely to interfere with cargo recognition by Apm2, consistent with a regulatory role for Mil1. Interestingly, other lipid-modifying proteins have been found to associate with AP µ subunits. AP-1 µ1B binds and activates the PI(4)P 5-kinase PIPKIγ, which may contribute to the formation of a PI(3,4,5)P3-enriched domain that favors further AP-1B recruitment (Ling et al., 2007; Fields et al., 2010). All three PI 4 phosphate 5-kinase isoforms bind the C-terminal 48  domain of AP2 µ subunit, and this does not require the AP2 µ subunit’s tyrosine-binding pocket (Krauss et al., 2006). We found predicted active site residues in Mil1 were not required for Apm2 recruitment, but were partially required for Apm2-mediated processes, suggesting Mil1 has a catalytic activity whose role is downstream of adaptor recruitment. Our discovery that Mil1 regulates vesicle trafficking in yeast indicates that its uncharacterized human homologue, TMCO4, may act in a similar process. The Apm2-binding region of Mil1 is not conserved in TMCO4, suggesting that this protein regulates other trafficking pathways in higher organisms. Further studies will be required to determine the extent to which lipid-modifying proteins interact with different adaptor protein complexes and other coat proteins to regulate vesicle transport. 2.6 Materials and Methods 2.6.1 Yeast strains and plasmids All strains from this study were made by homologous recombination as described (Longtine et al., 1998; Janke et al., 2004; Sheff and Thorn, 2004). Briefly, log-phase yeast were grown in YPD media, washed twice with dH2O and suspended in transformation mix (33% polyethylene glycol, 100 mM lithium acetate, 0.28 mg/mL single-stranded herring sperm DNA, 25 µL desired PCR product, total volume 180 µL), heat-shocked at 42°C for 40 min and plated on selective medium. Transformations involving positive selection included a 1-hour rescue step in 2x YPD to permit expression of the antibiotic selection cassette. Colonies were confirmed by colony PCR and/or western blotting.BY4741 and BY4742 yeast strains were purchased from GE Dharmacon (Lafayette, CO). Strains were propagated in rich media (YPD: 1% yeast extract, 2% peptone, 2% dextrose) or SD minimal media (0.17% yeast nitrogen base, 0.5% ammonium sulfate, 2% synthetic complete mix, 2% dextrose) supplemented with the appropriate amino acids, for plasmid selection. Plasmids were made by homologous recombination in yeast, rescued in E. coli and confirmed by sequencing. pHPH was made by PCR amplification of the hygromycin resistance gene HPH from pFA6a-hphNT1 and co-transformation of the PCR product with linearized pRS416. Proteins were tagged with either the bright GFP variant, GFP+ (Scholz et al., 2000), or with 49  GFP(envy) (Slubowski et al., 2015), a gift from C. Slubowski (University of Massachusetts Boston), by PCR amplification of the GFP::HIS cassette and transformation into yeast. Strains and plasmids used in this chapter are described in Supplementary Tables A1 and A2. 2.6.2 Chemical compounds A calcofluor white (fluorescent brightener 28; Sigma Aldrich, St. Louis, MO) stock solution was made in 0.5 M Tris-HCl pH 9.6 to a final concentration of 10 mg/mL. Stock solutions of sertraline hydrochloride (Cedarlane Laboratories, Burlington, ON, Canada), and myriocin (Cayman Chemical Company, Ann Arbor, MI)  of 25mg/mL and 1mg/mL respectively, were made in dimethyl sulfoxide (DMSO) and stored at -20°C until use. Myriocin was purchased from the Cayman Chemical Company (Ann Arbor, MI), resuspended to a final concentration of 1 mg/mL in DMSO and treated similarly. Hygromycin B (Hyg B; Roche Diagnostics, Indianapolis, IN) was purchased as a 50 mg/mL stock in PBS. All other chemicals were purchased from Sigma Aldrich and stored according to supplier’s recommendations. 2.6.3 Invertase assay Strains expressing the GSS construct were grown for 20 h in 2 mL YP-fructose (YPF) (final OD600 ≈10). Cultures were diluted to 3 OD600/mL and the liquid invertase assay was performed as described (Dalton et al., 2015). Briefly, 5 µL of yeast were added to 150 uL YPF liquid in 96-well plates, OD600 measurements taken, and 13 µL of 0.5 M sucrose solution added to wells. After 5 min incubation, 100 µL of glucostat reagent (0.1 M K2HPO4 pH 7.0, 1000 U/mL glucose oxidase, 1 mg/mL horseradish peroxidase, 20 mM n-ethylmaleimide, 10 mg/mL o-dianisidine) was added and incubated for 2 min, then was stopped by addition of 100 µL of 6 N HCl and read at OD540. All reactions took place at room temperature. Results are reported as nM glucose produced per 1 OD600 of culture, normalized to wild-type readings from the day. Glucose standards (ranging from 5-50 nM) and blanks were included in each experiment to ensure measurements were within the linear range.  50  2.6.4 Calcofluor White assay Calcofluor assay was performed as described (Burston et al., 2008). Briefly, knockout strains were plated in 1,536-array format onto YPD plates containing 50 μg/mL calcofluor white, using a Virtek automated colony arrayer (Bio-Rad Laboratories, Hercules, CA). After incubation at 30°C for 3 days, white-light images were acquired using a model 2400 flat-bed scanner (Epson, Nagano, Japan), 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 was used for densitometry of digital images.  2.6.5 Small molecule inhibitor assays  1 OD600/mL of log phase yeast grown under selective conditions to maintain plasmids (either SD-Leu or YPD + Hyg B) were spotted in 10x serial dilution on selective plates containing inhibitors or DMSO as a control, incubated at 30°C for 3 days, and imaged with a Canon Rebel T3I camera (Canon Inc., Tokyo, Japan).  2.6.6 Fluorescence microscopy Log-phase yeast in minimal selective media were imaged at room temperature with an Axioplan 2 fluorescence microscope (Carl Zeiss Inc., Jena, Germany) equipped with a Plan-Apochromat 100×/1.40 NA oil immersion objective lens (Zeiss) and a CoolSNAP camera (Roper Scientific, Tucson, AZ) using MetaMorph 7.7 software (MDS Analytical Technologies, Sunnyvale, CA). Alternatively, in panels 2C and 6, images were acquired with a DMi8 microscope (Leica Microsystems GmbH, Wetzlar, Germany) using an HC PL APO 63x/1.30 Glyc CORR CS, or an HC PL APO 100x/1.40 OIL STED WHITE objective (Leica) and an ORCA-Flash4.0 digital camera (Hamamatsu Photonics, Hamamatsu City, Japan). Images were adjusted using MetaMorph and Photoshop CS5 (Adobe, San Jose, CA). Maximum intensity z-projections were based on z-stacks of 9 planes with a 0.3m step size and made using MetaMorph after photobleaching correction based on histogram matching in Fiji (www.fiji.sc/Fiji). Cellular features were quantified by manually scoring images, except the Sec7-dsRed quantification which was 51  done by automated counting using in-house Metamorph-based algorithms. For total internal reflection fluorescence microscopy (TIRF) experiments, cells from early-log phase cultures were incubated in a final concentration of 200 μM LatA dissolved in DMSO for 30 min at 30°C. 200 μL of treated cells were spotted onto Concanavalin A-coated 8-well Lab-Tek dishes (Nalge Nunc International, Rochester, NY), and TIRF images were collected with a 3i Marianas microscope (Intelligent Imaging Innovations, Denver, CO) equipped with an α-Plan-Fluor 100x 1.45 NA objective lens and a Zeiss TIRF slider (Carl Zeiss Inc.). Images were acquired with 488nm and/or 561nm laser excitation, with GFP and RFP emission split between two Cascade II 512 EM cameras (Photometrics, Tucson, AZ) with an Optical Insights Dual Cam (Photometrics) with an exposure time of 750ms. TetraSpeck 100nm beads (Life Technologies, Carlsbad, CA) were used to align the two channels and for subsequent registration in software. Slide-Book 4.2® software (Intelligent Imaging Innovations) was used for image acquisition and dual channel image registration. Montages were created using the National Institute of Health ImageJ software (rsb.info.nih.gov/ij/) with the Kymograph plugins installed (www.embl.de/eamnet/html/kymograph.html).  2.6.7 Co-immunoprecipitation and Western Blotting  Log-phase cells in YPD media were converted to spheroplasts by digesting with zymolyase (MJS BioLynx, Brockville, Canada), and stored at -85°C until use, as described (Conibear and Stevens, 2000). 40 OD600 of cells were resuspended in co-IP lysis buffer [20 mM HEPES pH7.5, 100 mM NaCl, 2 mM EDTA, with 1% CHAPSO and 1 mM PMSF (phenylmethylsulfonyl fluoride) added before use] and centrifuged for 15 min at 4°C. Supernatant fractions were incubated at 4°C for 1 h with rabbit anti-HA (Santa Cruz Biotechnology, Dallas, TX) or rabbit anti-GFP (Molecular Probes, Carlsbad, CA) followed by Protein A Sepharose beads (GE Healthcare, Little Chalfont, United Kingdom) for 2.5 h. The beads were washed and proteins were eluted by heating at 95°C for 5 minutes. Samples were run on 10% SDS-PAGE gels. Proteins were transferred overnight to nitrocellulose membranes and blotted with either mouse anti-GFP (Roche) or mouse anti-HA (Covance, Princeton, NJ), then with goat anti-mouse antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch, West 52  Grove, PA). Blots were developed with the enhanced chemiluminescent West Pico (Pierce, Rockford, IL) and West Femto (Pierce) and exposed to Amersham Hyperfilm (GE Healthcare).  2.6.8 Yeast two-hybrid assay PJ694a strains carrying pGBDU-C2-based plasmids were mated to PJ694α strains carrying pGAD-C2-based plasmids, each expressing the indicated full-length or truncation proteins (James et al., 1996). Positive two-hybrid interactions were scored on minimal media (SD-ura-leu-his+ade) containing 3, 5, or 10mM 3-AT. 2.6.9 Bioinformatic analyses All sequences are from Uniprot (www.uniprot.org). Homologs in other organisms were found using the Panther classification system (www.pantherdb.org). Visualization of MUSCLE-aligned sequences (Edgar, 2004) used Jalview (www.jalview.org). For 3D structure modeling, Phyre2 with “intensive” settings was used (Kelley and Sternberg, 2009). Apm2 tyrosine-motif recognition pocket residues were labelled based on sequence conservation with known tyrosine-pocket residues of AP complex µ subunits (Owen, 1998; Heldwein et al., 2004), and the predicted region involved in lipid-binding was extrapolated based on alignment with human µ1B (Fields et al., 2010). 2.6.10 Membrane fractionation The membrane fractionation protocol was adapted from Conibear and Stevens (2000). 60 OD600 of log-phase cells were spheroplasted, lysed and spun at 500g for 5 min at 4°C. The P13 fraction was pelleted at 13 000g for 10 min at 4°C and resuspended in SDS sample buffer (5% SDS, 50 mM Tris pH 6.8, 0.4 mg/mL bromophenol blue, 1% β-mercaptoethanol). The supernatant was centrifuged at 100 000g in a table-top ultracentrifuge for 60 min, to generate the P100 pellet which was resuspended in 1 mL lysis buffer as the P100 fraction, and the supernatant kept as the S100 fraction. For high-salt treatment of the P100 and S100 fractions, NaCl was added to a final concentration of 1M before the 100 000g spin. Samples were TCA precipitated, resuspended in SDS sample buffer and subjected to SDS-PAGE and immunoblotting. 53  Chapter 3: A yeast genome-wide screen for AP-1 trafficking regulators reveals that Fez proteins, aftiphilin and CLBA1 are all related to a fundamental Laa complex in yeast through binding to HEATR5-family members  A version of this chapter is a manuscript in preparation: Whitfield ST, Tam YYC, Sridhar V, Schluter C, Davey M, Mast F, Rachubinski RA, Conibear E. Fez proteins, aftiphilin and CLBA1 are all related to a fundamental Laa complex in yeast through binding to HEATR5-family members.  3.1 Synopsis This chapter describes our discovery of a new AP-1 regulatory complex in yeast, and the identification of related complexes in mammalian cells. To find new AP-1 regulators we performed a genome-wide screen based on the sorting of the model polytopic membrane protein Chs3 (chitin synthase 3). Appearance of Chs3 at the cell surface depends on the exomer complex, and when exomer is deleted Chs3 is retained intracellularly at endosomes in an AP-1-dependent manner. It is not known whether AP-1 acts in anterograde or retrograde trans-Golgi network/endosome sorting, and a full picture of the AP-1-associated trafficking machinery has been lacking. We took advantage of a “bypass” phenotype that occurs upon deletion of both exomer and the AP-1 pathway to address these issues. We then performed an epistatic mini-array (E-MAP) analysis of top hits to identify functionally-connected genes and integrated these results with other high-quality large-scale datasets to uncover functionally related genes. We identified the uncharacterized YBL010C (Laa2) and Slo1 as members of a complex with the AP-1 regulator Laa1 and showed that Laa2 contains an AP-1 γ appendage binding motif that allows it to bridge the interaction between Laa1 and AP-1. Based on homology, we hypothesized that there is a similar “Laa-like” complex in humans. We identified such a complex, consisting of components previously thought to be involved in separate trafficking pathways – Fez1/2 and SCOC in synaptic vesicle trafficking and autophagy, and HEATR5A in AP-1-mediated trafficking. This mammalian “Laa-like” complex is distinct from the previously-identified aftiphilin/γ-54  synergin/HEATR5B complex involved in AP-1 function and is conserved in yeast, suggesting a fundamental role in protein trafficking that may involve multiple pathways. Fez1/2, aftiphilin and the aftiphilin-related CLBA1 all share a conserved site for binding HEATR5-family proteins, providing a potential link between many different trafficking pathways. 3.2 Introduction Selective sorting of proteins between cellular compartments is critical to cell survival and functioning. At the Golgi/endosome compartment, the AP-1 adaptor protein (AP) complex selects cargo proteins for inclusion into clathrin-coated vesicles (CCVs). It also coordinates the machinery necessary for vesicle formation, providing an essential hub for regulatory inputs to ensure a high level of specificity. Accessory proteins play important roles in AP-1 function and vesicle trafficking (Duncan et al., 2003; Hirst et al., 2015; Chamberland et al., 2016). Currently, far fewer accessory proteins have been identified for AP-1 than for the endocytic adaptor complex AP-2, and for those that have been found a biological function or mechanism is often missing. Identifying and characterizing the full complement of AP-1 accessory proteins is necessary for a proper understanding of CCV trafficking from Golgi/endosomes. AP-1 is a heterotetramer consisting of two large subunits (β1 and γ), one medium subunit (µ1) and one small subunit (σ1). Many AP-1 accessory proteins bind to the AP-1 γ “appendage” domain, a C-terminal extension that projects from the rest of the complex on a long, flexible linker (Lui et al., 2003; Kent et al., 2002; Shiba et al., 2002; Neubrand et al., 2005; Takatsu et al., 2000; Mattera et al., 2004). These proteins contribute to different stages of CCV trafficking. NECAP2 facilitates recruitment of AP-1/clathrin to early endosomes (Chamberland et al., 2016). EpsinR (also known as enthoprotin, CLINT1 or epsin4) is thought to be a cargo-selective adaptor (Hirst et al., 2004; Miller et al., 2007) that stimulates clathrin assembly on membranes (Wasiak et al., 2002). Gadkin (formerly γ-BAR) regulates endosomal membrane traffic by connecting the kinesin-1 motor protein KIF5 to AP-1 (Schmidt et al., 2009). Cyclin-G-associated kinase (GAK, also called auxilin 2) acts as a cofactor in Hsc70-mediated uncoating of clathrin-coated vesicles (Eisenberg and Greene, 2007), but also recruits clathrin and adaptors to 55  clathrin-coated pits (Lee, 2005) and activates the endocytic AP-2 complex (Conner and Schmid, 2003). Less is known about the mechanism of action of aftiphilin and γ-synergin, which each contain numerous γ-ear binding motifs and bind the HEAT repeat protein p200/HEATR5 to form a complex that may act as a cargo-specific adaptor and/or link to the cytoskeleton (Hirst et al., 2005). Mammalian p200 is a largely uncharacterized but well-conserved HEAT-repeat-containing protein of approximately 200kDa that has two paralogs in human cells, p200a/HEATR5B and p200b/HEATR5A, which share 58% amino acid identity. HEATR5B forms a complex with aftiphilin and γ-synergin that is required for sorting of AP-1-dependent cargoes (Hirst et al., 2005) and for maturation of Weibel-Palade bodies, specialized secretory granules involved in blood clotting (Lui-Roberts et al., 2008). It is not clear whether HEATR5A also participates in this complex, and if HEATR5A and 5B provide redundant functions (Hirst et al., 2005; Lui-Roberts et al., 2008). Intriguingly, the yeast Saccharomyces cerevisiae lacks apparent aftiphilin or γ-synergin homologs but has a p200/HEATR5 homolog, Laa1, that contributes to proper AP-1 localization (Fernández and Payne, 2006). This function is shared with Laa1/HEATR5 homologs in flies (D. melanogaster- CG2747; Le Bras et al., 2012) and worms (C. elegans- SOAP-1; Gillard et al., 2015).  In this study, we used a yeast genome-wide screen and epistatic mini-array profile (E-MAP) approach to uncover new protein trafficking components involved in sorting the AP-1 cargo chitin synthase 3 (Chs3). We showed that the AP-1 accessory protein Laa1 is part of a previously-undiscovered complex with Laa2 (the product of the uncharacterized ORF YBL010C) and Slo1, and that all these components contribute to AP-1 recruitment lifetime on membranes. We then identified a corresponding complex in mammalian cells, composed of HEATR5A, Fez1/2 and SCOC. Fez1 and Fez2 are kinesin-1 cargo adaptors that form a complex with SCOC to regulate synaptic vesicle trafficking and autophagy (Gindhart, 2003; Su et al., 2006; Bloom and Horvitz, 1997; McKnight et al., 2012; Spang et al., 2014) but have not been linked to AP-1. We found a conserved HEATR5-binding domain in Laa2, Fez2, aftiphilin and the aftiphilin-related protein CLBA1, suggesting that HEATR5 family members are involved in an unexpected diversity of trafficking processes in humans. 56  3.3 Results 3.3.1 A genome-wide screen identifies candidate AP-1 regulators We initially performed a yeast genome-wide screen to gain a global, comprehensive picture of genes involved in the AP-1-dependent trafficking of the model polytopic membrane protein chitin synthase 3 (Chs3). Chs3 is the primary chitin synthase in yeast (Shaw et al., 1991) and its activity at the cell surface can be visualized using the fluorescent dye calcofluor white (CW), which stains chitin in the yeast cell wall. Deletion of the exomer subunit Chs6 causes intracellular retention of Chs3 and decreased CW fluorescence (Figure 3.1A), which can be “bypassed” by additional deletion of AP-1 pathway components (Liu et al., 2008; Valdivia et al., 2002). We exploited this retention-bypass phenotype to uncover new AP-1 regulators, using synthetic genomic array (SGA) technology (Tong, 2001) to cross a chs6Δ mutation into the MATa and MATα yeast gene deletion collections (Giaever et al., 2002) and measuring the fluorescence of CW-stained double mutant colonies.  Plotting a ranked list of average Z-scores generated from duplicate screens of the two collections (Figure 3.1B) shows that approximately 150 genes exhibited a robust chs6Δ bypass phenotype (Z-score >1.5). The top hits from our screen included both AP-1 subunits and dubious ORFs predicted to overlap with AP-1 subunits (Gorynia et al., 2012), demonstrating that our approach was specific for AP-1-mediated trafficking components. This suggested that some of the other top hits might be novel AP-1 regulators.   57   Figure 3.1 A genome-wide screen identifies candidate AP-1 regulators. A) Although yeast colonies grown on calcofluor white plates appear similar under white light (left panels), chs6Δ cells exhibit decreased fluorescence under UV light (right panel) due to intracellular retention of the chitin synthase Chs3. Deletion of the AP-1 subunit Apl2 bypasses this retention and restores cell-surface Chs3 and fluorescence. B) Plot representing all yeast knockout strains ranked by Z-score with rank of AP-1 subunit genes indicated. C) Cytoscape representation of correlations between genes with a chs6 bypass Z-score >1, arranged by approximate cellular location. This figure is not intended to distinguish between AP-1 roles at the TGN/endosomes. Node size is mapped to strength of the chs6 bypass phenotype; values for clusters and components are given in Table B2. Colour of edge lines denotes the dataset used for correlation analysis. Knockouts of dubious ORFs that are assumed to affect a known gene on the opposite strand are given in the format [dubiousORF]/[known ORF], e.g. YKL136W/APL2.  58  3.3.2 Refining top hits using genetic interaction profiles To help us focus on connections between contributors to Chs3 trafficking, we performed an epistatic miniarray profile (E-MAP) experiment (Collins et al. 2010). E-MAP allows for dissection of functional connections between genes while providing a higher signal-to-noise ratio because of rational selection of the panel. We selected the top ~250 genes, corresponding to the genes with a Z-score above 1 in our Chs6 bypass screen (Supplementary Table B1), to avoid missing true but weaker connections. Whereas the traditional E-MAP approach uses a synthetic lethality readout from all mutants crossed against themselves, we crossed our selected mutants against a panel of trafficking-related genes and measured the CW fluorescence of the resultant double mutants to gain more information about functional trafficking connections in this context.  To assess connections between genes of interest, we integrated genetic correlations from three independent but complementary screening approaches. Our E-MAP screen focused on the relationship between gene pairs in the specific context of CW fluorescence and CHS6 deletion. The Cellmap synthetic genomic array (SGA) dataset (Usaj et al., 2017) provides information regarding the functional overlap between two genes based on their general fitness, while the Novartis chemicogenomics dataset (Hoepfner et al., 2014) assesses the fitness of single gene mutants in response to a battery of small molecules. For each dataset, we computed correlations between top hits from our initial Chs3 genome-wide screen (see Methods section for full detail), and performed clustering on the combined correlation data using the MCL (Markov Cluster Algorithm) through the Cytoscape Clustermaker2 analysis suite (Morris et al., 2011). Figure 3.1C shows a representation of clusters arranged by approximate cellular localization; AP-1 is proposed to function at both the trans-Golgi network (TGN) and endosomes and has been represented at the Golgi for graphical convenience. However, recent work by Day et al. requires a re-evaluation of established definitions of yeast TGN/endosomes and the roles of AP-1 at these compartments Node size indicates strength of the chs6 bypass phenotype and edge colour indicates the dataset giving the correlation between genes. CW phenotype median values were also calculated for each MCL cluster and for functional groups within each cluster (Supplementary Table B2).  59  A persistent question is whether AP-1 acts in anterograde or retrograde routes between the trans-Golgi network and endosomes (reviewed in Hinners and Tooze, 2003). With regards to Chs3 trafficking, deletion of exomer and AP-1 could cause a bypass at the level of the TGN or endosomes. In our screen the top clusters largely represent endosomal, endosome-Golgi recycling, and AP-1 trafficking machinery, supporting a role for AP-1 in retrograde sorting of Chs3 (or of another component necessary for its trafficking, such as the flippase Drs2). Arf1 (Valdivia et al., 2002), Irc6 (Gorynia et al., 2012; Babu et al., 2012) and Drs2 (Liu et al., 2008) work with AP-1, and also exhibit the chs6 bypass phenotype, but could act at either the TGN or endosomes. However, additional machinery such as the Arf1-regulators Gcs1 and Age2 (Zendeh-Boodi et al., 2013), recycling machinery Rcy1, Ypt31 and Cdc50 (which work with Drs2) (Hanamatsu et al., 2014; Chen et al., 2006; Furuta et al., 2006) and the GARP tethering complex (Conibear and Stevens, 2000) work primarily in endosome-TGN recycling.  Components of endosome transition/maturation, including endosomal Rabs and GEFs (Vps9, Vps21, Pep12) and the CORVET and HOPS complexes, provided a larger overall contribution than previously-identified Chs3 trafficking machinery like ESCRT and retromer (Arcones et al., 2016; Cui et al., 2017). This could reflect the importance of the former in establishing a compartment distinct from other early or late endosomes (the “chitosome”), or in general endosome homeostasis. HOPS has also been implicated in AP-3 function (Angers and Merz, 2009); AP-3 was suggested to have overlapping roles with AP-1 in Chs3 sorting (Starr et al., 2012). Meanwhile, ESCRT and retromer may provide a backup Chs3 recycling mechanism when the AP-1/Ent3/Ent5-mediated recycling pathway has been overwhelmed (Arcones et al., 2016). The requirement for vacuolar Rabs and SNARES and the V-ATPase machinery is not entirely unsurprising, since depletion of the V-ATPase can cause formation of aberrant/nonfunctional clathrin-coated structures (Kozik et al., 2013), which may essentially mimic an AP-1 deletion phenotype and indirectly affect Chs3 trafficking.  60  The high stringency of our clustering may have resulted in some fragmentation of clusters. For example, RCY1, YPT31, CDC50 and DRS2 are implicated in a common pathway for recycling from endosomes (Furuta et al., 2006) but were placed in three different groups in our analysis. Collectively, these analyses suggest that AP-1 is involved in sorting of Chs3 at the level of endosomes. 3.3.3 Laa1 and Laa2 (YBL010C) co-localize and form a complex We were particularly interested in genes that clustered with AP-1 and encode known or candidate AP-1 regulators: IRC6, LAA1 and YBL010C. The ORF YBL010C was the only uncharacterized gene that clustered with AP-1 in our network analysis. We observed that this ORF, which we will refer to as LAA2, was linked with LAA1 through a chemicogenetic interaction from the Novartis dataset (Figure 3.1C). To see whether Laa1 and Laa2 are linked to AP-1, we first examined the cellular localization of Laa1 (Figure 3.2A) and Laa2 (Figure 3.2B) by fluorescence microscopy. Laa1 co-localized with the AP-1 medium subunit Apm1 more than with Gga2 (35.5 ± 0.75% vs 25.9 ± 0.24%), another Golgi-localized protein, perhaps indicating a greater specificity for the AP-1 complex. These numbers are generally consistent with the previous study of Laa1, which showed greater overlap of Laa1 with AP-1 than with Gga2 (Fernández and Payne, 2006). The lower percentage of co-localization could reflect differences in counting technique: our automated co-localization uses area overlap as a measure of co-localization, and compared to hand-counting may under-estimate the true extent of co-localization if the two puncta are not perfectly overlapping. Laa2 co-localized similarly with Laa1 and with (26.0 ± 0.61%), and with the AP-1 medium subunit Apm1 (Laa2: 28.0 ± 1.31%). Laa2 puncta are often small and dim, so all we can conclude here is that Laa2 is found at similar compartments as Laa1 and Apm1. Laa1 and Laa2 co-localized poorly with Snx3-Ruby (Laa1-GFP: 3.43 ± 0.25%, Laa2-GFP: 4.90 ± 0.88%), which is found at endosomes (Strochlic et al., 2007). To assess the dependence of Laa1 and Laa2 recruitment on AP-1 and on each other, we performed microscopy of Laa1-GFP and Laa2-GFP in apl2Δ, laa1Δ and laa2Δ strains (Figure 3.2C). Neither Laa1 nor Laa2 appeared to be dependent on the large AP-1 subunit Apl2 for localization. Automated counting showed a reduction in the number of 61  Laa1-GFP puncta when LAA2 was deleted (Figure 3.2D) (p<0.0001, two-tailed t-test, n=8), and vice versa (Figure 3.2E) (p=0.01).   Figure 3.2 Laa1 and Laa2 co-localize and form a complex. A) By fluorescence microscopy, Laa1 co-localizes to a higher extent with AP-1 than with other Golgi proteins (Gga2) or late endosome markers (Snx3). TdT: TdTomato red fluorescent tag. All proteins were genomically tagged. Automated quantification shown to right of images. Data represent the average of three separate experiments, >100 cells each, error bars are standard error of the mean (S.E.M.). One-way ANOVA with Tukey post-hoc, ****: p<0.0001. Scale bar, 2µm. B) Laa2 co-localizes to a higher extent with Laa1 and AP-1 than with Snx3. Same data analysis as in A. N.S.: No significance. C) Representative fluorescence microscopy of Laa1-GFP or Laa2-GFP strains with indicated genomic deletions. Deletion of LAA1 but not APL2 slightly decreases the number of Laa2-GFP puncta visible by eye. Scale bar, 2µm. D) Automated quantification of Laa1-GFP puncta with indicated deletions or plasmid complementation. Data represent the average of eight separate experiments, error bars are S.E.M. Unpaired two-tailed t-test, ****: p<0.0001, ***:  p<0.001, **:  p<0.01, N.S.: No significance. E) Same as in D but with Laa2-GFP. *:  p<0.05. F) Deletion of LAA1 reduces the abundance of Laa2-GFP by western blot. G) Co-immunoprecipitation shows that Laa1 binds Laa2 but also binds Laa1, suggesting that Laa1 is a homodimer.  62  The decrease in Laa1 and Laa2 puncta revealed by automated counting could be partially due to protein degradation. Deletion of LAA1 decreased Laa2-GFP levels by western blot, but not vice versa (Figure 3.2F). The instability of Laa2 upon deletion of LAA1, combined with our microscopy results, suggested that Laa1 and Laa2 may form a complex. Removal of one component of a tight complex may affect the stability of its other members; the stability of the Laa1 ortholog HEATR5B is compromised by knockdown of its binding partners aftiphilin and γ-synergin (Hirst et al. 2005). We tested whether Laa1 and Laa2 could interact by co-immunoprecipitation (co-IP) (Figure 3.2G). We detected Laa2-Laa1 and Laa1-Laa1 interactions and could not detect any Laa2-Laa2 dimerization (data not shown). We also tested whether Laa1 and Laa2 were found in the same fractions after gel filtration (Supplementary Figure B1). Laa1 and Laa2 co-eluted, with a peak in abundance in fraction 14 corresponding to approximately 450 kDa, supporting the idea that they form a complex. One explanation for the observed size is that Laa1 (~230 kDa) and Laa2 (~33 kDa) are present in 2:1 stoichiometry. These results indicate the existence of a “Laa complex”, likely containing two copies of Laa1 and one copy of Laa2.  3.3.4 Laa2 bridges the interaction between AP-1 and Laa1 via an FGxF motif We next investigated the connection between the Laa complex and AP-1. Fernández and Payne (2006) had found that AP-1 mislocalized in a laa1Δ strain, with a decreased intensity and frequency of Apl2-GFP puncta. We reproduced this finding, although the phenotype was less pronounced than previously reported. For a more sensitive assessment we turned to lifetime imaging, tracking the movement of each GFP-tagged protein signal over time. Plotting the mean lifetimes of all observed puncta (Figure 3.3A) showed that both LAA1 and LAA2 deletions decreased the mean lifetime of Apl2-GFP puncta. Laa1-GFP mean lifetimes were not significantly affected by loss of APL2 or LAA2, suggesting that Laa1 association with membranes is independent of AP-1.  63   Figure 3.3 Laa2 has an FGxF motif and bridges the interaction between AP-1 and Laa1. A) Laa1 and Laa2 contribute to mean lifetime of AP-1 puncta by fluorescence lifetime microscopy. Laa1-GFP mean lifetime is unaffected by deletion of LAA2 or APL2. Error bars represent the upper and lower 95% confidence intervals of the mean. Dunn’s multiple comparison test, ***:  p<0.001. B) Co-immunoprecipitation showing that interaction between Apm1 and Laa1 is disrupted when LAA2 is deleted. C) Sequence alignment of N-terminal regions of selected yeast species showing the conserved FGxF motif. D) Yeast two-hybrid showing that whereas the AP-1 accessory factor Irc6 binds to the Apl4 core region, wild-type Laa2 binds the Apl4 ear region. Laa2 with a mutated FGxF motif (F24GNF>AANA) can no longer bind the Apl4 ear. E) Co-immunoprecipitation showing that Apl4 and Laa2 normally interact, but no longer interact when either the Apl4 ear is removed (Apl4Δear) or the Laa2 FGxF motif is mutated (F24GNF>AANA). F) Calcofluor-based plate assay showing that the FGxF>AANA Laa2 mutant mimics the “bypass” phenotype of a LAA2 delete. Data represent the average of five separate experiments, error bars are standard error of the mean (S.E.M.). Unpaired two-tailed t-test, ****: p<0.0001, ***:  p<0.001. G) Summary figure: The γ-ear domain of AP-1 binds the FGxF motif of Laa2, allowing Laa1 to interact with AP-1 and recruit it or stabilize it on membranes. 64  Although Apm1 interacted with Laa1 under normal conditions, deletion of Laa2 disrupted this interaction (Figure 3.3B). We hypothesized that Laa2 could link Laa1 and AP-1, and identified a conserved FGxF motif (F24GNF) at the N-terminus of Laa2 (Figure 3.3C) that is reminiscent of a γ-adaptin ear (GAE) binding motif of consensus (ψ)G(P/D/E)(ψ/L/M), where ψ is an aromatic residue (Mattera et al., 2004). Yeast two-hybrid showed that Laa2 specifically interacts with the ear domain of the large AP-1 subunit Apl4 (Figure 3.3D), as compared with the AP-1 accessory factor Irc6 which binds the “core” region of Apl4 (Babu et al., 2012). Mutation of the Laa2 FGxF motif to alanines (F24GNF>AANA) had the same effect as removing the Apl4 “ear” region, as judged by both yeast two-hybrid (Figure 3.3D) and co-immunoprecipitation (Figure 3.3E). Stability of Laa2 was unaffected by this mutation, as shown by equal levels of Laa2 in the GFP lysate of Figures 3.5E and 3.5F and western blotting of the Laa2 yeast two-hybrid constructs (data not shown). The chs6Δ laa2FGxF mutant had an increase in fluorescence on calcofluor white plates compared to chs6Δ alone, similar to LAA1 or LAA2 deleted cells (Figure 3.3F). These results strongly suggest that Laa1 acts on the AP-1 complex through the FGxF motif of Laa2 (Figure 3.3G), with consequences for sorting of Chs3 and likely other cargo. 3.3.5 Laa2 is the central component of a yeast complex consisting of Laa1-Laa2-Slo1 To gain evolutionary insight into the Laa complex, we performed bioinformatic analyses to identify non-yeast homologs of Laa2. BLAST searches did not immediately identify any candidates so we turned to HHPred, which was specifically developed to identify remote homologs of proteins of interest (Soding et al., 2005). Searching the Homo sapiens proteome with Laa2 as a query identified fasciculation and elongation protein zeta 2 (Fez2) and Fez1 as top hits; two main regions of homology were shared between the three proteins (Figure 3.4A). Interestingly, regions from aftiphilin and the aftiphilin-related protein CLBA1 (C14orf79) also aligned with Laa2, albeit at lower confidence.  65  Laa2 was the top hit when we searched the S. cerevisiae proteome for Fez2 or Fez1 homologs. Fez1 has been more intensively studied than Fez2 due to its enrichment in brain tissue (Honda et al., 2004) and links to HIV infectivity (Malikov et al., 2015) and to neurological disease through its interaction with DISC-1 (Disrupted in Schizophrenia 1) (Miyoshi et al., 2003). Both human Fez proteins have a common ancestor in Unc-76, which is thought to function as a kinesin-1 activator and cargo adaptor in C. elegans and D. melanogaster and is important for axonal development (Bloom and Horvitz, 1997; Gindhart, 2003; Toda et al., 2008).  One of the better-studied conserved interactions of Unc-76/Fez1 is with the short coiled-coil protein Unc-69/SCOC. The yeast SCOC homolog Slo1 binds Arl3, a GTPase involved in tethering at the Golgi (Panic et al., 2003), which would also be the correct location for Slo1 to interact with Laa2. The yeast genome-wide deletion collection, which we used to make our chs6ΔgeneXΔ mutants for our calcofluor plate assay, does not contain a SLO1-delete strain because Slo1 has a very small ORF (85 amino acids) that was below the 100-amino acid threshold for ORFs that was used for annotation of the Saccharomyces cerevisiae genome and creation of the collection (Panic et al., 2003). A freshly-constructed chs6Δslo1Δ strain showed a small but significant Chs3 bypass phenotype (Supplementary Figure B2A), suggesting that Slo1 may be involved in Chs3 trafficking.  We tested whether Laa2 could interact with Slo1, as would be predicted if Laa2 is a yeast form of Fez1/2. We found that Slo1 could bind Laa2 (Figure 3.4B) and also Laa1 (Figure 3.4C) by co-IP, although this second interaction depended on LAA2. Deletion of LAA1 or SLO1 affected the stability of Laa2 but not its interaction with Slo1 (Figure 3.4B) or Laa1 (Figure 3.4D), respectively. SLO1 deletion also decreased the number of Laa1-GFP and Laa2-GFP puncta in steady-state microscopy (Supplementary Figure B2B). These data suggest that the Laa complex contains Laa1, Laa2 and Slo1, with Laa2 bridging the interaction between the other two proteins.   66   Figure 3.4 Laa2 is the central component of a yeast complex consisting of Laa1-Laa2-Slo1. A) MUSCLE sequence alignment of selected yeast, worm (C. elegans) and human (H. sapiens) sequences. Laa2 has regions of homology (tan, orange) with human Fez1 and Fez2. Green represents predicted coiled-coil region. Top of figures B, C and D: cartoon diagrams showing which proteins are being tested for interaction; transparency indicates protein deletion. B) Co-immunoprecipitation (co-IP) showing that Slo1 interacts with Laa2. LAA1 deletion destabilizes Laa2 but otherwise does not affect the co-IP between Slo1 and Laa2. C) Co-IP showing that Slo1 interacts with Laa1 and that this interaction depends on Laa2. D) Co-IP showing that the interaction between Laa2 and Laa1 does not depend on Slo1, although loss of SLO1 destabilizes Laa2. E) Co-IP showing that the Laa2 Y91A mutation selectively disrupts the interaction between Laa2 and Laa1, whereas Laa2 F24GNF>AGAA and L237P do not disrupt this interaction. F) Co-IP showing that the Laa2 L237P mutation selectively disrupts the interaction between Laa2 and Slo1, whereas Laa2 F24GNF>AGAA and Y91A do not disrupt this interaction.  67  To identify the Laa1 and Slo1 binding sites on Laa2, we performed alanine mutagenesis of highly conserved residues. Mutation of tyrosine 91 of Laa2 (Laa2Y91A) selectively abolished its interaction with Laa1 by co-IP (Figure 3.4E). A previously-reported mutation of leucines 254 and 260 of HsFez1 disrupts its interaction with SCOC (McKnight et al., 2012). Mutation of a similar leucine in Laa2 (Laa2L237P) disrupted the interaction between Laa2 and Slo1 (Figure 3.4F). Identification of these binding residues strengthens the idea that Laa2 is the yeast homolog of mammalian Fez proteins. 3.3.6 A mammalian form of the Laa complex contains Fez2, SCOC and HEATR5A Our results thus far identify Laa2, Slo1 and Laa1 as members of a “Laa complex” in yeast cells that promotes AP-1 membrane association. Laa1 is well conserved throughout eukaryotes and is homologous to human HEATR5A and HEATR5B (Fernández and Payne, 2006; Hirst et al., 2005). HEATR5B partners with the γ-ear binding proteins aftiphilin and γ-synergin to form a complex that is involved in AP-1-mediated transport, but it is unclear whether HEATR5A also participates in this kind of complex (Hirst et al., 2005; Lui-Roberts et al., 2008). We hypothesized that HEATR5A may bind SCOC and Fez1/2, forming a complex more similar to the yeast Laa complex than the aftiphilin-γ-synergin-HEATR5B complex. In HEK293T/17 cells over-expressing SCOC (isoform 5) and Fez2 (Figure 3.5A), HEATR5A bound SCOC and Fez2, whereas HEATR5B bound γ-adaptin, γ-synergin and aftiphilin. We found a similar albeit weaker interaction between HEATR5A and Fez1 (Supplementary Figure B3A). Since these results suggested the yeast Laa complex was conserved in mammalian cells as Fez2-SCOC-HEATR5A, and we had already found an amino acid residue on Laa2 necessary for Laa1 binding (Laa2Y91A), we tested whether an analogous mutation in Fez2 would disrupt its interaction with HEATR5A. Mutation of W91 in Fez2 largely disrupted the interaction between Fez2 and HEATR5A by co-IP (Figure 3.5B). These results support the idea that there are two distinct complexes in mammalian cells that are related to the yeast Laa complex: the AP-1-associated 68  aftiphilin-γ-synergin-HEATR5B complex that associates with AP-1 and the HEATR5A-SCOC-Fez2 complex that likely participates in other pathways.  Figure 3.5 A mammalian form of the Laa complex contains Fez2, SCOC and HEATR5A. A) Co-immunoprecipitation (co-IP) showing that SCOC, Fez2 and HEATR5A interact with each other but not with AP-1 (y-adaptin), aftiphilin, y-synergin or HEATR5B. HEK293T cells were transiently co-transfected with GFP-SCOC and Flag-Fez2. α-F indicates anti-Flag antibody used for pulldown; α-G indicates anti-GFP; α-A indicates anti-adaptin γ; IgG indicates immunoglobulin G negative control. B) Co-IP showing that the Fez2 W91A mutation selectively disrupts the interaction between Fez2 and HEATR5A. Same transfection and co-IP strategy as above. C) HEATR5A co-IPs with Fez2, GFP-SCOC and myc-UVRAG. α-M indicates anti-Myc antibody used for pulldown. Same transfection and co-IP strategy as above. D) UVRAG interaction with Fez2 and SCOC does not depend on HEATR5A. The Fez2 W91A mutation selectively disrupts the interaction between Fez2 and HEATR5A but not Fez2 and UVRAG. Same transfection and co-IP strategy as above. Having identified HEATR5A as a component of a Fez-SCOC complex, we were interested in whether it participates in previously-studied Fez-SCOC pathways. The Fez-SCOC complex is thought to regulate macroautophagy (hereafter referred to simply 69  as autophagy) by binding and sequestering the phosphatidylinositol (PI) 3-kinase component UVRAG (McKnight et al., 2012), preventing it from promoting autophagosome maturation. More recently, Fez2 has also been linked to autophagy (Spang et al., 2014). We tested whether HEATR5A was present when Fez1/2 interacts with UVRAG. We detected UVRAG, SCOC, Fez1/2 and HEATR5A in a complex upon reciprocal co-IPs (Figure 3.5C, Supplementary Figure B3B). Pulldowns using our Fez2W91A mutant showed that UVRAG binding to Fez2/SCOC appeared to be unaffected; whereas HEATR5A was no longer present, UVRAG levels were constant (Figure 3.5D). These results suggest that some HEATR5A is present but not required for Fez and SCOC binding to UVRAG.  3.3.7 Laa2, Fez2, Aftiphilin and CLBA1 share a conserved HEATR5-family binding domain In our HHPred analysis, we noticed homology of Laa2 with a region in aftiphilin and the aftiphilin-related protein CLBA1/C14orf79. The region of homology maps to the Laa1-binding site of Laa2 (Figure 3.6A, tan region). Since we had also found that the corresponding region of Fez2 is involved in binding HEATR5A (Figure 3.5B), we hypothesized that this represents a common domain for binding to HEATR5-family proteins and is the site where aftiphilin binds HEATR5B. Mutating the analogous conserved tryptophan (W702A) in aftiphilin selectively disrupted interaction with HEATR5B (Figure 3.6B), leaving γ-synergin, clathrin and AP-1γ binding seemingly unaffected. The nearby Y716QW>SQS mutant previously implicated in clathrin binding (Hirst et al., 2005) had similar effects to the W702A mutant, surprisingly affecting HEATR5B binding but not the interaction with clathrin.  70   Figure 3.6 Laa2, Fez2, Aftiphilin and CLBA1 share a conserved domain for HEATR5-family binding. A) Laa2 has a region of homology with Fez2, aftiphilin and CLBA1 (tan). Residues mutated during this study are indicated. B) Co-immunoprecipitation showing that the aftiphilin W702A and Y716QW>SQS mutations selectively disrupt the interaction between aftiphilin and HEATR5B. HEK293T cells were transiently co-transfected with indicated GFP-aftiphilin constructs. Clathrin HC denotes clathrin heavy chain. C) Co-immunoprecipitation showing that the CLBA1 W197A mutation selectively disrupts the interaction between aftiphilin and HEATR5B. HEK293T cells were co-transfected with indicated constructs as above. CLBA1 was named “clathrin binding box of aftiphilin containing 1” based on its homology to this region of aftiphilin (Hirst et al., 2005), but is otherwise uncharacterized. Because this conserved region is shared with proteins that bind HEATR5-family members, we tested whether CLBA1 could interact with HEATR5B (Figure 3.6C). CLBA1 bound HEATR5B (but not HEATR5A; data not shown) and this interaction was 71  again dependent on the analogous conserved aromatic residue (W197). These results strongly support the idea of a common binding domain for binding to Laa1/HEATR5 proteins. 3.4 Discussion We have performed a yeast genome-wide screen for trafficking machinery that contributes to AP-1-mediated protein sorting. We strengthened our initial chs6Δ-based Chs3 sorting screen with epistatic miniarray profiling (E-MAP) of our top hits, which was integrated with other genome-wide data. By following up on a predicted interaction between Laa1 and YBL010c (Laa2) we identified a new yeast protein complex implicated in vesicle trafficking. This discovery also gave us insight into the mammalian trafficking machinery, as we demonstrated that there are multiple distinct HEATR5-containing complexes that likely evolved from the yeast Laa complex: the AP-1-associated aftiphilin-γ-synergin-HEATR5B complex, a CLBA1-HEATR5B complex and a Fez1/2-SCOC-HEATR5A complex (Figure 3.7). The presence of HEATR5A in the Fez1/2-SCOC-UVRAG complex suggests that it may contribute to autophagy or other UVRAG-associated processes. Laa2 is a new AP-1 accessory protein that links Laa1 and AP-1γ through its FGxF motif. This finding not only adds another protein to the small group of known yeast AP-1 accessory factors but also resolves a longstanding question about how Laa1 influences AP-1 at the molecular level. It was originally suggested that the interaction between Laa1 and AP-1 might be indirect (Fernández and Payne, 2006) but a candidate interactor was not evident: neither aftiphilin nor γ-synergin are present in yeast. Studies of Laa1 homologs in worms (Gillard et al., 2015) and flies (Le Bras et al., 2012) did not test whether aftiphilin or some other factor bridges the interaction, and unc-76/Fez was an unlikely candidate since it already has well-characterized roles in autophagy and kinesin-1 cargo adaptation (McKnight et al., 2012; Spang et al., 2014; Chua et al., 2012; Toda et al., 2008). 72   Figure 3.7 Model summarizing the yeast and mammalian Laa1/HEATR5-containing complexes. Our data suggest that there is a fundamental yeast Laa complex (2xLaa1-1xLaa2-1xSlo1) that regulates AP-1 through an FGxF motif (yellow square on Laa2). This complex is related to three distinct mammalian complexes whose functions have diverged but share a common site for binding HEATR5-family proteins. The aftiphilin-y-synergin-HEATR5B complex associates with AP-1, while the Fez1/2-SCOC-HEATR5A complex interacts with UVRAG. There is also potentially a CLBA1-HEATR5B complex that remains uncharacterized. Laa2 is likely a yeast member of the fasciculation and elongation zeta (Fez) family of proteins, supported by bioinformatic and biochemical similarities. This finding indicates that Fez-family proteins have a much more fundamental function than previously suspected. Unc-76/Fez1’s established role as a kinesin-1 adaptor (Chua et al., 2012; Toda et al., 2008; Alborghetti et al., 2011) in neuronal trafficking seems at odds with Laa2’s AP-1-facilitating role in yeast. It will be important to see whether Laa2 can work as a cargo adaptor in its own right. Laa2’s involvement in sorting of Chs3, a cargo enriched at the bud neck, could reflect a polarized trafficking pathway that was adopted for axonal trafficking. Laa2 may also participate in autophagy, a process that requires Fez1/2 and harnesses many different trafficking pathways. Slo1 interacts with Arl3 (Panic et al., 2003), and recently Arl3 has been linked to autophagy through regulation of Atg9 trafficking and the Cvt selective autophagy pathway (Wang et al., 73  2017). Alternatively, Laa2 may have roles traditionally associated with aftiphilin since we show that they both bear a common HEATR5-binding region.  Laa2, Fez1/2, aftiphilin and CLBA1 are all related, and use a conserved region to bind HEATR5 proteins. There is likely some specificity to the interaction, since we found no binding between Fez1/2 and HEATR5B nor between aftiphilin/CLBA1 and HEATR5A. We have not yet identified determinants of this specificity, nor whether HEATR5A/B can substitute in different complexes upon knockdown of the other. While loss of both HEATR5A and 5B together exacerbates missorting phenotypes (Hirst et al., 2005) it is unclear whether this is because of compensation of single knockdowns on the level of a particular complex or a different trafficking pathway. Another question is whether the complexes we have identified are stably assembled or dynamic. Dynamic competition between aftiphilin and CLBA1 for binding HEATR5B is an attractive mechanism for regulating distinct trafficking pathways.  Although the two HEATR5 isoforms in humans seem largely distinct, the existence of only one HEATR5 homolog in many organisms suggests it may govern multiple pathways in these organisms. Competition for a shared recruitment factor would allow for concurrent regulation of two pathways by the HEATR5-family protein, depending on which interaction it makes. Alternatively, one of the proteins may function independently of a HEATR5 complex in these organisms. We predict that there are forms of our Laa1-Laa2-Slo1 complex that are conserved in worms (SOAP-1,Unc-76,Unc-69) and in flies (CG2747,Unc-76,CG5934), although we cannot say that these are the only HEATR5-containing complexes. Supporting this hypothesis, the worm HEATR5 homolog SOAP-1 shares the uncoordinated (unc) phenotype with Unc-76 (Fez-like) and Unc-69 (SCOC-like) (Kamath et al., 2003), and its fly homolog CG2747 shares a defect in the sensory perception of pain with unc-76 and CG5934 (Neely et al., 2010), suggesting at minimum a common pathway. Arguing against an obligate aftiphilin-HEATR5 complex in worms, neither the worm forms of aftiphilin (Y45G5AM.9) nor γ-synergin (R10E11.6) contribute to E-cadherin sorting, whereas SOAP-1 is involved, having been suggested to provide an AP-1 recruitment role (Gillard et al., 2015).  74  HEATR5 family members likely contribute to their respective complexes by providing conserved scaffolding and recruitment functions. Interestingly, an N-terminal portion of Unc-76 that includes the conserved region involved in HEATR5-family binding is sufficient to target the protein to axons (Bloom and Horvitz, 1997), which may reflect the need for SOAP-1 for recruitment onto axon-bound vesicles. Laa1-family homologs are important for AP-1 recruitment in many organisms: budding and fission yeasts (Fernández and Payne, 2006; Yu et al., 2012), flies (Le Bras et al., 2012) and worms (Gillard et al., 2015). The exact basis of this recruitment remains unknown, however. Further investigation clarifying the role of HEATR5-family members and their mechanism for recruitment will be vital for understanding the functions of Laa2, Fez1/2, aftiphilin and CLBA1. The association of HEATR5A with the autophagy effector UVRAG suggests that it is needed for some UVRAG-associated processes. UVRAG is a multi-functional protein, regulating both autophagosome formation and maturation (Liang et al., 2008; He et al., 2013; Kim et al., 2015) and endocytic/endosomal trafficking (McKnight et al., 2014; Thoresen et al., 2010; Nakajima et al., 2017) through interaction with different machinery such as Beclin-1 and the HOPS tethering complex in these pathways. HEATR5A could contribute by providing a recruitment and scaffolding platform on autophagosomes/endosomes and facilitating connections with the microtubule motor machinery. We are working on knocking down HEATR5A to directly test whether it is implicated in autophagy or other UVRAG-associated processes. Our current results suggest that studies of UVRAG should consider the potential contribution of HEATR5A as part of the Fez1/2-SCOC complex.  3.5 Materials and Methods 3.5.1 Yeast strains and plasmids BY4741, BY4742, and the yeast genome-deletion collection were purchased from GE Dharmacon (Lafayette, CO). Strains from this study were made by homologous recombination as described (Longtine et al., 1998; Janke et al., 2004; Sheff and Thorn, 2004) or taken from the yeast deletion collection. Transformations were performed as 75  described in section 2.6.1. Mutants in Figures 3.4E and 3.4F were constructed by co-transformation of a laa2Δ strain with Laa2-GFP::HIS PCR products containing the desired mutations. Strains were propagated in rich media (YPD: 1% yeast extract, 2% peptone, 2% dextrose) or SD minimal media (0.17% yeast nitrogen base, 0.5% ammonium sulfate, 2% synthetic complete mix, 2% dextrose) supplemented with the appropriate amino acids or drugs for plasmid selection. Plasmids were made by homologous recombination in yeast, rescued in E. coli and confirmed by sequencing. Proteins were tagged with fluorescence or epitope tags by PCR amplification of the appropriate cassette and transformation into yeast. Strains and plasmids used in this chapter are described in Supplementary Tables B3 and B4. 3.5.2 Mammalian plasmids Flag-Fez1 (CAT#HG15551-NF) was purchased from Sino Biologicals (Beijing, China) through Cedarlane Laboratories (Burlington, ON, CA). Flag-Fez2 plasmid was obtained by PCR amplification of the Fez2 clone from the Mammalian Gene Collection (GE Dharmacon, catalog # MHS6278-202800929) and subcloning into pFLAG-NT (provided by Dr. Stefan Taubert, Centre for Molecular Medicine and Therapeutics, Vancouver, CA). GFP-SCOC plasmid was obtained by Gateway cloning from pDONR-SCOC (GE Dharmacon, catalog # OHS1770-202312830) to pDS_GFP-XB mammalian expression vector (ATCC ID 10326342). Myc-UVRAG plasmid was kindly provided by Dr. Sharon Tooze (The Francis Crick Institute, London, UK). GFP-aftiphilin plasmid was the kind gift of Dr. Jennifer Hirst (Cambridge Institute for Medical Research, Cambridge, UK). Myc-CLBA1 plasmid was purchased from Genscript (CloneID OHu07365). Fez2, aftiphilin and CLBA1 mutations were made by quick-change mutagenesis and verified by sequencing. 3.5.3 Mammalian cell culture HEK293T/17 cells (ATCC CRL-11268) were maintained in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) with 10% fetal bovine serum (FBS; Gibco). Cells were transfected using Lipofectamine 2000 (ThermoFisher Scientific, cat# 11668019) according to the manufacturer’s recommendations with a DNA:lipofectamine 76  ratio of 1:2.5. 24h post-transfection, cells were harvested with addition of lysis buffer and cell-scraping, or by trypsinization, washing with PBS and freezing pellets at 80°C until use.   3.5.4 Calcofluor-white based genomic screen and E-MAP MATa or MATα yeast knockout collections were mated with a chs6Δ strain using the SGA technique (Tong et al., 2001) and pinned in 1,536-array format onto YPD plates containing 50 μg/mL calcofluor white (fluorescent brightener 28, Sigma Aldrich, St. Louis, MO), using a Virtek automated colony arrayer (Bio-Rad Laboratories, Hercules, CA). After incubation at 30°C for 3 days, white-light images were acquired using a model 2400 flat-bed scanner (Epson, Nagano, Japan), 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). Two replicates of each screen (MATa or MATα) were performed. For the E-MAP screen, pairwise deletions of all combinations of the top hits of the genome-wide screen (minus mitochondrial and particularly slow-growing hits) were generated by SGA technology (resulting in yfg1Δ yfg2Δ chs6Δ triple mutants) and plated on calcofluor white-containing plates as before. E-MAP strains were run in six batches. Small-scale testing of strains in Figure 3.3F and Supplemental Figure B3A was done as above but images were captured on a Syngene PXi imager (Syngene, Frederick, MD) using the Epi White and Epi UV light source with a 525nm filter. The formerly open-source program GridGrinder was used for densitometry of digital images, using an in-house script. 3.5.5 Screen data analysis and clustering Each of the four chs6 screens (two MATa and two MATα) were normalized by centering on the median plate intensity. Results were filtered for poor growth (CW growth < 0.4) and combined by averaging. Z-scores were calculated from the averaged, filtered values. EMAP data were median-centered using the open-source program Cluster3.0, obtained from bonsai.hgc.jp/~mdehoon/software/cluster/software.htm. A list of genes with a Z-score >1 in the chs6 bypass screen was used to filter our E-MAP, Cellmap SGA and Novartis chemicogenomic data. Correlations between genes in these screens 77  were computed with R (www.r-project.org) using a custom script. Data were visualized in Cytoscape (www.cytoscape.org) and clustered using the MCL algorithm through the clustermaker v2 app, with a weighting factor of 0.5 applied to E-MAP data so as not to overwhelm the other datasets.  3.5.6 Fluorescence microscopy Log-phase yeast in minimal selective media were imaged at room temperature on concanavalin A-treated glass bottom MatriPlates (Brooks Automation Inc., Chelmsford, MA) with a DMi8 microscope (Leica Microsystems GmbH, Wetzlar, Germany) using an HC PL APO 63x/1.30 Glyc CORR CS, or an HC PL APO 100x/1.40 OIL STED WHITE objective (Leica) and an ORCA-Flash4.0 digital camera (Hamamatsu Photonics, Hamamatsu City, Japan) using MetaMorph 7.7 software (MDS Analytical Technologies, Sunnyvale, CA). Alternatively, in Figure 2, chitin staining was performed in cells fixed with 3.7% formaldehyde, incubated in 100 μg/ml CW in 0.5 M Tris pH 9.6 for 30 min at 30ºC, and washed twice prior to analysis. images were taken on an Axioplan 2 fluorescence microscope (Carl Zeiss Inc., Jena, Germany) equipped with a Plan-Apochromat 100×/1.40 NA oil immersion objective lens (Zeiss) and a CoolSNAP camera (Roper Scientific, Tucson, AZ). Images were adjusted using MetaMorph and Photoshop CS5 (Adobe, San Jose, CA). Puncta quantification and co-localization was done by automated counting using custom MetaMorph 7.8 using in-house journals written by Dr. Björn D.M. Bean. Briefly, live cells were identified by using several iterations of the Count Nuclei function. Puncta number and size were identified with the Granularity function. Puncta attributed to dead cells and extracellular noise were masked by using the LogicalAND function, and co-localization was measured with the built in Co-localization application. Significance was determined by Student’s t test or ANOVA followed by Tukey’s multiple comparison test. Data distribution was assumed to be normal.  3.5.7 Gel filtration 100 OD600 of yeast spheroplasts were lysed in lysis buffer (20mM HEPES, pH 7.4, 1% Tween 20, 50 mM KCl, 100 mM potassium acetate, protease inhibitors), cleared of cell 78  debris by spinning at 13000g for 10 min, then spun at 100000g for 1h. Supernatant was passed over a Superose 6 10/300 column (GE Healthcare) at a flow rate of 0.35 mL/min on an ÄKTA FPLC (GE Healthcare), fractions collected and used for immunoblotting. 3.5.8 Co-immunoprecipitation Log-phase cells in YPD media were converted to spheroplasts by digesting with zymolyase (MJS BioLynx, Brockville, Canada), and stored at -80°C until use, as described (Conibear and Stevens, 2000). 50 OD600 of cells were resuspended in co-IP lysis buffer (20mM HEPES, pH 7.4, 1% Tween 20, 50 mM KCl, 100 mM potassium acetate, protease inhibitors) and centrifuged for 10 min, max rpm at 4°C. Mammalian cells were lysed in HNE buffer (50mM HEPES pH 7.4, 150mM NaCl, 1mM EDTA) + 1% TX-100 and protease inhibitors and centrifuged. Supernatant fractions were incubated at 4°C for 1 h with antibodies for immunoprecipitation (Supplementary Table B5) followed by Protein A or Protein G Sepharose beads (GE Healthcare, Little Chalfont, UK) for 1 h. Beads were washed 3x with lysis buffer without detergent, re-suspended in 1x SDS-PAGE sample buffer and proteins eluted by heating at 70°C for 5 minutes.  3.5.9 Immunoblotting Samples were run on 8% or 10% SDS-PAGE gels. Proteins were transferred overnight to nitrocellulose or PVDF membranes and blotted with indicated primary antibodies, then with goat anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA). 5% milk solution was used as blocking agent for yeast samples and 3% bovine serum albumin for mammalian samples. Antibodies are given in Supplementary Table B5. Blots were developed with the enhanced chemiluminescent West Pico (Pierce, Rockford, IL), ECL Prime (GE Healthcare) and West Femto (Pierce) and exposed to Amersham Hyperfilm (GE Healthcare). Alternatively, Alexa488-conjugated secondary antibodies were used and signals acquired on a Typhoon Trio scanner (GE Healthcare) using the blue laser (excitation 488 nm) with a 520BP40 emission filter. Signals were acquired in the linear range. 79  3.5.10 Lifetime fluorescence measurements  The time-lapse sequences were collected on an inverted Zeiss LSM510 Meta confocal microscope using a 63x PlanApo objective. 488nm and 543nm laser light was used to excite the fluorophores (green fluorescent protein and tdTomato, a tandem dimer red fluorescent protein) and the reflected light was collected using a 505-530nm bandpass filter for GFP and a 600nm longpass filter for mRFP. The dimension of a single voxel is 114nm by 114nm by 300nm. For the rapid collection of z-stacks a piezo actuator was used to drive continuous z movement during acquisition. A complete z-stack consisted of 12-15 slices and was captured every 2sec. Images were deconvolved using a Classic Maximum Likelihood Estimation algorithm in Huygens Professional software (Scientific Volume Imaging, Hilversum, The Netherlands). A photobleaching algorithm was also applied using this software. Tracking was done using Imaris software (Bitplane, Zurich, Switzerland). For the tracking of fluorescent structures an autoregressive-tracking algorithm was used that models a path based on both continuous movement and stochastic behaviour. Significance was determined using ANOVA followed by Dunn’s multiple comparison test. Data distribution was assumed to be normal. 3.5.10 Yeast two-hybrid PJ694a strains carrying pGBDU-C2-based plasmids were mated to PJ694α strains carrying pGAD-C2-based plasmids, each expressing the indicated full-length or truncation proteins (James et al., 1996). Diploids were assessed for positive two-hybrid interactions (activation of the HIS3 reporter) by growth on minimal media (SD-his).    80  Chapter 4: Discussion and conclusions 4.1 Summary of key findings These studies enhance our understanding of AP-1 functions in yeast and humans. We have investigated both intrinsic aspects of AP complex regulation such as inclusion of different subunits and extrinsic aspects such as binding to regulatory proteins. We show that differential inclusion of the AP-1 µ subunits Apm1 and Apm2 results in two functionally distinct AP-1-related complexes in yeast, AP-1 and AP-1R, and confers differential cargo sorting and the ability to bind different regulators. We identify the predicted lipase Mil1 as an Apm2-specific regulator, joining the growing literature demonstrating that lipid-modifying proteins associate with AP complex µ subunits (Ling et al., 2007; Fields et al., 2010; Krauss et al., 2006).  We also discover a new AP-1-associated “Laa complex” consisting of Laa1-Laa2-Slo1, and its unexpected conservation in humans. The uncharacterized ORF YBL010C codes for Laa2, a protein that is related to the fasciculation and elongation protein zeta (Fez)/Unc-76 family in higher eukaryotes that was not previously linked to AP-1 functioning. Surprisingly, such seemingly diverse trafficking proteins as Laa2, Fez1/2, aftiphilin and the aftiphilin-related protein CLBA1 all share a common region for binding Laa1/HEATR5 family members. The two isoforms of Laa1 in humans, HEATR5A and HEATR5B, are distinct and participate in different complexes. These studies underscore the complexity of AP-1-mediated trafficking and suggest that conserved accessory proteins operate at many steps of vesicle transport but their functions may have diverged. 4.2 AP-1 µ subunits Apm1 and Apm2 are functionally distinct in yeast  We found that Apm1 and Apm2 contribute differently to cargo sorting (Chs3 vs Snc1) and bind different regulators. We favour a model where interaction with different regulatory proteins is the primary distinction between Apm1 and Apm2. In this model, regulator-mediated recruitment onto particular membrane subdomains allows Apm1 and 81  Apm2 to “see” different cargo but they otherwise function similarly. Addressing this by microscopy might be challenging because the membrane subdomains of interest may be below the limit of light microscopy, so double-labeling immunoelectron microscopy (Hagiwara et al., 2010) may be the most promising imaging approach to pursue.  Another way of testing whether Apm1 and Apm2 are segregated into different membrane domains and are near specific cargo proteins would be to use a yeast dihydrofolate reductase (DHFR)-based assay (Tarassov et al., 2008). Yeast DHFR screening is a protein complementation assay similar to yeast two-hybrid that gives information on the spatial relationship between two proteins (a “bait” and a “prey” tagged with complementary fragments of the DHFR molecule) in vivo. This technique may be useful not only in determining whether Apm1 and Apm2 are in close proximity on the membrane but also in identifying candidate cargo specific for one µ subunit or the other, a prerequisite for subsequent biochemical assessment of differential binding affinities. Further, it may reveal new AP-1 or AP-1R-specific regulators, and allow us to test in a different way the hypothesis that Mil1 recruits Apm2 to a specific subdomain. 4.3 The predicted lipase is Mil1 is a specific regulator of Apm2 Recruitment activity Mil1 likely regulates Apm2 by directly recruiting it to the membrane through Mil1’s conserved WQEMP motif. Although we mapped binding of this motif to the B subdomain of Apm2 we did not determine the exact binding site. This information would help in understanding whether Mil1 contributes to AP-1R activation as well as recruitment. In the closed, inactive AP complex, part of the µ subunit is inaccessible and buried in the core (Heldwein et al., 2004). A conformational change results in an opening and activation of the complex, and some regulatory proteins specifically recognize this form (Beacham et al., 2018). It would be interesting to see whether Mil1 specifically binds an active (phosphorylated) form of Apm2. One approach would be to test whether Mil1 interacts preferentially with mock phosphorylated (Thr>Asp mutants) or non-phosphorylatable (Thr>Ala mutants). This could help in distinguishing between Mil1 82  recruiting Apm2 to a membrane versus stabilizing an active conformation at the membrane.  Catalytic activity Mil1 catalytic activity is important in AP-1R-mediated sorting but not AP-1R recruitment, suggesting that Mil1’s catalytic activity is needed at another step of protein trafficking. Determining the substrate of Mil1 and its human homolog TMCO4 could shed light on their trafficking functions. An initial approach might be to compare the lipid profile of mil1Δ yeast or CRISPR-deleted TMCO4 human cells with their wild-type counterparts using lipidomics (Yang and Han, 2016). This approach depends heavily on the sensitivity of the technique, since TMCO4 may perform highly localized lipid modification, but it may narrow down the number of substrates we will need to test. Since the conserved domain matches most with lipases, it is plausible that the catalytic activity serves to cleave one of the acyl chains of a phospholipid to make a lyso-phospholipid. This activity would be reasonable considering the association of Mil1 in trafficking pathways, which need mechanisms to facilitate membrane curvature. Ultimately, only recombinant expression, purification and testing of these proteins in biochemical assays (for instance, using p-nitrophenyl esters as colorimetric substrates (Gupta et al., 2003; Hasan et al., 2009)) will demonstrate whether they are true lipid-modifying proteins.      Possible roles in humans The Mil1 catalytic site is much more broadly conserved than the WQEMP AP-complex-interaction site, which seems to be present only in yeast species. What could be the function of TMCO4 in humans? One of the few studies mentioning TMCO4 found that it interacts with the vascular endothelial growth factor (VEGF) receptor FLT1 by cross-linking mass spectrometry and may inhabit lipid raft domains at the cell surface in podocytes (Jin et al., 2012). TMCO4 may help to generate or maintain lipid rafts, similar to our suggested role of Mil1 for TGN/endosome domains. More detailed study of its localization is needed, and co-localization with AP complexes and clathrin will indicate whether TMCO4 plays a role in CCV trafficking or other processes. Further identification 83  of TMCO4 binding partners will provide the most evidence for its inclusion into a particular trafficking pathway. 4.4 The yeast Laa complex (Laa1-Laa2-Slo1) is related to a human HEATR5A-Fez1/2-SCOC complex Composition and binding partners of the Laa complex Laa2 seems to need to bind other proteins for stability, as demonstrated by the decrease in Laa2 levels upon deletion of either Slo1 or Laa1.This suggests that the yeast Laa complex is at minimum an obligate tetramer (Laa1-Laa1-Laa2-Slo1), although this does not necessarily extend to unc-76 or Fez proteins. However, we cannot exclude the idea that there is also a pool of Laa1 in the cell that is not bound to Laa2 at a given time. This would be worth pursuing in more detailed microscopy experiments, since assembly of the Laa complex on membranes under particular cellular conditions would facilitate transport in a more regulated fashion. We were not able to determine whether Laa2 or Slo1 dimerize, as has been shown for their mammalian counterparts (Lanza et al., 2009; Alborghetti et al., 2013; Behrens et al., 2013); we also do not know whether HEATR5A and HEATR5B can homo- or hetero-dimerize similar to Laa1. This information would allow us to refine our models regarding how many different binding partners the complex can interact with at once. While Fez proteins likely have many binding partners (Fujita et al., 2007; Alborghetti et al., 2011; Watanabe et al., 2014) we do not know if this binding is mutually exclusive with HEATR5-binding. If Laa1 contains membrane-recruitment domains, a Laa1 dimer would increase association with the membrane or better recognize coincidence signals. Two copies of Laa2 would present two FGxF motifs for AP-1 binding. Do Fez proteins associate with AP complexes?  We identified Laa2 through a screen for AP-1 regulators and found an FGxF motif required for its binding to the GAE domain, a common site for regulator binding. However, the human homologs of Laa2 have not been previously been linked to AP-1 activity. We have noticed a possible ΨxxΨ motif in the N-termini of Fez2 and Fez1, so these proteins may potentially interact with an adaptor protein complex in humans. 84  Although we could not detect an interaction between Fez1/2 and AP-1 there could be still be a weak binding below our limit of detection; in this case, aftiphilin and γ-synergin may have been more easily detected because they both contain multiple FGxF motifs and therefore have a higher affinity for AP-1. There could also be a regulated association, with the ΨxxΨ otherwise occluded. This binding could be useful in adapting the vesicle coat to motor proteins, since Fez-family proteins are thought to be cargo adaptors (Bloom and Horvitz, 1997; Gindhart, 2003; Toda et al., 2008; Butkevich et al., 2016b; Chua et al., 2012). In this regard, Fez proteins might function similarly to the AP-1 accessory protein Gadkin, which provides a link between AP-1 and kinesin-1 (Schmidt et al., 2009). Fundamental role of the complex  Although the Laa complex is implicated in AP-1-mediated Chs3 trafficking, it exhibits a much weaker bypass phenotype than AP-1 mutants and also has a relatively small effect on AP-1 recruitment. This could indicate that the complex plays additional roles in trafficking, separate from its AP-1 role. Could there be a fundamental function for the Laa complex that unifies its involvement in AP-1-mediated Chs3 trafficking with the previously-identified roles of Fez-family members as cargo adaptors and autophagy regulators?  One possibility is that the Laa complex mediates organelle positioning in a manner similar to FYCO1. Briefly, LEs bearing FYCO1 (FYVE and Coiled-Coil Domain Containing 1) are loaded onto kinesin-1 motor proteins in a contact-site-dependent manner (regulated by cholesterol availability and other signals), and repeated contacts promote translocation of these late endosomes to the cell periphery, where they fuse with the PM (Raiborg et al., 2015a). Specific transport of lysosomes into axons is also linked to autophagosome clearance and growth-cone dynamics (Farías et al., 2017). The Laa complex could facilitate the formation of membrane contact sites at particular organelles, restraining or promoting transport as needed. It is also possible that kinesin-based microtubule trafficking may play a minor or backup role in yeast under certain conditions, which remain to be discovered.  85  Comparison to AP complexes It is interesting to compare the yeast Laa complex with AP complexes in terms of composition and function of the various subunits: both contain two large-sized, HEAT-repeat-containing subunits with a membrane-recruitment and scaffolding role; one medium-sized subunit that is involved in binding to various cargo-type proteins (and is regulated by phosphorylation); and one small-sized subunit that may play a structural role. Further supporting the comparison, bioinformatics approaches show homology between HEATR5 and large adaptin subunits. The aftiphilin-γ-synergin-HEATR5B complex may also fit this model, as both aftiphilin and γ-synergin have EH domains that may enable them to act in selection of cargo containing NPF-type motifs  (Salcini et al., 1997; Page et al., 1999; Naslavsky and Caplan, 2011). Altogether, both the aftiphilin-γ-synergin-HEATR5B and the Fez1/2-SCOC-HEATR5A complexes may act in concert with other trafficking complexes, both recruiting them and expanding the variety of cargo that can be sorted. 4.5 Binding to HEATR5 family members is a common feature of Laa2, Fez1/2, aftiphilin and CLBA1 Different HEATR5-containing complexes We have found evidence for three distinct HEATR5-containing complexes in humans: HEATR5A-Fez1/2-SCOC, HEATR5B-CLBA1, and HEATR5B-aftiphilin-γ-synergin. Combinatorial assembly of components is an often-used mechanism to enhance the range of possible functions of a complex (see adaptor complex isoforms). Since the common factor is HEATR5, greater effort should be directed towards understanding its functions as they are likely to have the widest ramifications. It is likely that the HEATR5 isoforms act as recruitment platforms for machinery to localize to a particular cellular location. Identifying Laa1/HEATR5 membrane recruitment factors will be especially important. A first step would be to test Laa1 for physical interaction with a range of Rabs and Arf-related proteins and to map their binding sites. Laa1 localization is brefeldin A-sensitive, implicating Arf1 in Laa1 membrane association (Fernández and Payne, 2006), but a physical interaction between the two has yet to be demonstrated. 86  Slo1 and SCOC have already been shown to interact with Arl3 and ARL1, respectively (Van Valkenburgh et al., 2001). Another route would be to see whether Laa1 has a PI-binding pocket, similar to the large subunits of AP complexes. This would help to distinguish between recruitment and scaffolding roles for HEATR5, and indicate at which steps HEATR5 is needed.  Regulation of HEATR5 complex assembly Can the HEATR5-binding proteins act independently of HEATR5? While knockdown of aftiphilin significantly destabilizes HEATR5B the reverse is not true (Hirst et al., 2005) and aftiphilin and γ-synergin knockdowns clearly affect regulated secretion of WPBs but HEATR5 knockdowns do not (Lui-Roberts et al., 2008). This suggests that aftiphilin may have HEATR5-independent roles. Similarly, Fez protein homologs have not previously been linked to HEATR5A and neither SCOC nor HEATR5A are detected in immuno-isolated Fez1 vesicles (Butkevich et al., 2016a). It will therefore be important to determine what signals regulate the association of aftiphilin, CLBA1 and Fez1/2 with HEATR5, and whether binding HEATR5 vs other cargo is mutually exclusive.  What regulatory modifications may affect association with HEATR5? The best-characterized modification of Fez1/Unc-76 is its phosphorylation at a conserved serine (S58 in Fez1) (Malikov et al., 2015; Butkevich et al., 2016a; Malikov and Naghavi, 2017; Toda et al., 2008; Chua et al., 2012). This signal allows it to interact with kinesin-1 and bind cargo. Interestingly, this conserved serine residue in Fez1 is included in the HHPred-predicted (HEATR5-binding) region of homology, albeit just at its edge. Phosphorylation of Fez1 could disfavour interaction with HEATR5, providing a mechanism to switch between membrane recruitment and cargo/kinesin binding. Testing this hypothesis would be relatively straightforward, making mock-phosphorylated or dephosphorylated mutants of Fez1/2 and assessing their interaction with HEATR5. Ubiquitination of Fez1 is another modification of interest, having been suggested to be a regulatory signal governing dendrite growth in neurons (Watanabe et al., 2014) that is not based on altering Fez1 abundance (Okumura et al., 2004). Regulation of Fez1/2 levels could also influence its incorporation into the HEATR5 complex. Fez proteins seem to act differently based on abundance: a 87  threshold effect has been observed where high amounts of Fez1 are protective against HIV-1 (Haedicke et al., 2009) but lower levels promote viral infection (Malikov et al., 2015). Fez proteins may have a hierarchy of preferences for their many binding partners, with different levels of Fez successively allowing formation of new complexes. It will be interesting to test whether our Fez2W91A mutant exhibits enhanced interaction with other Fez binding partners because it can no longer work with HEATR5A, indicating that HEATR5 provides some steric hindrance. Whether this mutant can promote neurite differentiation in PC12 cells (Fujita et al., 2004) or rescue the uncoordinated phenotype exhibited by unc-76 mutant worms (Bloom and Horvitz, 1997) might indicate whether HEATR5 proteins play a role in these phenotypes. 4.6 Conclusion The AP-1 complex is a highly important component of the cellular protein trafficking machinery, and fully understanding its function is vital for a larger understanding of cell biology. AP-1 integrates many signals by participating in specific interactions at particular times, and there is still much we do not know about how the clathrin-coated vesicle machinery works with AP-1 and regulates it. Our studies contribute to our knowledge of this process by discovering new players in AP-1-mediated cellular protein trafficking: Mil1 and the Laa complex and their human homologs. 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(A) MUSCLE sequence alignment of Apm1- and Apm2-related sequences, showing conservation of the two tyrosine-binding pockets. Intensity of blue-highlighted residues corresponds to percent identity. Red-highlighted residues interact with the YxxΦ sorting signal. (B) Cellular levels of mutant and overexpressed forms of Apm1 and Apm2. The levels of genomically-tagged Apm1-GFP and Apm2-GFP were compared with plasmid-borne GFP-tagged Apm1tyr, and either Apm2tyr-GFP or Apm2OE-GFP expressed from the APM1 promoter. Samples were resolved by 10% SDS-PAGE on the same gel and detected by immunoblotting. PGK levels are shown as loading control. White gaps between panels indicate cropping of empty lanes from the same blot. 121   Supplementary Figure A2 Deletion of MIL1 does not affect Apm1-GFP recruitment or number of Golgi compartments. (A) Localization of Apm1-GFP in wild type or mil1 mutant strains quantified in a single slice. No significant differences between the means were found (unpaired t-test with wild-type). Error bars represent standard error of the mean (n=3). (B) Number of Sec7-dsRed puncta expressed as mean/cell. No significant differences between the means were found (unpaired t-tests with wild types). Error bars represent standard error of the mean (n=3). Supplementary Table A1 List of yeast strains used in Chapter 2. Strain ID Genotype Source PJ694a MATa  trp1-901 leu2Δ-3 ura3Δ-52 his3Δ-200 gal4Δ gal80Δ LYS::GAL1::HIS3 GAL2::ADE-2 met2::GAL7-lacz A. Merz (U. Washington) PJ694α MATα  trp1Δ-901 leu2Δ-3 ura3Δ-52 his3Δ-200 gal4Δ gal80Δ LYS::GAL1-HIS3 GAL2-ADE-2 met2::GAL7-lacz A. Merz BY4742 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 GE Dharmacon MDY122 BY4742 chs6Δ::NAT This study MDY869 BY4742 chs6Δ::NAT apl2∆::KANR This study MDY784 BY4742 chs6Δ::NAT apl2Δ::HPH This study MDY783 BY4742 chs6Δ::NAT apm1∆::KANR This study MDY786 BY4742 chs6Δ::NAT apm2∆::KANR This study CTY234 BY4742 can1Δ::STE2pr::LEU2 lyp1Δ::cyh2 C. Boone (U. Toronto) BY4741 MATa HIS3Δ1; leu2Δ0; met15Δ0; ura3Δ0 GE Dharmacon SWY85 BY4741 apl2Δ::KANR This study SWY89 BY4741 apm1Δ::KANR This study SWY91 BY4741 apm2Δ::KANR This study SWY97 BY4741 mil1Δ::KANR This study HBY577 BY4741 suc2Δ::GFP-SNC1-SUC2::URA3  This study HBY573 BY4741 apm1Δ::KANR suc2Δ::GFP-SNC1-SUC2::URA3  This study HBY574 BY4741 aps1Δ::KANR suc2Δ::GFP-SNC1-SUC2::URA3  This study HBY575 BY4741 apm2Δ::KANR suc2Δ::GFP-SNC1-SUC2::URA3  This study HBY576 BY4741 apl4Δ::KANR suc2Δ::GFP-SNC1-SUC2::URA3  This study 122  Strain ID Genotype Source HBY571 BY4741 mil1Δ::KANR suc2Δ::GFP-SNC1-SUC2::URA3   This study MDY613 yap1801Δ::KANR yap1802-3HA::HIS3 suc2Δ::GFP-SNC1-SUC2::URA3  This study LC1979 BY4741 APM4-GFP+::HIS3 CLC1-RFP::NAT This study HBY155 BY4741 APM4-GFP+::HIS3 CLC1-RFP::NAT sla2∆::URA3 This study LC1977 BY4741 APM2-GFP+::HIS3 CLC1-RFP::NAT This study LC1978 BY4741 APM2-GFP+::HIS3 CLC1-RFP::NAT sla2∆::URA3 This study CTY301 BY4741 MIL1-GFP+::HIS3  This study CTY564 BY4741 MIL1-GFP+::HIS3 APL4-3HA::KANR This study CTY574 BY4741 MIL1-GFP+::HIS3 APL4Δear-3HA::KANR This study CTY265 BY4741 MIL1-GFP+::HIS3 APM1-3HA::KANR This study CTY661 BY4741 MIL1-GFP+::HIS3 APM2-3HA::KANR This study MDY1114 BY4741 APM2-GFP(envy)::HIS3 This study MDY1133 BY4741 APM2-GFP(envy)::HIS3 APM1::tdTomato::NAT  This study MDY1136 BY4741 APM2-GFP(envy)::HIS3 ANP1-mRFP1.5::NAT This study SWY310 BY4741 APM2-GFP(envy)::HIS3 mil1Δ::KANR This study MDY1153 BY4741 MIL1-GFP(envy)::HIS3  This study MDY1155 BY4741 MIL1-GFP(envy)::HIS3 apm2Δ::KANR This study CTY157 BY4741 APM1-3HA::KANR This study HBY567 BY4741 APM2-3HA::HIS3 This study CTY15 BY4741 APM1-GFP+ This study CTY416 BY4741 APM1-GFP+ mil1Δ::NAT This study   Supplementary Table A2 List of plasmids used in Chapter 2. Plasmid ID Description Source pCS10 pSNF7-RFP1.5::URA (CEN) This study pSec7-dsRed pSEC7-dsRed::URA3 (CEN) S. Ferro-Novick (UCSD)  pRS415 pLEU2 (CEN) Sikorski and Hieter (1989)* pHFR80 pSNA2pr-SNA2(Y75A)-GFP-DXE::URA (CEN) P. Morsomme (U. Catholique de Louvain) pNR36 pHPH (CEN) This study pNR32 pAPM1::HPH (CEN) This study pMD177 pAPM1(F179A D181S)::HPH (CEN) This study pSW50 pAPM1(F179A D181S)-GFP+::HIS3::HPH (CEN) This study pMD178 pAPM2(F273A D275S)::HPH (CEN) This study pSW48 pAPM1pr-APM2-GFP+::HIS3::HPH (CEN) This study pSW60 pAPM1pr-APM2-3HA::HIS3::HPH (CEN) This study pNR39 pLC2632 pAPM1pr-APM2tyr-GFP+::HIS3::HPH (CEN) pMIL1::LEU2 (CEN) This study This study pLC1922 pMIL1-GFP+::HIS3::LEU2 (CEN) This study 123  Plasmid ID Description Source pLC2633 pMIL1(S759A)::LEU2 (CEN) This study pLC1912 pMIL1(D817A)::LEU2 (CEN) This study pLC1914 pMIL1(H858A)::LEU2 (CEN) This study pLC2604 pMIL1(W143QEMP)::LEU2 (CEN) This study pLC2646 pMIL1(W143QEMP)-GFP+::HIS3::LEU2 (CEN) This study pLC2605 pMIL1(F152NIY)::LEU2 (CEN) This study pMD158 pMIL1::HPH (CEN) This study pMD159 pMIL1(S759A)::HPH (CEN) This study pMD157 pMIL1(W143QEMP)::HPH (CEN) This study pGBDU-C2 pGBDU-C2::URA3 (2µ) P. James (U. Wisconsin) pCT11 pGBDU-C2-APL4::URA3 (2µ) This study pLC2643 pGBD-APM1::URA3 (2µ) This study pNR15 pGBDU-C2-APM2::URA3 (2µ) This study pNR16 pGBDU-C2-APM2(1-246)::URA3 (2µ) This study pNR18 pGBDU-C2-APM2(247-605)::URA3 (2µ) This study pNR19 pGBDU-C2-APM2(389-562)::URA3 (2µ) This study pGAD-C2 pGAD-C2::LEU2 (2µ) P. James  pCT15 pGAD-C2-APL4::LEU2 (2µ) This study pNR10 pGAD-C2-MIL1::LEU2 (2µ) This study pNR11 pGAD-C2-MIL1(1-450)::LEU2 (2µ) This study pLC2590 pGAD-MIL1(125-175)::LEU2  (2µ) This study pLC2644 pGAD-MIL1(125-175, W143QEMP>AAEAA)::LEU2  (2µ) This study pLC2645 pGAD-MIL1(125-175, F152NIY>ANAA)::LEU2  (2µ) This study *Sikorski, R.S., and Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27.  124  Appendix B. Supplementary material for Chapter 3  Supplementary Figure B1 Laa1 and Laa2 run in similar fractions by gel filtration. LAA2 deletion has no effect on which fractions Laa1 is found in.   Supplementary Figure B2 Slo1 deletion affects chs6Δ bypass and Laa complex puncta number. A) Calcofluor-based plate assay showing that a slo1Δ strain also has a chs6Δ bypass phenotype. Data represent the average of five separate experiments, error bars are standard error of the mean (S.E.M.). Unpaired two-tailed t-test, ****: p<0.0001, **: p<0.01. B) Automated quantification of Laa1-GFP or Laa2-GFP puncta with SLO1 deletions or plasmid complementation. Data represent the average of eight separate experiments, error bars are standard error of the mean (S.E.M.). Unpaired two-tailed t-test, *: p<0.05, N.S.: No significance. 125   Supplementary Figure B3 Fez1 also interacts with HEATR5A but not HEATR5B.  A) Co-immunoprecipitation showing that SCOC, Fez1 and HEATR5A interact with each other but not with AP-1 (y-adaptin), aftiphilin, y-synergin or HEATR5B. HEK293T cells were transiently co-transfected with GFP-SCOC and Flag-Fez1. α-F indicates anti-Flag antibody used for pulldown; α-G indicates anti-GFP; α-A indicates anti-adaptin γ. B) HEATR5A co-immunoprecipitates with Fez1, GFP-SCOC and myc-UVRAG. α-M indicates anti-Myc antibody used for pulldown; α-G indicates anti-GFP. Same transfection and co-IP strategy as above. Supplementary Table B1 Ranked list of average Z-scores >1 from duplicate screens of the MATa and MATα yeast gene deletion collections. Rank ORF Gene standard name CW fluor MATα avg CW fluor MATa avg CW fluor avg of avgs Z-score 1 YDR495C VPS3 2.893544 2.869042 2.881293 8.952486 2 YML097C VPS9 2.716229 2.538517 2.627373 7.752352 3 YPL259C APM1 2.759197 2.469363 2.61428 7.690467 4 YGL095C VPS45 2.028451 2.91683 2.47264 7.021018 5 YDL192W ARF1 2.530406 2.370082 2.450244 6.915163 6 YOR109W INP53 2.646937 2.199054 2.422996 6.786375 7 YKL136W Dubious/APL2 2.623291 2.131563 2.377427 6.570998 8 YKR001C VPS1 2.190107 2.542571 2.366339 6.518592 9 YER151C UBP3 2.564904 2.133 2.348952 6.436412 10 YDR456W NHX1 2.360577 2.28368 2.322128 6.309633 11 YLR242C ARV1 2.676687 1.939967 2.308327 6.244403 12 YIL044C AGE2 2.519519 2.0044 2.26196 6.02525 13 YPR029C APL4 2.569243 1.952828 2.261035 6.020881 14 YLR322W VPS65 3.003872 1.512489 2.25818 6.007388 15 YDR293C SSD1 2.204872 2.254297 2.229584 5.872229 16 YLR169W Dubious/APS1 2.308405 1.98547 2.146938 5.481607 17 YAL002W VPS8 2.090019 2.15989 2.124954 5.377705 18 YJL204C RCY1 2.150786 2.055325 2.103056 5.274202 126  Rank ORF Gene standard name CW fluor MATα avg CW fluor MATa avg CW fluor avg of avgs Z-score 19 YDL226C GCS1 1.618111 2.563974 2.091043 5.217423 20 YLR171W Dubious/APS1 2.439057 1.733822 2.086439 5.195666 21 YCR094W CDC50 2.373612 1.768498 2.071055 5.122953 22 YPR043W RPL43A 2.319712 1.80175 2.060731 5.074157 23 YOR036W PEP12 1.563985 2.556679 2.060332 5.072272 24 YOR089C VPS21 2.524628 1.592218 2.058423 5.063248 25 YNR051C BRE5 1.965398 2.036096 2.000747 4.790646 26 YGL212W VAM7 2.512281 1.427847 1.970064 4.645626 27 YDR027C VPS54 2.327187 1.563275 1.945231 4.528253 28 YAL026C DRS2 2.598775 1.267278 1.933026 4.470569 29 YNR006W VPS27 1.757475 2.086619 1.922047 4.418677 30 YDR080W VPS41 1.884894 1.932795 1.908844 4.356275 31 YFR043C IRC6 2.053387 1.751291 1.902339 4.325529 32 YLR170C APS1 2.238864 1.526496 1.88268 4.232611 33 YDR455C Dubious/NHX1 1.424451 2.331927 1.878189 4.211383 34 YOR270C VPH1 1.961541 1.779419 1.87048 4.174949 35 YDR392W SPT3 2.511915 1.193459 1.852687 4.09085 36 YCR037C PHO87 2.626361 1.040204 1.833282 3.999138 37 YGR261C APL6 1.853806 1.79529 1.824548 3.957857 38 YKL002W DID4 2.166667 1.482112 1.824389 3.957106 39 YKL041W VPS24 1.89854 1.643132 1.770836 3.703989 40 YDL077C VAM6 2.543269 0.989937 1.766603 3.683982 41 YPL195W APL5 2.189959 1.277001 1.73348 3.527429 42 YMR123W PKR1 2.107299 1.355226 1.731263 3.516948 43 YPL045W VPS16 1.699637 1.756473 1.728055 3.501787 44 YLR338W OPI9 1.542317 1.909685 1.726001 3.49208 45 YHR108W GGA2 1.510737 1.93722 1.723979 3.482522 46 YIL159W BNR1 0.89023 2.524038 1.707134 3.402906 47 YPR173C VPS4 1.814761 1.567052 1.690906 3.326207 48 YER187W YER187W 0.923824 2.450476 1.68715 3.308454 49 YNL297C MON2 1.869839 1.489277 1.679558 3.272569 50 YCR044C PER1 1.981296 1.351001 1.666149 3.209192 51 YBR288C APM3 1.763885 1.568269 1.666077 3.208854 52 YDR414C ERD1 1.785106 1.54202 1.663563 3.196972 53 YOL072W THP1 0.844671 2.460325 1.652498 3.144671 54 YJL062W LAS21 1.457006 1.846755 1.65188 3.141753 55 YBR078W ECM33 1.816825 1.47649 1.646657 3.117067 56 YJR037W Dubious/HUL4 1.07183 2.206216 1.639023 3.080985 57 YJR040W GEF1 1.706841 1.557004 1.631922 3.047423 58 YJL024C APS3 1.973305 1.27387 1.623588 3.008031 127  Rank ORF Gene standard name CW fluor MATα avg CW fluor MATa avg CW fluor avg of avgs Z-score 59 YDR157W YDR157W 1.013818 2.232248 1.623033 3.005407 60 YOR106W VAM3 1.680057 1.537912 1.608985 2.939009 61 YJR113C RSM7 1.33103 1.85042 1.590725 2.852707 62 YLR360W VPS38 1.990587 1.18194 1.586263 2.831618 63 YPL084W BRO1 1.986146 1.167691 1.576918 2.787451 64 YBR189W RPS9B 0.610295 2.519071 1.564683 2.72962 65 YDR389W SAC7 1.690213 1.430201 1.560207 2.708464 66 YPR045C THP3 1.017389 2.082718 1.550053 2.660474 67 YJR033C RAV1 1.708421 1.381787 1.545104 2.637083 68 YKL212W SAC1 1.461628 1.551092 1.50636 2.453962 69 YHR193C EGD2 0.951918 2.048013 1.499966 2.423739 70 YER066C-A Dubious/RGI1 0.893708 2.099291 1.4965 2.407359 71 YER110C KAP123 0.843063 2.118436 1.480749 2.332914 72 YPR044C OPI11 0.920921 2.030951 1.475936 2.310165 73 YFL003C MSH4 0.936144 2.00977 1.472957 2.296086 74 YKL138C MRPL31 1.467855  1.467855 2.271972 75 YML001W YPT7 1.574531 1.356889 1.46571 2.261832 76 YMR307W GAS1 1.508948 1.419118 1.464033 2.253906 77 YGR062C COX18 1.652134 1.271734 1.461934 2.243985 78 YBR181C RPS6B 2.044734 0.862099 1.453417 2.203728 79 YPR100W MRPL51 1.738993 1.167424 1.453209 2.202746 80 YGL124C MON1 1.407624 1.495875 1.451749 2.195847 81 YBR171W SEC66 1.677885 1.21206 1.444972 2.163816 82 YLR087C CSF1 1.486506 1.396305 1.441406 2.14696 83 YOL076W MDM20 2.559329 0.323223 1.441276 2.146347 84 YDL006W PTC1 1.839973 1.04047 1.440221 2.14136 85 YMR256C COX7 1.407253 1.460172 1.433712 2.110597 86 YOR065W CYT1 1.606997 1.257659 1.432328 2.104056 87 YCR003W MRPL32 1.463994 1.397396 1.430695 2.096338 88 YOL014W YOL014W 0.975847 1.881127 1.428487 2.085901 89 YOL106W YOL106W 1.038402 1.813116 1.425759 2.073005 90 YHR051W COX6 1.54954 1.301845 1.425692 2.072692 91 YDR379W RGA2 0.960851 1.886106 1.423478 2.062227 92 YDR375C BCS1 1.583984 1.25918 1.421582 2.053263 93 YLR358C Dubious/RSC2 2.679279 0.155711 1.417495 2.033946 94 YDR532C KRE28 1.899792 0.933425 1.416609 2.029757 95 YKL134C OCT1 1.612702 1.211355 1.412029 2.00811 96 YER031C YPT31 1.401483 1.421421 1.411452 2.005385 97 YKL016C ATP7 1.617951 1.201252 1.409601 1.996637 98 YFR019W FAB1 1.940591 0.862901 1.401746 1.95951 128  Rank ORF Gene standard name CW fluor MATα avg CW fluor MATa avg CW fluor avg of avgs Z-score 99 YDR194C MSS116 1.514337 1.270111 1.392224 1.914506 100 YDR406W PDR15 0.974362 1.806698 1.39053 1.906498 101 YMR085W YMR085W 0.996919 1.783428 1.390174 1.904815 102 YDR500C RPL37B 1.130227 1.648691 1.389459 1.901437 103 YDR377W ATP17 1.449462 1.329096 1.389279 1.900587 104 YDL005C MED2 1.388393  1.388393 1.896398 105 YGL133W ITC1 1.847527 0.922814 1.385171 1.881169 106 YHR194W MDM31 1.064852 1.703775 1.384313 1.877116 107 YMR257C PET111 1.279542 1.481924 1.380733 1.860194 108 YDL067C COX9 1.288462 1.472093 1.380277 1.858041 109 YNR037C RSM19 1.566705 1.182212 1.374458 1.830537 110 YKR020W VPS51 1.582082 1.159398 1.37074 1.812964 111 YER154W OXA1 1.357143 1.375611 1.366377 1.79234 112 YPL157W TGS1 1.811702 0.919635 1.365669 1.788994 113 YLR377C FBP1 1.606715 1.122945 1.36483 1.78503 114 YPL172C COX10 1.488981 1.230134 1.359558 1.76011 115 YGR076C MRPL25 1.584765 1.125122 1.354943 1.738302 116 YJR073C OPI3 1.326932 1.380168 1.35355 1.731717 117 YOL033W MSE1 1.630748 1.071998 1.351373 1.721425 118 YDR347W MRP1 1.456598 1.245963 1.35128 1.720989 119 YDR197W CBS2 1.507331 1.191991 1.349661 1.713335 120 YLR203C MSS51 1.3922 1.307112 1.349656 1.713312 121 YML112W CTK3 1.835242 0.854299 1.34477 1.690219 122 YHL038C CBP2 1.374718 1.30564 1.340179 1.668518 123 YMR231W PEP5 1.644326 1.030209 1.337268 1.654758 124 YDR049W VMS1 1.098723 1.566589 1.332656 1.632963 125 YBR290W BSD2 1.224666 1.435166 1.329916 1.620012 126 YGL024W YGL024W 1.660577 0.998725 1.329651 1.618759 127 YGR102C GTF1 1.455275 1.203175 1.329225 1.616744 128 YDR350C ATP22 1.541168 1.109916 1.325542 1.599337 129 YKL087C CYT2 1.694061 0.95699 1.325526 1.599261 130 YNL164C IBD2  1.322575 1.322575 1.585313 131 YBR119W MUD1 1.734547 0.910539 1.322543 1.585163 132 YGR136W LSB1 0.97387 1.670741 1.322305 1.58404 133 YGR022C YGR022C 1.578376 1.064249 1.321313 1.579349 134 YLR023C IZH3 0.967103 1.671219 1.319161 1.569178 135 YDL204W RTN2 1.009856 1.62572 1.317788 1.562687 136 YGL114W YGL114W 0.98283 1.650747 1.316788 1.557963 137 YMR286W MRPL33 1.490239 1.142463 1.316351 1.555899 138 YDR175C RSM24 1.452933 1.176483 1.314708 1.54813 129  Rank ORF Gene standard name CW fluor MATα avg CW fluor MATa avg CW fluor avg of avgs Z-score 139 YMR228W MTF1 1.614112 1.011305 1.312708 1.53868 140 YNR041C COQ2 1.391164 1.231651 1.311407 1.532531 141 YKR006C MRPL13 1.381131 1.241512 1.311322 1.532126 142 YBL010C YBL010C/LAA2 1.391725 1.228693 1.310209 1.526868 143 YMR064W AEP1 1.147376 1.468399 1.307888 1.515895 144 YDR296W MHR1 1.017768 1.59575 1.306759 1.51056 145 YOL112W MSB4 0.976118 1.632969 1.304543 1.500089 146 YGR150C CCM1 1.558303 1.04998 1.304142 1.49819 147 YDR417C YDR417C 1.496096 1.110344 1.30322 1.493835 148 YDR204W COQ4 1.349002 1.256311 1.302656 1.491169 149 YNL296W YNL296W 1.728666 0.871275 1.299971 1.478476 150 YPL013C MRPS16 1.373563 1.222301 1.297932 1.468842 151 YDR230W YDR230W 1.417404 1.17805 1.297727 1.467872 152 YLL033W IRC19 1.518632 1.076553 1.297592 1.467235 153 YNR042W YNR042W 1.495279 1.098157 1.296718 1.463103 154 YER001W MNN1 1.034149 1.555612 1.294881 1.454419 155 YBR134W YBR134W 1.456731 1.132689 1.29471 1.453613 156 YOL096C COQ3 1.337428 1.251356 1.294392 1.452109 157 YLR202C YLR202C 1.533294 1.054243 1.293769 1.449163 158 YML117W NAB6 1.572381 1.014795 1.293588 1.448309 159 YHR185C PFS1 1.527361 1.059504 1.293433 1.447576 160 YJL209W CBP1 1.619664 0.965587 1.292626 1.443761 161 YNL169C PSD1  1.290091 1.290091 1.431781 162 YMR008C PLB1 1.019186 1.559323 1.289254 1.427826 163 YLR386W VAC14 1.771256 0.805378 1.288317 1.423396 164 YGL243W TAD1 0.985662 1.586094 1.285878 1.41187 165 YJL178C ATG27 1.291489 1.279106 1.285297 1.409124 166 YDR484W VPS52 1.04533 1.523806 1.284568 1.405675 167 YLR067C PET309 1.424584 1.144085 1.284334 1.404572 168 YBL099W ATP1 1.452406 1.113061 1.282733 1.397006 169 YOR330C MIP1 1.536396 1.028952 1.282674 1.396726 170 YBR273C UBX7 1.132596 1.427869 1.280232 1.385185 171 YMR244W YMR244W 1.004637 1.553381 1.279009 1.379404 172 YGR222W PET54 1.175926 1.375244 1.275585 1.36322 173 YMR150C IMP1 1.384522 1.165107 1.274815 1.359578 174 YOL122C SMF1 1.091195 1.457901 1.274548 1.358318 175 YLR342W FKS1 0.273718 2.271678 1.272698 1.349573 176 YER173W RAD24 0.924779 1.61926 1.272019 1.346367 177 YHL002W HSE1 1.12338 1.41952 1.27145 1.343677 178 YOR115C TRS33 1.142593 1.397924 1.270258 1.338044 130  Rank ORF Gene standard name CW fluor MATα avg CW fluor MATa avg CW fluor avg of avgs Z-score 179 YJR125C ENT3 1.217467 1.310556 1.264011 1.308517 180 YGR183C QCR9 1.364357 1.163552 1.263954 1.308248 181 YMR035W IMP2 1.354698 1.173059 1.263879 1.30789 182 YLR119W SRN2 1.275765 1.249327 1.262546 1.301592 183 YBR037C SCO1 1.35072 1.1625 1.25661 1.273536 184 YDR372C VPS74 1.16 1.351772 1.255886 1.270113 185 YER017C AFG3 1.343463 1.166957 1.25521 1.266918 186 YMR238W DFG5 1.074031 1.435686 1.254858 1.265257 187 YNL315C ATP11 1.466967 1.035357 1.251162 1.247786 188 YJR044C VPS55 1.001432 1.499573 1.250502 1.244667 189 YLR201C COQ9 1.418749 1.081929 1.250339 1.243896 190 YPL079W RPL21B 0.856866 1.638734 1.2478 1.231897 191 YOR158W PET123 1.32739 1.165914 1.246652 1.226469 192 YML118W NGL3 0.991393 1.497123 1.244258 1.215155 193 YNL073W MSK1 1.455951 1.030609 1.24328 1.210532 194 YNL143C YNL143C 1.013377 1.470513 1.241945 1.204222 195 YML110C COQ5 1.183204 1.297081 1.240143 1.195704 196 YER116C SLX8 1.105769 1.36926 1.237515 1.183282 197 YDR203W YDR203W 1.196794 1.27085 1.233822 1.165829 198 YLR286C CTS1 1.217525 1.247496 1.232511 1.159633 199 YKL109W HAP4 1.225783 1.237668 1.231726 1.155921 200 YDR178W SDH4 1.349751 1.11087 1.23031 1.149231 201 YJL063C MRPL8 1.22539 1.235188 1.230289 1.149131 202 YEL063C CAN1 1.036915 1.423596 1.230255 1.148972 203 YBR044C TCM62 1.242887 1.215545 1.229216 1.14406 204 YMR282C AEP2 1.451067 1.006127 1.228597 1.141135 205 YBR122C MRPL36  1.227833 1.227833 1.137525 206 YJL166W QCR8 1.141204 1.312392 1.226798 1.132631 207 YPR170C YPR170C 1.147429 1.305688 1.226559 1.1315 208 YPR057W BRR1 1.027361 1.420823 1.224092 1.119842 209 YDR153C ENT5 1.208903 1.237722 1.223313 1.116158 210 YGL168W HUR1 1.395948 1.050012 1.22298 1.114586 211 YFR056C YFR056C 1.309977 1.135524 1.22275 1.113501 212 YPL104W MSD1 1.391593 1.049849 1.220721 1.10391 213 YPL262W FUM1 1.299007 1.13848 1.218744 1.094563 214 YBL102W SFT2 1.226291 1.210417 1.218354 1.09272 215 YOL011W PLB3 0.935479 1.500766 1.218123 1.091628 216 YMR097C MTG1 1.04846 1.387213 1.217836 1.090274 217 YGR112W SHY1 1.287322 1.147344 1.217333 1.087896 218 YMR202W ERG2 1.373987 1.059762 1.216875 1.085728 131  Rank ORF Gene standard name CW fluor MATα avg CW fluor MATa avg CW fluor avg of avgs Z-score 219 YDR363W ESC2 1.406064 1.027241 1.216653 1.08468 220 YHR011W DIA4 1.436188 0.993277 1.214732 1.075603 221 YOL017W ESC8 1.11131 1.317862 1.214586 1.07491 222 YCR071C IMG2 1.010713 1.416923 1.213818 1.071283 223 YDL107W MSS2 1.283105 1.142543 1.212824 1.066584 224 YKL211C TRP3 1.208041 1.214693 1.211367 1.059699 225 YKL003C MRP17 1.278544 1.143712 1.211128 1.058568 226 YDR486C VPS60 1.216346 1.203836 1.210091 1.053667 227 YCR024C SLM5 1.410432 1.008876 1.209654 1.051602 228 YJL117W PHO86 0.952986 1.465071 1.209028 1.048644 229 YNL136W EAF7 1.469705 0.944631 1.207168 1.039852 230 YML104C MDM1 1.070582 1.343315 1.206949 1.038814 231 YOR366W YOR366W 1.081279 1.33125 1.206264 1.03558 232 YJL193W YJL193W 1.023113 1.389157 1.206135 1.034967 233 YDR444W YDR444W 1.294161 1.114069 1.204115 1.025422 234 YLR137W RKM5 1.056791 1.350816 1.203803 1.023948 235 YKL148C SDH1 1.228134 1.177808 1.202971 1.020015 236 YDR268W MSW1 0.989823 1.413654 1.201739 1.01419 237 YMR191W SPG5 1.462475 0.940733 1.201604 1.013554 238 YDR337W MRPS28 1.387054 1.015401 1.201227 1.011774 239 YJR099W YUH1 1.050507 1.35114 1.200823 1.009864 240 YLR139C SLS1 1.060038 1.340583 1.200311 1.00744 241 YBR235W VHC1 1.293298 1.107254 1.200276 1.007276 242 YJL186W MNN5 1.294444 1.105774 1.200109 1.006488 243 YPL120W VPS30 1.15266 1.246699 1.199679 1.004457 244 YJL207C LAA1 1.327849 1.071369 1.199609 1.004125  Supplementary Table B2 Median values and group assignments of MCL-calculated clusters from Cytoscape analysis.  Light grey is used to highlight MCL cluster assignment. Rank MCL cluster Functional group CW median Genes # genes 1 Endosomal recycling  2.091  3   Endosomal recycling 2.103 RCY1 1   ARF GAP 2.091 GCS1 1   Rab 1.411 YPT31 1 2 Endosomal   2.070  4   Trafficking 2.262 AGE2 1   pH homeostasis 2.100 YDR455C/NHX1, NHX1 2   Other 1.294 NAB6 1 3 AP-1   1.994  10   AP-1 2.204 APS1, APM1, APL4, YKL136W/APL2, 6 132  Rank MCL cluster Functional group CW median Genes # genes YLR171W/APS1, YLR169W/APS1   AP-1 regulator 1.551 IRC6, LAA1 2   Unknown 1.310 YBL010C 1   Other 1.270 TRS33 1 4 Endosomal fusion   1.728  17   Rabs 2.343 VPS21, VPS9 2   Tethers (CORVET) 2.125 VPS3, VPS8, VPS16 3   SNAREs 2.060 PEP12 1   ESCRT 1.634 BRO1, VPS4, VPS24, HSE1, SRN2, VPS60, DID4, VPS27 8   PI3K complex 1.393 VPS30, VPS38 2   Other 1.330 BSD2 1 5 Vacuole fusion and AP-3   1.624  19   Tethers (HOPS) 1.767 PEP5, VPS41, VAM6 3   AP-3 1.700 APL5, APM3, APL6, APS3 4   SNAREs (Vac/Golgi) 1.609 SFT2, VAM7, VAM3 3   V-ATPase assembly 1.545 YDR203W/RAV2, RAV1, PKR1 3   Rabs 1.459 YPT7, MON1 2   Other 1.448 ENT5, ENT3, DRS2, GEF1 4 6 Cell wall/secretion   1.558  6   GPI anchoring 1.980 LAS21, ARV1 2   Secretion 1.554 ERD1, SEC66 2   Cell wall 1.464 GAS1 1   Other 1.256 VPS74 1 7 Ribosomes   1.476 OPI11/RPL43A, YDR417C/RPL12B, RPL43A 3 8 Incoming Golgi vesicles  1.439  8   Trafficking 2.408 ARF1, VPS1 2   GARP 1.371 VPS51, VPS52, VPS54 3   Lipid dynamics 1.300 YNL296W/MON2, ERG2, SAC1 3 9 Stress E-MAP   1.345 KRE28, YGR022C/MTL1, CTK3, PTC1, FAB1, THP1, SLX8, CAN1, HUR1/PMR1 9 10 Mitochondria   1.327  22   Mitochondrial 1.329 RSM24, MTG1, RSM7, ATP22, CYT1, AFG3, MHR1, MSD1, MSW1, ATP7, MRPL32, MRP1, ATP17, MRP17, DIA4, OCT1, MSE1, MRPL31, MDM31, MRPL13, GTF1 21   Unknown 1.298 IRC19 1 11 Mitochondria  1.298  37   DNA 1.542 SPT3, HAP4 2   Other 1.317 YGL114W 1 133  Rank MCL cluster Functional group CW median Genes # genes   Mitochondrial 1.297 YDR230W/COX20, YNR042W/COQ2, YLR202C/COQ9, CYT2, MSS51, COQ4, ATP11, AEP1, COX6, COQ5, COQ2, COQ9, FUM1, MSS116, COX9, BCS1, CBP2, SHY1, COX7, PSD1, COX10, MSS2, QCR9, SCO1, QCR8, PET111, CBP1, IMP1, COX18, OXA1, CBS2, AEP2, IMP2, PET54 34 12 Mitochondria   1.284 IMG2, MRPL51, MRPS28, RSM19, CCM1, MIP1, PET309, MTF1, MRPL25, SLS1, PET123, MRPL33, SLM5 13 13 Mitochondria   1.230 MRPL36, MRPL8, MRPS16 3   Supplementary Table B3 List of yeast strains used in Chapter 3. Strain ID Genotype Source PJ694a MATa trp1-901 leu2Δ-3 ura3Δ-52 his3Δ-200 gal4Δ gal80Δ LYS::GAL1::HIS3 GAL2::ADE-2 met2::GAL7-lacz A. Merz (U. Washington) PJ694α MATα trp1Δ-901 leu2Δ-3 ura3Δ-52 his3Δ-200 gal4Δ gal80Δ LYS::GAL1-HIS3 GAL2-ADE-2 met2::GAL7-lacz A. Merz (U. Washington) BY4741 MATa HIS3Δ1; leu2Δ0; met15Δ0; ura3Δ0 Open Biosystems BY4742 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 Open Biosystems CSY3366 BY4741 LAA1-3HA::KANR LAA2(Y91A)-GFP+::HIS3 This study CTY105 BY4741 LAA1-GFP+::HIS3 GGA2-mRFP1.5::NATR This study CTY15 BY4741 APM1-GFP+::HIS3 This study CTY297 BY4741 LAA2(F24GNF>AANA)-GFP+::HIS3 This study CTY339 BY4741 LAA2-GFP+::HIS3 This study CTY340 BY4741 LAA1-GFP+::HIS3 This study CTY402 BY4742 chs6Δ::KANR laa2Δ::NATR This study CTY403 BY4742 chs6Δ::KANR laa1Δ::NATR This study CTY409 BY4742 chs6Δ::KANR apl2Δ::NATR This study CTY414 BY4741 APM1-GFP+::HIS laa1Δ::NATR This study CTY421 BY4741 LAA2-GFP+::HIS3 laa1Δ::NATR This study CTY427 BY4741 LAA2-GFP+::HIS3 LAA1-3HA::KANR This study CTY429 BY4741 LAA1-GFP+::HIS3 laa2Δ::NATR This study CTY435 BY4741 LAA1-GFP+::HIS3 apl2Δ::NATR This study CTY497 BY4741 APM1-GFP+::HIS3 LAA2-3HA::KANR This study CTY499 BY4741 LAA1-GFP+::HIS3 LAA2-3HA::KANR This study CTY505 BY4741 LAA2-GFP+::HIS3 LAA1-3HA::KANR This study CTY545 BY4741 APM1-GFP+::HIS3 LAA1-3HA::KANR This study 134  Strain ID Genotype Source CTY548 BY4741 LAA1-3HA::KANR This study CTY558 BY4741 APM1-GFP+::HIS3 LAA1-3HA::KANR laa2Δ::NATR This study CTY562 BY4741 LAA2-GFP+::HIS3 APL4-3HA::KANR This study CTY566 BY4741 LAA2(F24GNF>AANA)-GFP+::HIS3 APL4-3HA::KANR This study CTY571 BY4741 LAA2-GFP+::HIS3 APL4(ΔEAR)-3HA::KANR This study CTY611 BY4741 LAA2-GFP+::HIS3 LAA1-mRFP1.5::NATR This study CTY685 BY4741 LAA2-GFP+::HIS3 APM1-tdTomato::NATR This study CTY694 BY4741 LAA1-GFP+::HIS3 APM1-tdTomato::NATR This study CTY788 BY4742 chs6Δ::KANR LAA2(F24GNF>AANA)-GFP+::HIS3 This study CTY826 BY4741 LAA1-GFP+::HIS3 This study MDY122 BY4742 chs6Δ::NATR This study MDY768 BY4742 chs6Δ::NATR, slo1Δ::HPH This study MDY938 BY4741 LAA1-3HA::KANR LAA2(L237P)-GFP+::HIS3 This study MDY941 BY4741 LAA1-3HA::KANR LAA2(F24GNF>AANA)-GFP+::HIS3 This study SWY427 BY4741 LAA2-GFP::HIS3 slo1Δ::HPH This study SWY439 BY4741 LAA1-GFP+::HIS3 SNX3-Ruby::KANR This study SWY440 BY4741 LAA2-GFP+::HIS3 SNX3-Ruby::KANR This study SWY448 BY4741 SLO1-GFP+::HIS3 laa1Δ::NATR This study SWY449 BY4741 SLO1-GFP+::HIS3 laa2Δ::NATR This study SWY450 BY4741 SLO1-GFP+::HIS3 laa1Δ::NATR laa2Δ::HPH This study SWY451 BY4741 SLO1-GFP+::HIS3 laa1Δ::NATR laa2Δ::HPH This study SWY454 BY4741 laa1Δ::NATR This study SWY455 BY4741 laa2Δ::NATR This study SWY461 BY4742 LAA2(F24GNF>AANA)-GFP+::HIS3 slo1Δ::HPH This study SWY463 BY4742 slo1Δ::HPH, LAA2(L237P)-GFP+::HIS3 This study SWY464 BY4741 LAA2-GFP+::HIS3 laa1Δ::NATR slo1Δ::HPH This study SWY494 BY4742 slo1Δ::HPH, LAA2(Y91A)-GFP+::HIS3 This study  Supplementary Table B4 List of plasmids used in Chapter 3. Plasmid ID Description Source pRS316 pURA3 (CEN) (Sikorski and Hieter, 1989) pCT37 pLAA1-3HA::URA3 (CEN) This study pCT36 pLAA2-3HA::URA3 (CEN) This study pGAD-C2 pGAD-C2::LEU2 (2µ) P. James (U. Wisconsin) pCT15 pGAD-C2-APL4::LEU2 (2µ) This study pCT19 pGAD-C2-APL4(1-618)::LEU2 (2µ) This study pCT21 pGAD-C2-APL4(717-832)::LEU2 (2µ) This study pCT39 pGAD-C2-GGA2::LEU2 (2µ) This study pGBDU-C2 pGBDU-C2::URA3 (2µ) P. James 135  Plasmid ID Description Source pCT9 pGBDU-C2-IRC6::URA3 (2µ) This study pCT23 pGBDU-C2-LAA2::URA3 (2µ) This study pCT27 pGBDU-C2-LAA2(F24GNF>AGAA)::URA3 (2µ) This study pMD72 pKANR (CEN) This study pMD109 pHA-Slo1::KANR (CEN) This study pLC1864 pDS_GFP-SCOC(iso 5) (Gateway expression vector) This study pLC2987 pCMV3-FLAG-FEZ1 Sino Biologicals pLC2144 pCMV3-FLAG-FEZ2 This study pMD284 pCMV3-FLAG-FEZ2(L243P) This study pMD293 pCMV3-FLAG-FEZ2(W91A) This study pLC3043 pDEST-myc-UVRAG S. Tooze (Francis Crick Institute) pLC3059 pEGFP-aftiphilin J. Hirst (Cambridge Institute for Medical Research) pLC3198 pEGFP-aftiphilin(W702A) This study pLC3199 pEGFP-aftiphilin(Y716QW>SQS) This study pLC3179 pcDNA3.1+-myc-CLBA1 Genscript pLC3212 pcDNA3.1+-myc-CLBA1(W197A) This study  Supplementary Table B5 List of antibodies used in Chapter 3. Host Target Clonality Source Mouse GFP Monoclonal Roche Mouse FLAG M2 Monoclonal Sigma-Aldrich Mouse Gamma adaptin Monoclonal BD Transduction Laboratories  Mouse HA.11 Monoclonal Covance Mouse Myc Monoclonal EMD Millipore Mouse Dpm1 Monoclonal Molecular Probes Rabbit GFP Polyclonal L. Berthiaume (University of Alberta) www.eusera.com Rabbit Gamma adaptin Polyclonal Abcam Rabbit Gamma synergin Polyclonal M. Robinson (U. Cambridge) Rabbit Aftiphilin Polyclonal M. Robinson (U. Cambridge) Rabbit HEATR5B Polyclonal M. Robinson (U. Cambridge) Rabbit FLAG Polyclonal Sigma-Aldrich Rabbit HEATR5A Polyclonal Abcam Rabbit HA Polyclonal Abcam Rabbit Myc Polyclonal Abcam Goat Mouse HRP conjugate Jackson ImmunoResearch Goat Rabbit HRP conjugate Jackson ImmunoResearch   

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