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The endoplasmic reticulum diffusion barrier and inter-organelle contact sites Chao, Jesse Tzu-Cheng 2013

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THE ENDOPLASMIC RETICULUM DIFFUSION BARRIER AND INTER-ORGANELLE CONTACT SITESbyJesse Tzu-Cheng ChaoB.Sc.Hon., Queen?s University, 2007A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES(Cell and Developmental Biology)THE UNIVERSITY OF BRITISH COLUMBIA(Vancouver)October, 2013? Jesse Tzu-Cheng Chao, 2013AbstractPolarization of cellular membranes into domains is an important mechanism to compartmentalize cellular activities within the membrane and establish cell polarity. Recent studies have uncovered that the endoplasmic reticulum (ER) is polarized by diffusion barriers, which in neurons controls glutamate signaling in dendritic spines, but the molecular identity of these diffusion barriers is unknown. In Chapter 2 we show that a direct interaction between integral ER protein Scs2 and septin Shs1 creates the ER diffusion barrier in yeast. We uncovered a new ER-associated polarisome subunit, Epo1, which is required for the tethering of ER to septins. The human homologue of Scs2, VAP-B, also interacts with Shs1 in yeast indicating that the tether may be conserved. As mutations in VAP-B cause amyotrophic lateral sclerosis, loss of ER polarization in dendritic spines is a potential mechanism underlying motorneuron disease.Synthesis of phospholipids, sterols and sphingolipids is thought to occur at contact sites between the ER and other organelles because many lipid synthesizing enzymes are enriched at contact sites. In only a few cases have the enzymes been localized to contacts in vivo and in no instances have the contacts been demonstrated to be required for enzyme function. In Chapter 3 we show that plasma membrane (PM) - endoplasmic reticulum (ER) contact sites in yeast are required for phosphatidylcholine synthesis and regulate the activity of a key enzyme, Opi3, whose activity requires a lipid binding protein, Osh3. Thus, membrane contact sites provide a structural mechanism to regulate lipid synthesis.iiAdditionally, phospholipid metabolism requires phospholipid transfer between ER and mitochondria at membrane contact sites. In Chapter 4 we show that a S. cerevisae strain missing multiple components of the conserved ER-membrane proteins complex (EMC) have reduced phosphatidylserine (PS) transport from the ER to mitochondria. These defects were corrected by expression of an artificial protein that tethers the ER to mitochondria. Our findings suggest that the EMC facilitates ER to mitochondria PS transport by promoting ER-mitochondria tethering and that phospholipid exchange between the ER and mitochondria and tethering of these organelles are essential processes.iiiPrefaceChapter 2: ?Polarization of the Endoplasmic Reticulum is Mediated by ER-Septin Tethering? is adopted from a first author manuscript. The authors are Chao JT, Wong AKO, Tavassoli S, Young BP, Fang N, Chruscicki A, Howe LJ, Mayor T, Foster, LJ and Loewen CJ. All experiments were designed by myself and Dr. Chris Loewen. I conducted the majority of experiments with the help of Andrew Wong. Adam Chruscicki (Howe Lab) and Nancy Fang (Mayor lab) carried out Epo1-TAP purification and identified binding partners by mass spectrometry. Dr. Young did SGA for SCS2. Dr. Foster helped with the SILAC analysis for Scs2?TM purification.Chapter 3: ?Plasma membrane?endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis? is based on a second author manuscript published in EMBO Reports. Tavassoli S, Chao JT, Young BP, Cox RC, Prinz WA, de Kroon AI and Loewen CJR, Plasma membrane-endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis, EMBO Rep. (2013), doi:10.1038/embor.2013.36. I conducted many experiments especially for the resubmission. My exact contributions are Fig. 3.1I, Fig. 3.4C, Fig. 3.5E, Fig. 3.6C, Fig. 3.8 A-F and Fig. 3.9B-C.Chapter 4:? A conserved ER-membrane complex facilitates phospholipid exchange between the ER and mitochondria?  is adopted from a co-first authored manuscript. The authors are Lahiri S, Chao JT, Tavassoli S, Wong A, Young BP, Loewen CJR,  and Prinz WA. I contributed equally and am co-first authors with Sujoy Lahiri and Shabnam ivTavassoli. The project was initiated by myself and Shabnam Tavassoli, and a collaboration with Dr. Prinz at the NIH was set up and Sujoy Lahiri carried out lipid measurements. My exact contributions are Fig. 4.4B, Fig. 4.9A-D, Fig. 4.10 and Fig. 4.11. vTable of Contents..........................................................................................................................Abstract  ii...........................................................................................................................Preface  iv..........................................................................................................Table of Contents  vi..................................................................................................................List of Tables x................................................................................................................List of Figures xi...................................................................................................List of Abbreviations xiii........................................................................................................Acknowledgments xv...................................................................................................................Dedication xvii1 ...........................................................................................Chapter 1: Introduction 11.1 ..............................................................................................................ER$structure$and$func/on$ 11.1.1 .............................................................................................................................ER$structure$ 11.1.2 ..........................................................................................ER$is$a$mul/5func/oning$organelle$ 21.2 ...............................................................................................................................ER$polariza/on$ 31.2.1 .................................................................................Polarity$establishment$in$budding$yeast$ 41.2.2 ..................................................................................ER$inheritance$during$polarized$growth$ 61.2.3 .....................................................................................................................Diffusion$barriers$ 81.2.4 .....................................................................................................................................Sep/ns$ 91.2.5 ...................................................................................................Localized$protein$synthesis $ 111.2.6 .......................................................................................................Migra/on$of$the$nucleus$ 111.3 ..................................................................................................................................ER$junc/ons$ 141.3.1 ..........................................................................................Structural$characteris/cs$of$ERJs:$ 141.3.2 ............................................................................................Molecular$components$of$ERJs:$ 161.3.3 ...................................................................................................................Func/ons$of$ERJs$ 171.4 .................................................................................................................Phospholipid$synthesis$ 201.4.1 ....................................................................................................Pathways$for$PC$synthesis $ 211.4.2 ......................................................................................................Non5vesicular$lipid$traffic$ 22vi2 Chapter 2: Polarization of the Endoplasmic Reticulum is Mediated by an ER-.....................................................................................Septin Tethering Complex 262.1 .................................................................................................................................Introduc/on$ 262.2 .......................................................................................................................................Methods$ 282.2.1 ....................................................................................Yeast$cell$culture$and$manipula/ons$ 282.2.2 .............................................................................................SILAC$proteomics$for$Scs2?TM$ 292.2.3 ..............................................................................................................Proteomics$for$Epo1$ 302.2.4 .............................................................................................................................LC/MS/MS$ 302.2.5 ...........................................................................Protein5fragment$complementa/on$assay$ 322.2.6 .............................................................................................................Image$quan/fica/on$ 322.2.7 ................................................................................................Photobleaching$experiments$ 332.2.8 .................................................................................................Pearson$correla/on$analysis$ 342.2.9 ...........................................................................................SGA$analysis$and$bioinforma/cs$ 342.2.10 .........................................................................................Recombinant$protein$purifica/on$ 352.2.11 ..........................................................................Recombinant$protein$in$vitro$binding$assay$ 362.2.12 .......................................................................................................Co5Immunoprecipita/on$ 362.3 .........................................................................................................................................Results$ 372.3.1 ...................................................................Epo1$localizes$Scs2$to$sites$of$polarized$growth$ 372.3.2 ......................................................................................Epo1$a^aches$ER$to$the$bud$cortex$ 382.3.3 ......................................................................................Epo1$interacts$with$the$polarisome$ 432.3.4 .........................................................................ER$diffusion$barrier$requires$Epo1$and$Scs2$ 482.3.5 ........................Epo1,$Scs2$and$Shs1$form$an$ER5sep/n$tethering$complex$at$the$bud$neck$ 552.3.6 .............................................................ER5sep/n$tethering$creates$the$ER$diffusion$barrier$ 622.3.7 .....................................ER5sep/n$tethering$creates$the$nuclear$envelope$diffusion$barrier$ 632.3.8 .............Polariza/on$of$the$integral$ER$protein$Ist2$is$mediated$by$the$ER$diffusion$barrier$ 672.3.9 ....................................................................Scs2$plays$a$role$in$S$phase$spindle$posi/oning$ 712.3.10 ...............................................................................................Scs2$recruits$Num1$to$pmaER$ 742.3.11 The$ER$diffusion$barrier$controls$the$localiza/on$of$Num1$and$the$/ming$of$nuclear$...............................................................................................................................migra/on$ 812.4 ....................................................................................................................................Discussion$ 852.5 ...........................................................................................................................................Tables$ 893 Chapter 3: Plasma Membrane-Endoplasmic Reticulum Contact Sites Regulate ............................................................................Phosphatidylcholine Synthesis 98vii3.1 .................................................................................................................................Introduc/on$ 983.2 .......................................................................................................................................Methods$ 993.2.1 .....................................................................................................Plasmids$and$yeast$strains $ 993.2.2 ............................................................................................................Yeast$growth$assays$ 1013.2.3 ....................................................................Array5based$genome5wide$suppressor$screen$ 1013.2.4 ....................................................................................................In$vivo$methyla/on$assay$ 1023.2.5 ...........................................................................................In$vitro$Opi3$methyla/on$assay$ 1033.2.6 .........................................................NBD5PC,$NBD5PE$and$FM4564$cellular$uptake$assays$ 1043.3 .......................................................................................................................................Results$ 1043.3.1 ......PM5ER$contacts$play$roles$in$the$methyla/on$pathway$of$phospholipid$biosynthesis.$ 1043.3.2 ..........................................The$PC$methyltransferase,$Opi3,$func/ons$at$ER5PM$contacts$ 1103.3.3 Pah1$is$a$mul/5copy$suppressor$for$PC$synthesis$defects$and$restores$PM5ER$contacts$in$...................................................................................................................?scs2?ice2$cells$ 1153.3.4 ....................................................................Opi3$may$func/on$in$trans$at$PM5ER$contacts$ 1223.4 ..................................................................................................................................Discussion$ 1264 Chapter 4: A Conserved ER-Membrane Complex Facilitates Phospholipid ...................................................Exchange Between the ER and Mitochondria 1294.1 ...............................................................................................................................Introduc/on$ 1294.2 ....................................................................................................................................Methods$ 1324.2.1 ....................................................................Yeast$strains,$plasmids,$and$gene/c$methods$ 1324.2.2 ..............................................Synthe/c$Gene/c$Array$(SGA)$Analysis$for$CHO2$and$EMC6$ 1334.2.3 ...................................................Protein$Subcellular$Localiza/on$by$Confocal$Microscopy$ 1344.2.4 ..........................................................................................In$vivo$labeling$with$[3H]serine.$ 1344.2.5 ......................................................Mitochondrial$extracts$and$in$vitro$[3H]serine$labeling$ 1354.2.6 ...........Mitochondrial$purifica/on$and$determina/on$of$mitochondrial$steady$state$lipid$ 1364.2.7 ..............................................................................................................................Psd$assay$ 1364.3 .......................................................................................................................................Results$ 1374.3.1 Gene/c$screen$for$components$that$mediate$transport$of$phospholipids$between$ER$and$.......................................................................................................................mitochondria$ 1374.3.2 ...........Gene/c$interac/ons$reveal$that$the$EMC$func/ons$in$the$phospholipid$synthesis$ 1424.3.3 ..............................................................................EMC$proteins$form$a$complex$in$the$ER$ 1454.3.4 ..........ER$to$mitochondria$PS$transport$decreases$in$cells$missing$mul/ple$Emc$proteins.$ 1484.3.5 Mitochondria$in$5x5emc$cells$are$nonfunc/onal$and$have$abnormal$phospholipid$levels.$152viii4.3.6 .....................5x5emc$cells$have$a$reduced$rate$of$ER$to$mitochondria$PS$transfer$in$vitro$ 1564.3.7 The$EMC$and$ERMES$complex$are$required$for$viability$and$ER$to$mitochondria$PS$..............................................................................................................................transport$ 1564.3.8 An$ar/ficial$ER5mitochondria$tether$restores$PS$transfer$in$cells$missing$mul/ple$Emc$.............................................................................................proteins$and$ERMES$proteins.$ 1574.3.9 ........................................The$EMC$interacts$with$Tom5$at$ER5mitochondria$contact$sites$ 1594.4 ..................................................................................................................................Discussion$ 1664.5 .........................................................................................................................................Tables$ 1705 .........................................................................................................Conclusions 1735.1 ......................................................................................................................Chapter$summary$ 1735.2 .................................................................................................................Common$discussions$ 1755.2.1 .....................................................................................The$significance$of$ER$polariza/on.$ 1755.2.2 ..........................................................................ER$Polariza/on$by$the$ER$diffusion$barrier$ 1765.2.3 ..........................................................................................The$significance$of$ER$junc/ons $ 176................................................................................................................Bibliography 179ixList of Tables....................................................Table 2.1 Scs2?TM proteomics, related to Figure 2.1 87...........................................................Table 2.2 Epo1 proteomics, related to Figure 2.2 88Table 2.3 SGA results for SCS2, ...................................................related to Figure 2.7 89Table 2.4 Aggravating genetic interactions identified in the ?scs2 SGA grouped ................................................according to gene ontology term, related to Figure 2.7 . 95Table 4.1 Genetic interactions with EMC6 ................................, related to Figure 4.2. 168xList of FiguresFigure 1.1 Structural organizations of ER in mammalian cells and yeast ........................ 2............................................Figure 1.2 Polarization during cell growth in budding yeast  5....................................Figure 1.3 3D tomography analysis of yeast ER during budding  7.........................................Figure 1.4. Molecular and structural organization of septins  10.......................................................Figure 1.5 The nuclear migration pathway in yeast  13.................................................................................................Figure 1.6 ER junctions  15..............................Figure 1.7 Pathways for phospholipid synthesis in eukaryotic cells  21Figure 1.8 Models of lipid transfer proteins facilitating non-vesicular lipid traffic at ERJs ........................................................................................................................................ 23......................Figure 2.1 Epo1 interacts with Scs2 and captures ER tubules in the bud 39.............................................................Figure 2.2 Epo1 is a Subunit of the Polarisome 44............................Figure 2.3 Epo1 and Scs2 are Required for the ER Diffusion Barrier 50................................Figure 2.4 Epo1 and Scs2 Interact with Shs1 in Septin Filaments 57...................Figure 2.5 Characterizing the role of Shs1 CTE in the ER diffusion barrier 65..................................Figure 2.6 The ER Diffusion Barrier Polarizes Ist2 Within the ER 69.........................Figure 2.7 Scs2 plays a role in the nuclear migration pathway in yeast 72.............................................................Figure 2.8 Num1 interacts with Scs2 on the ER 76.................................Figure 2.9 The ER Diffusion Barrier Controls Spindle Positioning 81Figure 3.1 Opi3 function is compromised in ?scs2?ice2 ..................................... cells. 105...................................................................................Figure 3.2 Related to Figure 3.1 107...................................................................Figure 3.3 PM-ER contacts regulate Opi3. 110xi...................................................................................Figure 3.4 Related to Figure 3.3 112.................................................................Figure 3.5 Pah1 regulates PM-ER contacts. 115...................................................................................Figure 3.6 Related to Figure 3.5 117...................................................................................Figure 3.7 Related to Figure 3.5 119Figure 3.8 PM-ER contacts affect PM stability in trans ................................................ 122...................................................................................Figure 3.9 Related to Figure 3.8 123Figure 4.1 Genome-wide screen for regulators of phospholipid synthesis................... 137................................Figure 4.2 The EMC genes function in phospholipid metabolism. 139.......................................Figure 4.3 EMC genes function in phospholipid metabolism 142Figure 4.4 Cells missing multiple EMC proteins have defects in PS transfer from the ER mitochondria 145Figure 4.5 Mitochondria from cells missing emc proteins have reduced levels of PS and .............................................................................................PE and are not functional 149Figure 4.6 5x-emc mmm1-1 cells are not viable and have a dramatic reduction in ER to ............................................mitochondria PS transfer at nonpermissive temperature. 152.......................Figure 4.7 The EMC interacts with Tom5 at ER-mitochondria contacts 153Figure 4.8 5x-emc mmm1-1 cells are not viable and have a dramatic reduction in ER to mitochondria PS transfer at nonpermissive temperature..............................................157Figure 4.9 The EMC interacts with Tom5 at ER-mitochondria contacts........................160Figure 4.10 Ale1 control for PCA between the EMC and Tom5....................................162Figure 4.11 Localization of ERMES subunit Mdm34 is not disrupted in EMC mutants.........................................................................................................................163xiiList of Abbreviations?C Degree Celsius? Delta- signifies deletionALS Amyotrophic Lateral SclerosisCa2+CalciumCCD Coiled-coil DomainCTE C-terminal ExtensionDAG DiacylglycerolDTT DithiothreitolEDTA Ethylene Diamine Tetraacetic AcidEGTA Ethylene Glycol Tetraacetic AcidEM Electron MicroscopyEMC ER Membrane protein ComplexER Endoplasmic ReticulumERAD ER Associated DegradationERJ Endoplasmic Reticulum JunctionsERMES ER-Mitochondria Encounter StructureFFAT Two-Phenylalanines in an Acidic TractFig FigureGFP Green Fluorescent ProteinGST Glutathione-S-TransferaseGTP Guanosine TriphosphateIP ImmunoprecipitationIP3 Inositol 1,4,5-trisphosphateL LiterLC Liquid ChromatographyLTP Lipid Transfer ProteinsMBP Maltose Binding Proteinml MililitermM MilimolarMME MonomethylethanolaminemRNA Messanger RNAMS Mass SpectrometryMSP Major Sperm Proteinp Denotes PlasmidPA Phosphatidic AcidPC PhosphatidylcholinePCA Protein Complementation AssayPCR Polymerase Chain ReactionxiiiPDE Phosphatidyl-dimethyl-ethanolaminePE PhosphatidylethanolaminePEMT PE Methyl-TransferasePI PhosphatidylinositolPI4P Phosphatidylinositol 4-PhosphatePI4P Phosphatidylinositol 4-phosphatePIP PhosphoinositidePM Plasma MembranepmaER Plasma Membrane Associated ERPME Phosphatidyl-monomethyl-ethanolaminePS PhosphatidylserineRFP Red Fluorescent ProteinROI Region of InterestRyR Ryanodine ReceptorSD Synthetic DefinedSD Standard DeviationSDS Sodium Dodecyl SulfateSDS-PAGE SDS-Polyacrylamide Gel ElectrophoresisSEM Standard Error MeasurementsSGA Synthetic Genetic ArraySOCE Store-Operated Calcium Entryt1/2Half TimesTAP Tandem Affinity PurificationTM Transmembrane DomainUPR Unfolded Protein ResponseUPRE UPR ElementsVAPVesicle-Associated Membrane Protein-Associated ProteinVF Venus Fragmentw/v Weight to VolumeWB Western BlotWT Wild TypeYFP Yellow Fluorescent Protein? Alpha-signifies anti?g Microgram?M MicromolarxivAcknowledgmentsI would like to thank the people listed here who have helped me during my PhD studies. Their support was invaluable to completion of this work.First, I would like to thank Dr. Chris Loewen, my supervisor, mentor and friend. I am especially grateful to Dr. Loewen for his continual support and constant encouragement. As a friend, Dr. Loewen has given me countless advice, both on career and life.I would also like to thank my supervisory committee, Dr. Ivan Robert Nabi, Dr. LeAnn Howe and Dr. Leonard Foster for their insight, criticism and advice on my work. I want to acknowledge the past and present members of the Loewen lab, including Shabnam Tavassoli, John Shin, Barry Young, Leslie Chen and Jennifer McQueen. I would especially like to thank Andrew Wong for his essential contributions to this work. I would like to extend my gratitude to Adam Chruscicki (Howe lab) and Dr. Sujoy Lahiri (Prinz lab) for their productive collaboration that made this work possible. I want to extend my gratitude to the University of British Columbia for funding.xvMost importantly, I want to thank my parents, Shih-Lin Chao and Hsiu-Chuan Lee, as I would have never made it here if not for their sacrifices and dedication to bring me to Canada. My siblings, Werner, Glen and Willie, for our cohesive brotherhood. Finally, I have to thank my fianc?, Kay Pham, for giving me the love that no words can describe, and for making me the better person.For Laker and Mika, ruff ruff, woof, grr....lick lick lickxviDedication?Every kid starts out as a natural-born scientist, and then we beat it out of them. A few trickle through the system with their wonder and enthusiasm for science intact.?- Carl SaganI would like to dedicate this thesis to my parents. I am amongst the lucky ones whose parents have always encouraged me to be curious, considering I have been thrown out of class for asking too many questions before.xvii1 Chapter 1: Introduction1.1 ER structure and function1.1.1 ER structureStructurally, the endoplasmic reticulum consists of membrane sheets, linear tubules, and polygonal reticulum with three way junctions located in distinct compartments/domains within the cell (Voeltz et al., 2002). Functionally, it has been classified as smooth or rough, which play separate roles in calcium regulation and protein synthesis, respectively (Voeltz et al., 2002). In spite of its complex structures and diverse biochemical activities, the ER is one contiguous organelle that is capable of exchanging information between these regions. Advances are being made in identifying the molecular components required for structuring the ER, and work in budding yeast has resulted in numerous significant advances, including identification of proteins that tubulate the ER (Voeltz et al., 2006) or shape the sheets (Shibata et al., 2010), regulate nuclear envelope size (Kim et al., 2007; Santos-Rosa et al., 2005), and enable interaction of ER with the cytoskeleton (Du et al., 2004). In Saccharomyces cerevisiae, the ER is morphologically organized into distinct domains, including the nuclear envelope (NE) that surrounds the nucleus, and plasma membrane associated ER (pmaER) that forms a reticular network beneath the plasma membrane (PM) characteristic of all eukaryotes (Figure 1.1) (Voeltz et al., 2002).1    Figure 1.1. Structural organizations of ER in mammalian cells and yeast. (A) Confocal images of a COS cells expressing GFP fused to Sec61?, a subunit of the translocon complex. (B) Confocal images of an yeast cell expressing Sec63-GFP. Top: the microscope was focused on the centre to show the pmaER in the cell cortex and NE. Bottom: focused on the periphery to show the reticular ER. Images taken from Voeltz et al., 2006.1.1.2 ER is a multi-functioning organelleThe ER has a multitude of functions. First, it is responsible for the synthesis and facilitates the transport of membrane bound and secreted proteins. Its role in protein synthesis also includes quality control, where misfolded proteins are targeted for destruction by the ubiquitin proteasome system, in a pathway called ERAD for ER associated degradation. Additionally, the ER is sensitive to environmental and intracellular stress and can mount stress responses to maintain cellular homeostasis. 2A GFP-Sec61? B Sec63-GFPPeripheral ERZoomFigure 1.1Stressors such as heat or drug treatments (DTT, tunicamycin) often cause the accumulation of misfolded proteins in the ER lumen, leading to the activation of the highly conserved unfolded protein response (UPR). Upon activation, a signaling cascade leads to the upregulation of UPR elements (UPRE), which typically encodes for ER chaperones and phospholipid synthesizing enzymes, and their products work together to expand the ER membrane, to increase the protein folding capacities of the ER and to degrade misfolded proteins. Second, the ER is responsible for the biosynthesis of all lipid precursors (Du et al., 2004; Lowe and Barr, 2007). A large number of lipid synthesizing enzymes, including those for sterols, sphingolipids and phospholipids are localized to the ER (Natter et al., 2005). Lastly, the ER harbours the largest pool of Ca2+ within the cell and as such, regulates intracellular Ca2+ signaling. Ca2+ released from the ER activates a variety of transcriptional and translational cascades for protein folding, UPR, ERAD and apoptosis (Berridge, 2002). Notably, the regulation of Ca2+ release from and entry to the ER is crucial to cellular physiology. In mammalian cells, store-operated calcium entry (SOCE) relies on the physical interactions between ER and PM, and will be discussed further in section 1.3 ER Junctions. In short, the ER is a protein and lipid synthesis machinery, as well as a signaling organelle.1.2 ER polarizationEukaryotes localize proteins to specific membrane-bound organelles to concentrate and segregate biochemical activities. Establishing cell polarity enables spatial specialization of physiological functions and is a property of all cells, such as the dendrites and axons in neurons, or apical and basolateral surfaces of epithelial cells. 3The budding yeast Saccharomyces cerevisiae is an excellent model for studying eukaryotic cell polarity because it grows vegetatively by asymmetric budding. Similar to mammalian cells, yeast establishes polarity using the highly conserved Ras and Rho-like family of GTPases, particularly Cdc42 (reviewed in (Pruyne et al., 2004)). When a new bud forms, ER is arguably the first organelle to be inherited in the growing bud followed by the inheritance of Golgi and mitochondria (Frederick et al., 2008; Reinke et al., 2004). Localization of mRNAs to the yeast bud tip is also dependent on ER inheritance  (Schmid et al., 2006) and is guided by similar polarity factors such as Cdc42 and the exocyst subunit Sec4 (Aronov et al., 2007).1.2.1 Polarity establishment in budding yeastLike all eukaryotic cells, budding yeast can polarize in response to external cues such as mating factors to direct shmoo (mating projections) formation, as well as to internal cues such as during cell replication. The actin cytoskeleton is important for both processes. For polarized cell growth, once a bud site is selected Cdc42 recruits a yeast-specific polarity complex called the polarisome, comprising Pea2, Spa2, Bud6 and a formin, Bni1, which nucleates actin cables in the bud (Figure 1.2). Type V myosin motor proteins then mediate directional transport of exocytic vesicles on these cables resulting in polarized growth of the bud (Figure 1.2C). Polarity establishment also requires myosin-dependent transport of mRNA, mitochondria, vacuoles, ER, trans Golgi, and astral microtubules to the growing bud (Figure 1.2C). Therefore, work in budding yeast has made major contributions in our understanding of how proteins such as Rho and Cdc42 establish polarity, in organelle inheritance and distribution, and in identifying the exocyst complex and elucidating its functions in polarized secretion.  45BFigure 1.2PolarisomeFigure 1.2. Polarization during cell growth in budding yeast(A) A diagram of the yeast morphogenic cycle with major cellular components contributing to polarization labeled. (B) The Rho-GTPase Cdc42 recruits the polarisome at the bud tip and septins (blue) at the bud neck to establish polarity. The polarisome organizes actin cables (orange) in the mother-bud axis and the septins organize a contractile actin ring at the bud neck for cytokenesis. Red patches indicate the localizations of formins (Bni1 at the bud tip and Bnr1 and the neck). (C) Polarized transport of organelles in yeast. The formins of the polarisome polarizes actin cables, on which the myosin motors transport (1) peroxisome (2) mitochondria (3) vacuoles (4) Golgi (5) microtubule plus ends for orientation of the nucleus (6) mRNA (7) cortical ER and (8) post-Golgi secretory vesicles (SV). (A and C) are adapted from (Pruyne et al., 2004), and (B) from  (Moseley and Goode, 2006).1.2.2 ER inheritance during polarized growthER biogenesis in yeast has been studied at high resolution by 3D electron tomography (Figure 1.3) (West et al., 2011). As previously implied from light microscopy studies (Fehrenbacher et al., 2002; Prinz et al., 2000), electron tomography shows that as the bud emerges, a few ER tubules that originate from a central cisternal ER domain in the mother project into the bud along the polarity axis. As the bud grows, these tubules form a ?nexus? near the centre of the bud and project toward the bud cortex, where they are captured on the PM (West et al., 2011). From these contacts, a reticular network forms, consisting of three-way junctions and flattened cisternae, which are 6Figure 1.3. 3D tomography analysis of yeast ER during budding. A to F: increasing sized bud. Left to right: merged images showing all ER domains (left), yellow cisternal ER and green ER tubules (middle), and pmaER (blue). Adopted from West et al., 2011.7Figure 1.3similar to those observed in mammals (compare to Figure 1.1A) (Voeltz et al., 2006). However, in yeast this network lies just beneath the PM and is called PM-associated ER (pmaeER). pmaER is compartmentalized into bud and mother domains, which are connected by only a few ER tubules that pass through the neck (West et al., 2011). Thus, structurally, the ER in yeast is polarized.1.2.3 Diffusion barriersNot only is the structure of the ER polarized, but the distributions of certain ER proteins also appear polarized. For example, the yeast ER protein Ist2 was discovered to be asymmetrically enriched in the bud due to polarized delivery of its mRNA and localized translation there (Takizawa et al., 2000). Subsequently, 22 mRNAs were found to be polarized to the bud, and many of their protein products have bud-enriched localizations (Shepard et al., 2003). The polarized localization of Ist2 was found to be dependent on membrane diffusion barrier mediated by septins at the neck (discussed in the next section). Therefore, ER polarization may be achieved not only by actively polarizing its protein components to the bud, but also by preventing their diffusion back to the mother.The diffusion barrier concept encompasses the idea that membrane proteins diffuse freely within one domain, such as within the mother cell, their diffusion across domains are restricted, such as between the mother and bud. In yeast, this is achieved through the action of septins, which are known to polarize the PM in yeast by creating a diffusion barrier that separates bud and mother (Barral et al., 2000). Compartmentalization by septins is a common mechanism for creating polarized PM domains in many specialized cell types, including neurons, epithelial cells and 8spermatozoa (Saarikangas and Barral, 2011). The molecular constituents of septins will be discussed further in the next section.Accumulating evidence has suggested that the ER is also compartmentalized into separate mother and bud domains in budding yeast (Luedeke et al., 2005). In yeast, Ives Barral discovered that when the bud ER is repeatedly photobleached there is little fluorescence loss in the mother ER, and vice versa (Luedeke et al., 2005). Intact septin rings are required to prevent mixing of integral ER proteins between bud and mother (Luedeke et al., 2005), although the molecular mechanism is unclear. The master regulator of polarity establishment in yeast, the polarisome complex has also been found to play a role. Nevertheless, how the polarisome interacts with septins, and how septins interact with the ER is not well understood. 1.2.4 Septins Septins are highly conserved large GTPases that assemble into 10 nm filaments, and form continuous PM-associated cytoskeletal structures that physically restrict lateral diffusion of PM proteins. In the G1 phase of the cell cycle, septins form a ring-like structure at the incipient bud site through which the bud emerges. As the bud grows in S and G2, the septin ring expands to form a ?collar? at the neck, which compartmentalizes the bud PM (Figure 1.4) (Barral et al., 2000). Near the end of M phase the collar splits into two rings located on either side of the plane of division which localize cortical factors required for completion of cytokinesis (Dobbelaere and Barral, 2004). Continuous septin structures are found in close contact with the PM, usually within a distance of 10 nm (Bertin et al., 2012). In yeast, septins are essential for cytokinesis and are encoded by CDC11, CDC12, CDC3, CDC10 (Figure 1.4). Together, they assemble 9in hetero-oligomers that polymerize to form filaments (McMurray et al., 2011). A non-essential septin, SHS1, was later identified (Mino et al., 1998). Shs1 can substitute for Cdc11 in septin oligomers (Figure 1.4), and different phosphomimetic mutants of Shs1 form a gauze-like meshwork in vitro, suggesting that septins may form additional complex higher-order structures than straight filaments in vivo, and this process may be regulated by phosphorylation (Garcia et al., 2011). It is important to note that ER polarization has been found to be required for normal septin organization (Loewen et al., 2007), suggesting that the two processes may utilize similar molecular components, but the mechanism is still undefined.Figure 1.4. Molecular and structural organization of septins(A) Recombinant septin subunits organize to form octameric rods in vitro, with Cdc11 at the ends. Shown is a representative class average of EM images. (B) Shs1 can substitute for Cdc11 at the ends of octamers. (C) Septin rods join end-to-end and laterally to form a filamentous ring in vitro. (D) A 50-nm section of the yeast bud neck showing septin filaments (red arrow). (A-C) are adopted from (Garcia et al., 2011) and (D) from (Bertin et al., 2012).10BA C DFigure Localized protein synthesisER polarization could have a deeper function such as localized protein synthesis. At least 24 mRNAs are specifically translated in the bud (Shepard et al., 2003). One such example is the integral ER protein Ist2. When fused to GFP, Ist2 is present in the mother ER but is curiously absent from S phase buds; it appears in G2/M phase buds later in the cell cycle (Takizawa et al., 2000). Moreover, Ist2 expression in the bud ER depends on its mRNA being trafficking to the bud via polarized actin cables and myosin motors (J?schke et al., 2004), and its restriction there depends on the septin barrier (Takizawa et al., 2000). Adittionally, two targeted mRNAs encode lipid metabolic enzymes of the ER (Shepard et al., 2003). ERG2 encodes a C-8 sterol isomerase of the ER that catalyzes the isomerization of the delta-8 double bond to the delta-7 position in ergosterol biosynthesis (Ashman et al., 1991). LCB1 encodes a component of the serine palmitoyltransferase that is responsible for the first step in sphingolipid synthesis (Buede et al., 1991). Polarized targeting of ERG2 and LCB1 mRNAs further supports the concept that polarized synthesis of integral ER proteins may support localized lipid synthesis in the bud.1.2.6 Migration of the nucleusNuclear migration is the process of first positioning the nucleus in the correct axis towards the bud, followed by the separation of the nucleus into the bud (Figure 1.5A). In yeast, nuclear migration appears to also be a polarized process, and the ER appears intimately involved. The two processes also share similar polarization machinery such as the polarisome and actin (Figure 1.5B). Since budding yeast undergoes closed mitosis in which the nuclear envelope never breaks down (De Souza and Osmani, 112007), the NE must interact with polarized cytoskeletal machinery to deliver the nucleus into the bud. In fact, the spindle pole body, which is the yeast equivalent of the centrosome or microtubule organizing centre, is embedded in the nuclear envelope and interacts with both intranuclear and cytoplasmic (astral) microtubules (Siller and Doe, 2009). In G2/ M phase, tubular ER emanates from the mother nuclear envelope and extends into the bud (Fehrenbacher et al., 2002), setting the stage for nuclear migration. Recently, exocyst mutants have been shown to have nuclear migration defects (Kirchenbauer and Liakopoulos, 2013), although the mechanism is unclear.  Therefore, nuclear migration may be closely linked to ER inheritance and controlled by similar polarization components.Nuclear migration in yeast can be divided into two stages: (1) An early stage (pre-anaphase) that orients the old spindle pole body towards the bud and positions the nucleus at the neck; and (2) A late stage (anaphase) that reels in the nucleus through the neck into the bud (Figure 1.5)  (Pruyne et al., 2004). These two stages appear genetically overlapping, since defects in either process result in a low frequency of binucleate mothers (Revardel and Aigle, 1993), and double mutants between components of the two pathways are synthetic lethal (Costanzo et al., 2010), indicating functional redundancies between pathways. We have discovered similar genetic interactions between components in the ER polarization process and those in nuclear migration, suggesting a role for ER polarization in nuclear migration. This will be discussed further in Chapter 2.1213Bbudded cell, and contributes to the forces required to pullthe nucleus and chromatin DNA through the aperturebetween mother cell and bud [15]. Dynein is also requiredfor the prominent microtubule sliding that is seen at thisstage of the cell cycle [16]. Dynein is symmetricallydistributed along the length of both mother and daughtercell microtubules, and is unlikely to provide directionalcues itself. The challenge in the field has been tounderstand how dynein generates any motive force, and inparticular how it is responsible for microtubule slidingalong the cortex.There are numerous protein candidates that anchordynein to cortical (or other) sites. The most notableinclude the intermediate and light chains of the dyneincomplex itself, and components of the dynein-associateddynactin complex ? in particular, Nip100 (p150), Jnm1and Act5 (Arp1). Unfortunately for these models, thedynein heavy chain, Dhc1/Dyn1, localizes to the cytoplas-mic microtubules, and despite the effort of several labora-tories, there is no evidence for the localization of dynein tothe cortex in yeast. Similarly, several dynactin componentshave been localized to the spindle pole body [17,18]. Dyn-actin at the pole may mediate interactions betweendynein and components at the neck, but this does nothelp us understand dynein?s role in microtubule slidingalong the cortex.Cortical anchors for dyneinNum1, like many of the proteins involved in nuclearmigration, contributes to the efficiency of nuclear migra-tion but is not required for cell viability. It has takencareful inspection in live cells, protein localization usinggreen fluorescent protein (GFP) fusions and studies ofprotein?protein interactions to reveal the role of Num1 innuclear migration. The Num1 protein is initially localizedin the cortex of the mother cell [19]. But using Num1?GFPfusions, two groups [5,6] have recently observed Num1accumulation in the bud of large budded cells. Num1?GFPfirst appears in medium-sized buds as stationary corticalspots [5]. The sessile nature of Num1, as well as its pre-dominance in the mother cortex, distinguishes it fromother proteins implicated in nuclear migration and cellpolarity, including Bud6, Bni1 and Kar9.Num1 is thus well positioned to be a cortical anchor forcytoplasmic dynein. Direct evidence supporting this ideahas come from analysis of microtubule sliding [16].Microtubule sliding along the cortex, as visualized withtubulin?GFP, is abrogated in the absence of functionalNum1 [5]. Genetic interactions confirm that num1 mutantsbehave as if they are missing dynein function [6]. In partic-ular, num1 dynein double mutants behave like either singlemutant, and conversely the double mutants num1 kar9,num1 bni1 or num1 kip3 behave, respectively, like dyneinkar9, dynein bni1 or dynein kip3 [6,12,15,20]. These resultsplace Num1 on the dynein pathway of nuclear migration.What then is the specific evidence that Num1 provides acortical anchor for dynein? Direct physical interactionsbetween Num1 and components of the dynein complexwere examined by co-immunoprecipitation experiments[6]. Num1 was found to co-immunoprecipitate with thedynein intermediate chain Pac11, and with the alphatubulin Tub3. Furthermore, Num1 co-immunoprecipi-tates with Bni1 and Kar9. The interactions with Pac11 andDispatch R327Figure 1Kar9-assistedmicrotubulesearch and captureNuclear migrationto the neckDynein, anchored tocortical sites by Num1,powers the spindlethrough the neckSpindle elongationto distal end ofmother and budded cellCytokinesis and returnto microtubulesearch and captureCurrent Biology   MG1 AO LA G1Num1 anchors cytoplasmic dynein and contributes to spindleelongation in anaphase. The nucleus (blue sphere) is propelled byastral microtubules (green lines) pushing against the cell periphery(G1). Cytoplasmic dynein fused to GFP (not shown) decorates theastral microtubule lattice. Migration to the neck of budded cells isfacilitated by Kar9 (gray spheres in G1 and M phase cells), whichserves as a linker between actin and microtubule cytoskeletons (seetext). Num1 (red sphere) is present in the cortex of unbudded cells,and appears in the bud of medium to large budded cells (M phaseand anaphase onset, AO). Num1 is a cortical protein that bindstubulin and dynein (AO and late anaphase, LA). If dynein isimmobilized by Num1, the minus-end directed translocation ofmicrotubules by dynein would result in movement of the spindle poleand nucleus to cortical sites (late anaphase). Upon spindledisassembly, astral microtubule growth propels the nucleus for thenext cycle.AFigure 1.5  Figure 1.5. The nuclear migration pathway in yeast(A) A general overview of the nuclear migration process in yeast. AO, anaphase onset. LA, late anaphase. Adapted from (Bloom, 2001). (B) The molecular components involved in nuclear migration. Top, early stage: (1) After the SPBs duplicate, the old SPB is destined for the bud. (2) Kip2 promotes the transport of Kar9p from the SPB to the MT plus end. (3) Kar9 binds Myo2 (myosin motor). Myo2 drags the plus end of the microtubule to the bud tip by walking along actin cables to their sites of anchorage at the polarisome. (4) the nucleus is pulled to the bud neck. At this stage, the cortical anchor for the spindles, Num1, is present only in the mother cell. Bottom, late stage: (5) Dynein and dynactin are at the plus end of cytoplasmic microtubules, and probes for and binds to Num1, which is now present in the bud. (6) Num1 anchors the spindles in the cell cortex and minus-end-directed motor activity by dynein pulls the nucleus across the bud neck. Taken from (Pruyne et al., 2004). 1.3 ER junctions1.3.1 Structural characteristics of ERJs:ER makes direct physical contacts with most of the organelles within the cell, including plasma membrane (PM), mitochondria, Golgi and nucleus (Levine and Loewen, 2006); and are collectively called ER junctions (ERJs). For instance, PM-ER Junctions (PM-ERJs) are regions where the PM closely associates with pmaER, and mitochondria-ERJs (mito-ERJs) are where the outer mitochondrial membrane associates with the ER. ERJs have been visualized by electron microscopy between ER 14and Golgi, mitochondria and PM among others (Figure 1.6) (Achleitner et al., 1999; Ladinsky et al., 1999; Pichler et al., 2001) and involve close apposition of membranes (<10 nm), but in most cases there is no membrane fusion. Mito-ERJs and PM-ERJs have been isolated biochemically and are enriched for lipid synthesising enzymes (Pichler et al., 2001; Vance, 1990). Interaction of the sarcoplasmic reticulum with PM is extensive in muscle cells and creates a structure important for excitation contraction coupling, known as the triad junction (Levine, 2004). In budding yeast, the most extensive ERJ is with the PM with over 1100 contacts per cell, almost 15 times as many as between ER and mitochondria, the second most extensive ERJ in this organism (Pichler et al., 2001). Therefore, yeast is an ideal organism to study ERJs.15Concluding remarks: MCSs make the ER a hub fortrafficking of small moleculesThe first identified MCS components tend to confirm thatMCSs are involved in lipid trafficking between organelles.Targeting of a lipid-transfer protein to an MCS can besimply achieved by the presence of targeting determinantsfor the two different membranes. An interesting conse-quence of targeting both membranes simultaneously(Figure 4c) is that the lipid-transfer protein reversiblybecomes a structural MCS component, that is, it canenhance the connection between the two organelles, andso might modulate other MCS functions.Included within each MCS is a region of the ER, aremarkably heterogeneous organelle [59,60]. MCSs con-sist of these portions of ER, a narrow cytoplasmic gap anda part of the apposed organelle. To reflect the centralimportance of the ER, I suggest the term ?ER junction?(ERJ) as a different way to refer to MCSs. This namingsystem provides a framework for referring to those MCSsthat are currently largely not described (i.e. all exceptMAM), and can be used to classify MCSs (Figure 5). Thecommon presence of the ER as one partner in all thesejunctions has the important functional implication thatmolecules can diffuse rapidly between any of theseorganelles by crossing two MCSs. The lumen andmembrane of the ER would act as a conduit for thediffusion of ions and lipids, respectively, linking togetherall the other organelles.To date, cell biologists working in the separate fields ofthe intracellular trafficking of Ca2C or the intracellulartrafficking of lipids have tended to work in isolation fromeach other. Although there might turn out to be nomolecular connection between Ca2C trafficking and lipidtrafficking, the hypothesis that there is a shared substrateencourages future collaboration on one central unresolvedquestion: what are the structural components of MCSs? Aseparate question that needs to be addressed is whether,by spanning across MCSs, lipid-transfer proteins andother peripheral membra e proteins might act to modu-late MCS function.AcknowledgementsI thank Chris Loewen, Steve Moss and Sean Munro for helpful commentsregarding the manuscript.References1 Wirtz, K.W. (1991) Phospholipid transfer proteins. Annu. Rev.Biochem. 60, 73?992 Wirtz, K.W. (1997) Phospholipid transfer proteins revisited. Biochem.J. 324, 353?3603 Schouten, A. et al. (2002) Structure of apo-phosphatidylinositoltransfer protein alpha provides insight into membrane association.EMBO J. 21, 2117?21214 Sha, B. et al. (1998) Crystal structure of the Saccharomyces cerevisiaephosphatidylinositol-transfer protein. Nature 391, 506?5105 Tsujishita, Y. and Hurley, J.H. (2000) Structure and lipid transportmechanism of a StAR-related domain. Nat. Struct. Biol. 7, 408?4146 Flucher, B.E. and Franzini-Armstrong, C. (1996) Formation ofjunctions involved in excitation?contraction coupling in skeletal andcardiac muscle. Proc. Natl. Acad. Sci. U. S. A. 93, 8101?81067 Takeshima, H. et al. (2000) Junctophilins: a novel family of junctionalmembrane complex proteins. Mol. Cell 6, 11?228 Putney, J.W., Jr. et al. (2001) Mechanisms of capacitative calciumentry. J. Cell Sci. 114, 2223?22299 Ma, H.T. et al. (2000) Requirement of the inositol trisphosphatereceptor for activation of store-operated Ca2C channels. Science 287,1647?165110 Yuan, J.P. et al. (2003) Homer binds TRPC family channels and isrequired for gating of TRPC1 by IP3 receptors. Cell 114, 777?78911 Rizzuto, R. et al. (1998) Close contacts with the endoplasmic reticulumas determinants of mitochondrial Ca2C responses. Science 280,1763?176612 Csordas, G. et al. (1999) Quasi-synaptic calcium signal transmissionbetween endoplasmic reticulumandmitochondria.EMBOJ. 18, 96?10813 Filippin, L. et al. (2003) Stable interactions between mitochondria andendoplasmic reticulum allow rapid accumulation of calcium in asubpopulation of mitochondria. J. Biol. Chem. 278, 39224?3923414 Xu, C. et al. (2003) A permease-like protein involved in ER to thylakoidlipid transfer in Arabidopsis. EMBO J. 22, 2370?237915 Milla, P. et al. (2002) Yeast oxidosqualene cyclase (Erg7p) is a majorcomponent of lipid particles. J. Biol. Chem. 277, 2406?241216 Voelker, D.R. (1989) Reconstitution of phosphatidylserine import intorat liver mitochondria. J. Biol. Chem. 264, 8019?802517 Simbeni, R. et al. (1990) Intramitochondrial transfer of phospholipidsin the yeast, Saccharomyces cerevisiae. J. Biol. Chem. 265, 281?28518 Vance, J.E. (1990) Phospholipid synthesis in a membrane fractionassociated with mitochondria. J. Biol. Chem. 265, 7248?725619 Gaigg, B. et al. (1995) Characterization of a microsomal subfractionassociated with mitochondria of the yeast, Saccharomyces cerevisiae.Involvement in synthesis and import of phospholipids into mito-chondria. Biochim. Biophys. Acta 1234, 214?22020 Achleitner, G. et al. (1999) Association between the endoplasmicreticulum and mitochondria of yeast facilitates interorganelle trans-port of phospholipids through membrane contact. Eur. J. Biochem.264, 545?55321 Vance, J.E. (2003) Molecular and cell biology of phosphatidylserineand phosphatidylethanolamine metabolism. Prog. Nucleic Acid Res.Mol. Biol. 75, 69?11122 Yaffe, M.P. and Kennedy, E.P. (1983) Intracellular phospholipidmovement and the role of phospholipid transfer proteins in animalcells. Biochemistry 22, 1497?1507PtdEtnTRENDS in Cell Biology MAM or mitochondrial ERJGolgi ERJEndosomal ERJPlasmamembraneERJLipid-droplet ERJPtdEtnLysosomalERJ  NucleusFigure 5. A transport network based on the endoplasmic reticulum (ER). Differentmembrane contact sites (MCSs) (green lines) can be categorized on the basis of theorganelle that partners the ER, with each one forming a different type of ER junction(ERJ). Curved arrows indicate non-vesicular trafficking of small molecules at ERJs.The universal involvement of the ER enables non-vesicular trafficking of moleculesbetween two compartments that form ERJs. For example, phosphatidylethanol-amine (PtdEtn) that is synthesized in the inner mitochondrial membrane efficientlyaccesses the plasma membrane by a non-vesicular route (broken arrows) thatinvolves both mitochondrial ERJs [mitochondrial-associated ER membrane(MAM)] and plasma membrane ERJs [plasma-membrane-associated membrane(PAM)].Opinion TRENDS in Cell Biology Vol.14 No.9 September 2004 489www.sciencedirect.comPEPEpmaERFigure 1.6Figure 1.6. ER junctions (A)  An overview of a variety of ERJs that have been observed in eukaryotic cells. Notice that phosphatidylethanolamine (PE) trafficking is hypothesized to occur at both PM-ERJs and mito-ERJs based on the localizations of the up- and down-stream enzymes (see also Figure 1.7). Adapted from (Levine, 2004). (B) Serial ultra-thin sections of EM images showing a strand of pmaER contacting the PM multiple times and contacting mitochondria once. The authors refer to pmaER as PAM for PM associated membrane. CW, cell wall. Taken from (Pichler et al., 2001).1.3.2 Molecular components of ERJs:Work in yeast has robustly identified molecular components for several ERJs. Formation of ERJs generally involves interactions between integral membrane proteins on the ER and those on the target organelle, although there are exceptions. For example, the formation of nuclear envelope-vacuole junctions (NVJs) in yeast involves heterotypic interactions between Nvj1, a outer nuclear envelope protein, and Vac8, a vacuolar membrane protein (Pan et al., 2000). When NVJ1 is deleted, cells appear to have lost all NVJs by electron microscopy (Pan et al., 2000). For mito-ERJs, a bridging complex called ERMES for ER-mitochondria encounter structure has been discovered. ERMES contains an ER component, Mmm1; mitochondrial membrane proteins Mdm10 and Mdm34; and a soluble protein, Mdm12, that bridges the two membranes (Kornmann et al., 2009). The exceptional case is the recent identification of the PM-ERJ tether. The PM-ER tethering proteins include the VAP homolog Scs2, phosphatidylinositol (PI) phosphatase Sac1, and pmaER-localized proteins Ist2, Tcb1, 16Tcb2 and Tcb3, all of which have transmembrane domains embedded in the ER (Manford et al., 2012). How could they participate in PM-ERJ formation if there are no PM proteins involved? Interestingly, Ist2 contains a polybasic tail region and Tcb proteins have repeats of C2 domains, and both of these domains interact with acidic lipids in the PM (Maass et al., 2009; Schulz and Creutz, 2004). As a result, Ist2 and Tcb proteins localize specifically to pmaER instead of general ER, thereby bridging the junctional space (Toulmay and Prinz, 2012).In mammalian cells, the most well-characterized bridging complex so far is for PM-ERJs, where STIM1 (stromal-interacting molecule) in the ER interacts with the PM Ca2+ channel Orai (Carrasco and Meyer, 2011). Their functions will be discussed in detail below.1.3.3 Functions of ERJs1.3.3.1 Calcium signalingSeveral physiological functions have been discovered for ERJs, such as Ca2+ signaling , phosphoinositide metabolism, phospholipid synthesis, and non-vesicular lipid trafficking. In mammalian cells, the best documented role for PM-ERJs is in Ca2+ signaling, including Store-Operated Calcium Entry (SOCE) and Ca2+ release in muscle contraction. In response to a decrease in intracellular Ca2+ levels, STIM proteins, which are normally evenly distributed in the ER, polarize to PM-ERJs and activate the Orai Ca2+ channels on the PM, triggering Ca2+ entry into the cell across the junctional space (Carrasco and Meyer, 2011). The STIM-Orai interaction has recently been implicated in cancer cell migration (Dingsdale et al., 2013). 17In striated muscle cells, PM-ERJs form elaborate structures called triad (skeletal muscle) and dyad (cardiac muscle) junctions, and specialize in excitation-contraction coupling whereby electrical stimulation in the PM induces Ca2+ release from the ER (Fill and Copello, 2002). In this signaling cascade the information flows in the opposite direction to SOCE. Excitation-contraction coupling begins with the rapid arrival of an action potential, triggering the plasma membrane voltage-gated Ca2+ channel DHPR (dihydropyridine receptor) to interact with the RyR (ryanodine receptor) in the sarcoplasmic reticulum, which then releases its Ca2+ store, causing muscle contraction (Fill and Copello, 2002). ER-associated degradation (ERAD)GP78, also known as Autocrine Motility Factor Receptor (AMFR), is an ER ubiquitin ligase involved in ERAD. EM and confocal microscopy studies using the 3F3A antibody reveal that Gp78 is enriched in the smooth ER that makes extensive contacts with mitochondria, suggesting a specialized ERAD domain at mito-ERJs (Fairbank et al., 2009; Goetz et al., 2007; Wang et al., 2000). Consistently, Gp78-mediated ubiquitylation is found to occur in the peripheral ER (St-Pierre et al., 2012). Overexpression of Gp78 saturates co-localization with mitochondria, indicating that its localization to mito-ERJs is specific (Goetz et al., 2007). Also, cytosolic Ca2+ concentration plays a role in modulating the interaction between Gp78-bound smooth ER and mitochondria (Goetz et al., 2007; Wang et al., 2000). Together, these data suggest a role for mito-ERJs in ER quality control. Autophagy Autophagy is a ?self-eating? process in which the cell degrades its own organelles in order to recycle its own nutrients during starvation conditions. Where the autophagosomes originate from has been an important question. EM tomography has shown that autophagosomes are in close opposition with the ER (Hayashi-Nishino et al., 2009), and proteomics studies have identified ER proteins in autophagosome membranes (Axe et al., 2008). Additionally, in starvation-indued autophagy, the outer mitochondrial membrane is shown to participate in forming autophagosomes, and that disrupting mito-ERJs by depleting mitofusin2 interferes with autophagosome biogenesis (Hailey et al., 2010). Recently, autophagosomal marker ATG14 has been shown to localize to mito-ERJs in starvation conditions by microscopy and subcellular fractionation experiments. More importantly, ATG14 is recruited to mito-ERJs by the ER protein syntaxin 17 (Hamasaki et al., 2013). Taken together, these lines of evidence clearly demonstrates a role for mito-ERJs in autophagy. Lipid metabolismIn yeast, ERJs are shown to be involved in lipid synthesis. Our laboratory has recently demonstrated a role for PM-ERJs in regulating the synthesis of phosphatidylcholine (PC). A conserved enzyme for making PC, called phosphatidylethanolamine (PE) methyl-transferase (PEMT) or Opi3 in yeast resides in the ER; however, we found that in vitro it could methylate in trans, suggesting that in vivo Opi3 may act on plasma membrane PE across PM-ERJs (Tavassoli et al., 2013). We identified a double mutant strain with severe defects in PM-ERJs that also could not make PC, and Opi3 is mislocalized (for more details, see Chapter 3). In addition, PM-19ERJs are implicated in  PI4P metabolism. The oxysterol binding protein (OSBP) homolog Osh3 localizes to PM-ERJs and stimulate the PI phosphatase enzyme Sac1 in the ER to metabolize PI4P in the PM to PI (Stefan et al., 2011). Consistently, when all six components of the aformentioned PM-ER tether (IST2, SCS2, SCS22, SAC1, TCB1, TCB3 AND TCB3) are deleted, cells lose PM-ERJs, are unable to turnover PI4P and exhibit elevated levels of ER stress (Manford et al., 2012). Moreover, mutants of the ERMES complex that constitute mito-ERJs are found to have decreased levels of cardiolipins, but the mechanism is largely unknown (Kornmann et al., 2009). All in all, ERJs are involved in multiple aspects of lipid metabolism. The role for ERJs in non-vesicular lipid traffic will be discussed in Section Phospholipid synthesis Phospholipids are essential components of all eukaryotic cells that function in the compartmentalization of biochemical reactions. Constituting eukaryotic membranes are three major amino glycerolphospholipids: PS, PE and PC. PC is the most abundant phospholipid in eukaryotic membranes. In mammals, PC is also the major phospholipid in bile, plasma lipoproteins and lung surfactant (Jobe et al., 1989; Vance, 2008). In the brain, PC is the precursor for choline in the biosynthesis of the neurotransmitter acetylcholine (Li and Vance, 2008).The synthesis and metabolism of phosphatidylcholine (PC), the major bilayer-forming lipid, is highly regulated in all eukaryotic cells.20Cho2Opi3Figure 1.7. Pathways for phospholipid synthesis in eukaryotic cells.The Kennedy pathway, or the salvage pathway, can synthesize PE or PC from exogenous ethanolamine or choline. The methylation pathway, on the other hand, synthesizes phospholipids de novo. The Kennedy pathway can complement defects in the methylation pathway and vice versa, allowing for versatile genetic studies. PA, phosphatidic acid. PE, phosphatidylethanolamine. PC, phosphatidylcholine.1.4.1 Pathways for PC synthesis In yeast and humans, phospholipids are synthesized by two partially redundant pathways, which are the methylation and Kennedy pathways (Figure 1.7) (Carman and Henry, 1999). The Kennedy pathway synthesizes PC by using exogenous choline. On the other hand, the methylation pathway synthesizes PC via step-wise methylation of PE by PE methyltransferases (PEMT). In yeast Saccharomyces cerevisiae, two genes, CHO2 and OPI3, which encode PEMTs have been shown to work in concert for PC synthesis. In Saccharomyces cerevisiae and humans, the methylation pathway for de novo synthesis of phospholipids is compartmentalized and involves discrete lipid transport steps between organelles. For the production of PC, PS is made in the ER and is trafficked to mitochondria where it is converted to PE by PS decarboxylase. 21methylation PathwayEthanolamine CholineKennedy PathwayPACDP-DAGPS PE PCDAGMitochondrial PE is trafficked back to the ER where it is converted to PC via three sequential methylations by CHO2 and OPI3. Nevertheless, how lipid molecules traffic from the site of synthesis, the ER, to target membranes, such as the PM, remains unclear. We hope to provide some insights with our recent findings presented in Chapter Non-vesicular lipid trafficEukaryotic cells have organelles enclosed in lipid membranes to spatially segregate enzymatic processes. As a result, the cell must actively facilitate exchange of lipids between organelles due to their hydrophobic nature. Vesicular traffic transports newly synthesized cell surface proteins from the ER through the Golgi to the PM via lipid enclosed vesicles, and these lipids may equilibrate between the organelles connected by vesicular traffic. However, metabolic labeling using radioactive serine in vesicular trafficking mutants has revealed an alternative way of lipid traffic that is independent of vesicular traffic, termed non-vesicular traffic (Daum et al., 1986). Treating mammalian cells with brefeldin A, which impedes vesicular traffic, only slightly slows the movement of newly synthesized cholesterol from ER to PM, further supporting a role for non-vesicular traffic (Daum et al., 1986; Heino et al., 2000; Kaplan and Simoni, 1985). Therefore, non-veiscular traffic has long been speculated to be a method for moving lipids inside a cell.However, the mechanism for non-vesicular traffic has been elusive. It is possible that lipid monomers can move by spontaneous diffusion via the cytosol, but their hydrophobic nature does not allow this to happen in most cases. Nevertheless, recent 22effort has identified a family of lipid transfer proteins (LTPs) that facilitate the non-vesicular traffic of lipids (D'Angelo et al., 2008; Holthuis and Levine, 2005). Strikingly, the structure of the simplest LTP for sterols, Osh4 of the OSBP family in yeast, is comprised of a hydrophobic pocket with a flexible lid (Im et al., 2005). Other LTPs, such as Osh1, contain domains to target two membranes (ER and Golgi) simultaneously (Figure 1.8A) (Levine and Munro, 2001). Some LTPs have been localized to ERJs. Osh3 has been localized to PM-ERJs and Osh4 can subsittue for Osh3 functions (Figure 1.8B) (Stefan et al., 2011). In vitro, Osh3 has been shown to facilitate phosphoinositide metabolism on the surface of purified liposomes, suggesting its role as an LTP at PM-ERJs (Stefan et al., 2011). Thus, the ability to localize to regions of close donor and acceptor membrane contacts allows LTPs to transfer lipids most efficiently. This mechanism eliminates the need for LTPs to diffuse across the cytosol (Figure 1.8B).23AB* * Targeting of structural MCS proteinsStructural proteins of an MCS are only known in one case,which is found in budding yeast. The MCS is called thenucleus?vacuole junction (NVJ), and lies between theouter nuclear envelope (part of the ER) and the degrada-tive vacuole (equivalent to a lysosome) [50]. Nothing wasknown of its structure at the molecular level untilGoldfarb and coworkers discovered that the NVJ is formedby a simple protein bridge: Vac8p, an armadillo repeatprotein that binds the vacuolar-limiting membrane, bindsdirectly to the cytoplasmic domain of Nvj1p, a type Iintegral membrane protein of the nuclear envelope [51]. Inthe absence of either Vac8p or Nvj1p, the vacuole andnucleus separate slightly. The NVJ is topologically similarto junctions between the ER and endosomes or lysosomesin higher eukaryotes [52,53] (Figure 1d). Extrapolatingfrom these findings, MCSs might be established by simple,stable protein bridges, to which other components can berecruited (Figure 4b).Cargo selection proteins at MCSsWhy might cargo selection proteins be targeted to MCSs?A thought experiment shows that any protein transferringcargo between two organelles might tend to localize to anMCS between those membranes. This analysis starts withthe idea that, like other peripheral membrane proteins,lipid-transfer proteins are only truly active if they canbind to membranes [54]. In this case, the transfer proteinmust bind to both the correct donor membrane and thecorrect acceptor membrane to enable specific shuttling ofcargo, rather than equilibration among all the membranesof the cell (Figure 4a). However, any cytoplasmic proteinwith determinants for two different membranes would bepredicted to diffuse to those parts of the cell where the twomembranes are closest together, this being at the MCSbecause it is here that the concentration of receptors ishighest (Figure 4b). Once a transfer protein is at the MCS,a mechanism that would powerfully retain it there wouldbe the binding of both membrane receptors simul-taneously (Figure 4c). Such dual binding is likely toincrease the overall avidity for membranes many-fold,similar to that of bivalent antibody compared with amonovalent Fab fragment.This model suggests that cargo shuttling proteins canact at a site where, instead of diffusing across largecytoplasmic gaps, they do not have to shuttle any distanceat all, enhancing activity greatly. However, what evidenceis there that lipid-binding proteins can target MCSs inthis fashion? One large family of lipid-transfer proteinsthat are similar to CERT in structure and that have beenlocalized clearly to MCSs is the family of oxysterol-bindingprotein (OSBP) homologues. OSBP has the same con-stellation of domains as CERT, with the same twotargeting domains for both the ER and the Golgi, anda C-terminal domain that binds lipid [32,33,55]. Many ofthe homologues of OSBP, including three OSBP homol-ogues in yeast (called Osh proteins), target the ER andan organelle other than the Golgi: Osh1p to the NVJ,and Osh2p and Osh3p to the plasma membrane [56].Notably, these three yeast proteins also have a particu-lar affinity for MCSs: Osh1p to the NVJ, and Osh2p andOsh3p to ER?plasma membrane MCSs [33]. Using thecombination of two membrane-targeting domains, theseOSBP homologues might not only be close to twodifferent membranes (Figure 4b), but might actuallybind both simultaneously (Figure 4c). Another class oflipid-transfer proteins that could lend support to thismodel are those that have transmembrane domains[57,58]. Permanent anchoring of a lipid-transfer protein toone membrane, which prevents any transfer function thatrelies on diffusion across cytoplasmic gaps, would enhancerather than reduce activity according to the modelpresented here.Thus, the recent data on membrane targeting bylipid-binding proteins indicate that these cytoplasmicproteins might attain maximal activity only whenrecruited to MCSs. This model can be tested in severalways. First, what is the precise localization of proteinssuch as CERT and OSBP? Are they found over the transGolgi as a whole, or are they restricted to the ER?GolgiMCS? Second, what is the lipid-transfer activity ofproteins that are anchored permanently to membranesby transmembrane domains?TRENDS in Cell Biology (a)(b)1 2(c)FFFFFigure 4. Models of lipid-transfer proteins functioning at a membrane contact site(MCS). (a) Dual membrane targeting is required for efficient lipid transfer. In theexpanded views of an MCS, a lipid-transfer protein (black) is shown shuttling fromone side of anMCS to the other, indicated by the positions 1 and 2. The two differenttargeting domains facilitate the exchange of lipid down a concentration gradientbetween two specific compartments. By contrast, without specific targeting, a lipid-transfer protein might be expected to distribute its lipid ligand equally to all cellmembranes. (b) Proteins on each organelle bind tightly to each other, as in thenucleus?vacuole junction, to form a bridge that gives an MCS structural integrity.The bridge might enhance recruitment of other MCS components because theproximity of the two membranes increases the local concentration of membranereceptors. (c) Lipid-transfer proteins might span an MCS. A lipid-transfer proteinmight bind to both of its membrane receptors simultaneously, producing a hugegain in avidity and activity. Compartments and molecules embedded in them arecoloured: acceptor, red; donor, blue; lipid-transfer domains, black. Curved arrowsand their thickness indicate equilibration of lipid and the efficiency of transfer,respectively.Opinion TRENDS in Cell Biology Vol.14 No.9 September 2004488www.sciencedirect.comFigure 1.8. Models of lipid transfer proteins facilitating non-vesicular lipid traffic at ERJs.(A) Certain LTPs, su h as Osh1, contain motifs for targeting dual membr nes. Osh1 may shuttl  between two membranes to transport lipids. However, such shuttling may be inefficient. (B) Altern tively, a bridging complex (denoted by *) may bring t  two m mbranes close together forming an ERJ, allowing the LTP to target both membranes 24simultaneously and efficiently transfer lipids. This is the more favoured hypothesis. Diagram adapted from (Levine, 2004).252 Chapter 2: Polarization of the Endoplasmic Reticulum is Mediated by an ER-Septin Tethering Complex2.1 Introduction Establishment of cell polarity is a fundamental aspect of biology that enables cells to spatially segregate their functions and to divide. Cell polarization is most often achieved through cytoskeleton-based directional transport of cargo to polarized domains and through the establishment of molecular diffusion barriers that compartmentalize such domains.The budding yeast Saccharomyces cerevisiae is an excellent model for studying eukaryotic cell polarity because it grows vegetatively by asymmetric budding. Once a bud site is selected, Cdc42 recruits the polarisome, comprised of Pea2, Spa2, Bud6 and a formin, Bni1, which nucleates actin cables in the bud, and myosin-dependent transport moves mRNA, mitochondria, vacuoles, ER, trans Golgi, and astral microtubules to the growing bud (Pruyne et al., 2004). During budding, Cdc42 also initiates the formation of a cortical diffusion barrier at the neck comprised of septins, that compartmentalizes the bud from the mother (Faty et al., 2002; Versele and Thorner, 2005). Compartmentalization by septins is a common mechanism for creating polarized PM domains in many specialized cell types, including neurons, epithelial cells and spermatozoa (Saarikangas and Barral, 2011).  Like the PM, the ER is one continuous membrane system that is compartmentalized into separate domains. Diffusion of integral ER proteins is restricted between the bud and mother ER, whereas diffusion of luminal ER proteins is not 26(Luedeke et al., 2005; Shcheprova et al., 2008). These data suggest that a bidirectional diffusion barrier exists on the cytoplasmic side of the ER membrane to restrict the movement of integral ER proteins. In yeast, the ER diffusion barrier is dependent on the septin Shs1 and on two components of the polarisome, Bud6 and Pea2 (Luedeke et al., 2005). In neurons, ER in dendritic spines and at dendritic branch points is compartmentalized (Cui-Wang et al., 2012), and these structures are also dependent on septins (Tada et al., 2007), suggesting that a similar diffusion barrier is present in the ER in metazoans. Septins are involved in compartmentalizing the two ER domains (Luedeke et al., 2005), suggesting that septins might restrict diffusion of ER proteins through these tubules in the neck and create the ER diffusion barrier. It has been proposed that direct contact between the ER and septins creates the ER diffusion barrier (Luedeke et al., 2005); however, its molecular nature is not known. We previously discovered a role for the highly conserved tail-anchored ER protein Scs2 in the capture of ER tubules at the bud cortex and formation of PM-associated ER (Loewen et al., 2007). Scs2 is enriched within the ER at sites of polarized growth suggesting a role for these sites in ER polarization. Here, we have identified proteins that interacted with Scs2 at sites of polarized growth. We uncovered a novel subunit of the polarisome, called Epo1, which captures ER tubules at the bud cortex. At the bud neck, Epo1 and Scs2 interact with the septin Shs1. Together, they tether the ER to septins and create the ER diffusion barrier. The ER-septin tether restricts diffusion of integral and peripheral proteins from mother to bud and is required for proper spindle positioning during S phase.272.2 Methods2.2.1 Yeast cell culture and manipulationsAll yeast cultures were grown at 30?C with shaking in synthetic defined (SD) media with the appropriate dropouts and 2% dextrose unless otherwise stated. All yeast cells expressing GFP fusion proteins were tagged endogenously in haploids unless otherwise indicated. C-terminally tagged GFP strains were constructed by standard methods involving single-step gene replacement using the pKT128 (SpHIS5) plasmid (Sheff and Thorn, 2004) in the Y7043 background (Tong and Boone, 2006). N-terminally tagged GFP strains were made by the same method using pUPG (pURA3MX6 with constitutive PHO5 promoter upstream of GFP; gift of T. Levine). The Pea2-GFP strain was purchased from Invitrogen. Deletion strains were obtained from freezer stocks of the haploid yeast deletion collection (BY4741, Mat a, KanMX, a gift from C. Boone) unless otherwise stated. Temperature sensitive strains (BY4741, Mat a, KanMX) were a kind gift of C. Boone. Other gene deletions were constructed using the PCR method with the heterologous markers S. pombe HIS5 (pKT128), K. lactis URA3 (pKT209) or NatR (p4339). Double deletion strains were derived from the meiotic products of heterozygous diploids with at least three spores of each genotype being compared. The SILAC control strain was constructed by crossing ?arg4-KanR (BY4741, ura3 his3 leu2 met15) to BY4742 (ura3 his3 leu2 lys2) and isolating ?arg4?lys2 haploids by tetrad dissection. TAP-tagged strains were constructed as published using pBS1479 (Puig et al., 2001) and protein expression was verified by Western blot analysis. Spot assays were performed to determine the growth requirements of single and double deletion strains. Mid-log phase yeast cultures (OD600=0.5) were diluted in 10-fold 28serial dilutions and spotted onto plates using a metal pin-frogger (Sigma). For growth assays with the ?epo1 mutant, yeast were grown on YP (yeast extract, peptone) plus 2% galactose. To synchronize cells in G1 alpha factor (10?g/ml) was added to log phase cultures grown in YP plus 2% dextrose liquid medium for 2 hours. Similarly, to arrest cells in S phase, 0.3M hydroxyurea was added to liquid cultures for a minimum of 4 hours. To stain nuclear DNA, log phase yeast cells were fixed in 70% ethanol at room temperature for 20 minutes and washed with distilled water twice. The cell pellet was re-suspended in 100 ?l of 50 ng/ml DAPI solution (diluted in PBS from a 50 ?g/ml DAPI stock, Sigma) at room temperature for 10 minutes. Cells were washed with distilled water to remove excess DAPI and were imaged by UV laser.2.2.2 SILAC proteomics for Scs2?TMSILAC labeling and IgG purification were done as previously described (Chao et al., 2009; Foster et al., 2003). In brief, both the control strain and the Scs2?TM-TAP bait strain were grown to log phase and harvested by centrifugation. The control strain was grown in medium containing L-lysine-4,4,5,5-D4 and L-arginine-13C6 (Cambridge Isotopes). The two cultures were matched by wet pellet weight, resuspended in lysis buffer (10 mM sodium phosphate buffer (pH 7.2), 150 mM NaCl,1% NP-40, 50 mM NaF, 0.1 mM Na3VO4, 1mM DTT) with 1/2,000 of Protease Inhibitor Cocktail (Sigma)), mixed and lysed by grinding in liquid N2. Cell lysate was then cleared of cell debris by centrifugation and incubated with IgG beads (GE Healthcare) and eluted using TEV protease (Invitrogen). Eluted proteins were precipitated using the ethanol/acetic acid method and in-solution digested with trypsin as previously described (Foster et al., 2003) prior to preparation for LC/MS/MS analysis.292.2.3 Proteomics for Epo1 Epo1 and its associated proteins were purified using a one-step TAP-tag basedapproach (Lambert et al., 2009). Briefly, 1L of each of wild type (no tag) and Epo1-TAP strains were grown to OD600 of 1 in YPD at 30?C, lysed by bead beating in 100 mM HEPES pH 8, 20 mM Mg acetate, 300 mM Na acetate, 10% glycerol, 10 mM EGTA, 0.1 mM EDTA). Following gentle sonication, 1% NP40 was added. Immuno-purification was performed using rabbit-IgG cross-linked to magnetic beads (Invitrogen Dynabeads M-270 Epoxy) and eluted with SDS lysis buffer. After SDS-PAGE, three fractions were collected (MW>50 kDa; MW<50 kDa; and IgG heavy and light chains) from each sample (IP and control) for in-gel trypsin digestion (Shevchenko et al., 2000). Digest buffer contained 50mM ammonium bicarbonate. Gel pieces were dehydrated using neat ethanol and incubated at 56?C for 45 minutes in 10mM DTT followed by 30-minute incubation in 55 mM chloroacetamide at room temperature. Extracted peptides were purified on C-18 stage tips prior to LC/MS/MS (Ishihama et al., 2006; Rappsilber et al., 2003; 2007).2.2.4 LC/MS/MSPurified peptides were analyzed using a linear-trapping quadrupole - Orbitrap mass spectrometer (LTQ-Orbitrap Velos; ThermoFisher Scientific) on-line coupled to an Agilent 1200 Series HPLC using a nanospray ionization source (ThermoFisher Scientific) including a 2-cm-long, 100-?m-inner diameter fused silica trap column, 50-?m-inner diameter fused silica fritted analytical column and a 20-?m-inner diameter fused silica gold coated spray tip (6-?m-diameter opening, pulled on a P-2000 laser puller from Sutter Instruments, coated on Leica EM SCD005 Super Cool Sputtering 30Device).  The trap column is packed with 5 ?m-diameter Aqua C-18 beads (Phenomenex, while the analytical column is packed with 3 ?m-diameter Reprosil-Pur C-18-AQ beads (Dr. Maisch,  The HPLC system included Agilent 1200 series Degaser, Pump, Autosampler and Thermostat. The LTQ-Orbitrap was set to acquire a full-range scan at 60,000 resolution from 350 to 1600 Th in the Orbitrap and to simultaneously fragment the top ten peptide ions in each cycle in the LTQ (minimum intensity 1000 counts) and parent ions were then excluded from MS/MS for the next 30 sec. The Orbitrap was continuously recalibrated using lock-mass function. For analysis of mass spectrometry data centroided fragment peak lists were processed with Proteome Discoverer v. 1.2 (ThermoFisher Scientific) MSQuant after conversion by an in-house script. The Mascot search was done against the SGD protein sequence database ( with the following parameters: peptide mass accuracy 10 ppm; fragment mass accuracy 0.6 Da; trypsin enzyme specificity; fixed modifications ? carbamidomethyl; variable modifications ? methionine oxidation, lysine acetylation, serine/threonine/tyrosine phosphorylation; and appropriate SILAC modifications. Only those peptides with IonScores exceeding the individually calculated 99% confidence limit (as opposed to the average limit for the whole experiment) were considered as accurately identified. For SILAC quantitation, the elution peaks for the heavy and light forms of each identified peptide were autocentered and the MS intensity was quantitated across the entire peak to give a peptide average ratio. Further averaging across all peptides for a given protein yielded the final average ratios described in Tables 3.1 and 3.2. 312.2.5 Protein-fragment complementation assayThe Venus-YFP variant of PCA was used in this study to examine protein-protein interactions in live yeast. Unless otherwise stated, endogenous proteins were tagged in haploid yeast by the PCR method with either VF1 or VF2 in the BY4741 and Y7043 strains, respectively. Correct integration and expression was confirmed by colony PCR and Western blot analysis with anti-myc antibodies (Sigma) for each fusion protein. The VF1 and VF2 strains to be assayed were then crossed, and haploid meiotic progenies with both alleles were recovered by random spore analysis or tetrad dissection. Finally, the PCA was visualized in log phase yeast by confocal microscopy.2.2.6 Image quantificationThe ImageJ software (National Institute of Health) was used for all quantifications. To quantify protein polarization within a cell we generated a surface plot of the confocal image and estimated the peak fluorescent intensities from the plot. Non-specific background fluorescence was subtracted for each confocal image. This analysis was performed for a single confocal image per cell and values were averaged over the total cells measured for each localization (minimum of 25 cells). To enable direct comparisons, wild type and mutant cells were imaged on the same day with identical microscope settings for each experiment. Progression through the cell cycle was arbitrarily classified as follows: G1, cells with no buds; S, bud area less than 1/3 of the mother; G2, bud area greater than 1/3, but less than 2/3 of the mother; and M phase, bud area greater than 2/3 of the mother. Cells were binned according to these parameters to determine cell-cycle dependency of localizations. Bud to mother size ratios were determined by tracing bud and mother cell perimeters on the corresponding 32transmission images and measuring the area. Spindle length was quantified by measuring the distance between two in-focus spindle pole bodies in confocal images of cells expressing GFP-Tub1 for at least 250 cells per strain. Spindle position was determined relative to the bud neck in comparison to the corresponding transmission images. To quantify the bud:mother fluorescence ratios of GFP-Ist2 and Num1-GFP, the brush selection tool was used to measure the average pixel intensities in the cell cortex in the bud and in the mother cells, and the cytoplasmic fluorescence was subtracted. All error bars represent standard error measurements (SEM).2.2.7 Photobleaching experiments In all photobleaching experiments except those involving bleaching the nuclear envelope, cells in S phase with small (less than 1/3 the area of the mother) sized buds were chosen. For nuclear envelope bleaching experiments, M phase cells with ?dumbbell? shaped nuclei that crossed the bud neck were chosen. Fluorescence loss was monitored within the bleached region of interest (ROI). For all diffusion barrier photobleaching experiments, three unbleached ROIs in the peripheral ER were monitored in the mother of each bleached cell. Photobleaching during data acquisition was corrected for by monitoring ER in a neighboring unbleached cell and general nonspecific background was subtracted. Fluorescence intensity was normalized to the prebleached (T0) frame, which was set to 100%. Normalized fluorescence data were analyzed using one phase association nonlinear regression analysis using Prism software (GraphPad), which fitted all data except for barrier experiments conducted on wild type and ?sac1 cells, which did not show significant decreases in fluorescence in 33the unbleached ROIs. Mobile fractions and t1/2 values were calculated as previously described (Reits and Neefjes, 2001).2.2.8 Pearson correlation analysis To quantify the colocalization between Num1-GFP and Tcb3-RFP, we used an image processing package of ImageJ, Fiji. Prior to quantification, the red and green channels were separated and three Z-stack images taken at 0.5 ?m apart for each channel were combined. The brush selection tool was used to select ROIs of individual cells. The ?Coloc 2? function was then used to determine the Pearson Correlation Coefficient between Num1 and Tcb3 within the ROI. Automatic thresholding was used and below-threshold correlation coefficients were verified to be near to zero. At least 150 cells per strain were quantified.2.2.9 SGA analysis and bioinformaticsSGA analysis was performed as previously described (Tong and Boone, 2006). Briefly, a ?scs2::URA3 strain was constructed using standard techniques and crossed to the yeast haploid deletion mutant array (DMA) using a Singer RoToR HDA robot. Following diploid selection, spots were replicated three times and sporulated for 5 days. Haploids were germinated on SD-media lacking histidine, arginine and lysine supplemented with thialysine and canavanine (both at 100 ?g/ml). Control sets of single deletion strains were generated by plating on media containing 5-fluoroorotic acid to counter-select for the ?scs2::URA3 allele and G418 sulfate (200 ?g/ml) to select for the DMA strain; while double mutants were selected for by plating on media lacking uracil and containing G418 sulfate. After a further round of selection on the same media, spots  34were imaged using a flatbed scanner. Balony software ( was used to measure spot sizes, determine cut-off values for genetic interactions and define strains the showed a statistically significant growth defect. Gene ontology analysis was performed using the GO Term Finder at the Saccharomyces Genome Database ( 2.2.10 Recombinant protein purification GST tagged fusion constructs were made by cloning into pGEX-6P-2 (GE Healthcare Life Sciences). MBP tagged constructs were made by cloning into pMAL-C2X (New England Biolabs). The residue identities of the cloned fragments are as follows: CCDEpo1, 803-943; CCDPea2, 249-416; Shs1-CTE, 349-551; Shs1-extended-CTE, 272-551; Num1 FFAT motif, 306-330. GST and MBP tagged fusion constructs were transformed into BL21(DE3) cells. An 800 mL culture was then grown to OD600 ~0.7, induced with 0.25 mM IPTG for 4 hours, then harvested by centrifugation. Pellets were incubated with lysozyme (0.25 mg/mL, 30 min) then sonicated. The lysate was centrifuged (40k x g, 25 min), and the tagged proteins were purified on glutathione (GSH) beads (Sigma) or amylose beads (New England Biolabs). GST proteins were stored immobilized on beads in 50 mM Tris pH 7.5, 150 mM NaCl, 0.05% NP-40, 1 mM AEBSF, and 1 mM DTT. MBP proteins were eluted with 25 mM HEPES pH 7.4, 150 mM KCl, 25 ?M CaCl2, 20 mM maltose, 1 mM AEBSF, and 1 mM DTT. GST (non-fusion construct) was expressed from pGEX-6P-2 directly.352.2.11 Recombinant protein in vitro binding assay Purified GST constructs bound to GSH beads (10 - 30 ?L of slurry) were incubated (overnight, 4 ?C) with purified MBP constructs (50 ?L) in binding buffer (25 mM HEPES pH 7.4, 150 mM KCl, 25 ?M CaCl2, 20 mM maltose, and 0.1% NP-40), in a total reaction volume of 0.375 - 1 mL. The beads were centrifuged, and the supernatant saved (the "flow-through"). Beads were then washed with 3 x 1 mL of binding buffer, resuspended in SDS-sample buffer and eluted by heating at 65 ?C for 15 minutes. Samples were analyzed on a SDS-PAGE gel (8%), and either stained with Coomassie-Blue or transferred to a PVDF membrane and blotted with an anti-MBP antibody (New England Biolabs). Membranes were then stained with Ponceau-S. Flow-throughs were run on a separate gel and stained with Coomassie-Blue, and represent between 0.8-2.0 % of the total reaction volume.2.2.12 Co-Immunoprecipitation Log phase culture (OD600= 0.6-0.8) was harvested, washed once with dH2O and resuspended in NP-40 buffer (10mM sodium phosphate buffer pH 7.2, 150 mM NaCl, 1% NP-40, 1mM DTT) with 1/2000 protease inhibitor cocktail (Sigma) acid-washed glass beads. Cells were lysed by bead bashing for 10 min, at 4?C, as well as all subsequent steps. Cell debris was pelleted by centrifugation at 10,000 x g, and the supernatant was mixed with IgG beads (GE) previously prepared in 1:1 slurry in NP-40 buffer and incubated overnight. The beads were then washed three times in NP-40 buffer with 300 mM NaCl and 0.1% Triton X-100. The proteins were eluted from the beads with SDS sample buffer and heating at 65?C for 10 minutes. Samples were 36analyzed by SDS-PAGE, transferred to a PVDF membrane and blotted with either anti-TAP (Open Biosystems) or anti-GFP (Roche) antibodies.2.3 Results2.3.1 Epo1 localizes Scs2 to sites of polarized growth We previously found that a soluble version of Scs2 lacking the C-terminal transmembrane domain and tagged with GFP (Scs2?TM-GFP) localized to sites of polarized growth (Loewen et al., 2007). Now, to identify proteins responsible for polarizing Scs2, we employed a SILAC (Stable Isotopic Labeling of Amino acids in Cell culture) quantitative proteomics approach (Chao et al., 2009; Ong et al., 2003). We replaced the C-terminal transmembrane domain of endogenous Scs2 with the TAP tag and used this fusion protein as the bait for affinity purification (Figure 2.1A). One protein identified as a binding partner for Scs2?TM (Figure 2.1B and Table 2.1), encoded by the ORF YMR124W, was found to localize to sites of polarized growth in a high throughput GFP-tagging study (Huh et al., 2003). We tagged the endogenous Ymr124w protein at the C-terminus and found it localized to the incipient bud site in G1, the tips of small and medium buds in S and G2, and the septum during cytokinesis (Figure 2.1C). These localizations overlapped with the localizations of Scs2?TM-GFP (Figure 2.1D), which additionally localized to the distal pole of mothers and unbudded cells, and to the nucleus. Thus, the Ymr124w protein was an excellent candidate for polarizing Scs2 and we named it Epo1 for ER Polarization. To characterize the Epo1-Scs2 interaction, we employed the protein-fragment complementation assay (PCA) using the Venus variant of YFP (Michnick et al., 2007). In 37this assay, a bait and prey protein are each tagged with a different half of Venus. If the bait and prey interact, they reconstitutes Venus fluorescence, allowing the precise location of the protein-protein interaction to be visualized by confocal microscopy in living cells. We found that Scs2?TM interacted with Epo1 by PCA at sites of polarized growth that overlapped with the localization of Epo1-GFP (Figure 2.1E). We did not detect an interaction by PCA between Epo1 and another abundant ER protein, Pho88 (Figure 2.1F), indicating that the Epo1-Scs2 PCA was specific. We mapped the Epo1 binding site in Scs2 to a highly conserved region in its MSP domain known to bind the FFAT motif (two phenylalanines in an acidic tract) in multiple protein families with ER-related functions (Figures 2.1 G-J) (Loewen et al., 2003). The Epo1-Scs2 interaction at polarized sites suggested that Epo1 might localize Scs2. We found that targeting of Scs2?TM-GFP to the tips of small and medium buds was dramatically reduced in ?epo1 cells (Figure 2.1K). Quantification of Scs2?TM-GFP showed significantly reduced localization to the G1 incipient bud site and targeting was virtually abolished at the tips of small buds (Figures 2.1L and M). Targeting to large G2 buds was also reduced (Figure 2.1N), but targeting in M phase was not significantly affected (Figure 2.1O). 2.3.2 Epo1 attaches ER to the bud cortex We previously found that loss of Scs2 prevented formation of PM-associated ER (pmaER) at the bud tip (Loewen et al., 2007) and we now tested for a role for Epo1. We localized full length Scs2-GFP, which appeared functional (Figure 2.1O), in wild type and ?epo1 cells and examined ER morphology. Scs2-GFP localized throughout the ER and was enriched at sites of polarized growth (Figure 2.1Q), similar to Scs2?TM-GFP. In 38?epo1 cells we observed loss of Scs2-GFP from the distal cortex of the bud during S and G2 phases, consistent with Epo1 localizing Scs2?TM-GFP to these sites (Figures 2.1R and S). Since this also suggested a defect in pmaER in the mutant, we compared ER in small buds of ?epo1 and ?scs2 cells using an abundant type I ER protein, Pho88, tagged with GFP. Both the ?epo1 and ?scs2 mutants showed a clear lack of pmaER at the distal bud cortex (Figure 2.1T). Thus, the interaction between Epo1 and Scs2 was required for capture of ER tubules at the distal bud cortex.3940NucleusTAPERPM?ERScs2?Scs2? PME 6FV?70;(SR3&$ F (SR;3KR3&$G GFP-MSPScs2 H (SR;063Scs23&$I Scs2T42A?70*)3 J (SR;6FVT42A?703&$ORF GeneName)ROG(QULFKPHQW<(5: 6&6 27<2/& 1%$ <'5: 180 <05: (32 A BC (SR*)3* S MG2 * S MG2D 6FV?70*)3Figure 2.141K 6FV?70*)3LQ?epo1Q 6FV*)3 R 6FV*)3LQ?epo1T 3KR*)3:7 ?epo1 ?scs2**W7?HSRLPixel Intensity,QFLSLHQW%XG6LWH'LVWDO3ROHRI0RWKHUUnbudded Cells (G1)0204060 ***MN OSmall Budded Cells (S) %XGWLS 'LVWDO3ROHRI0RWKHU0204060Large Budded Cells (G2) %XGWLS 'LVWDO3ROHRI0RWKHU%XGWLS 'LVWDO3ROHRI0RWKHU6HSWXP02040600204060100120140M Phase Cells W7?HSRW7?HSRW7?HSRP LQR LQR:7?VFV6FV*)36FV?70*)33L[HO,QWHQVLW\Scs2-GFP at Bud TipS:7 ?HSR200160120400*Figure 2.1 (continued)Figure 2.1 Epo1 Interacts with Scs2 and Captures ER Tubules in the Bud(A) Scs2 interacts with an unknown polarized protein at the bud tip even in the absence of its transmembrane domain.(B) Scs2?TM binding partners identified by SILAC mass spectrometry having polarized localizations. See also Table 2.1.(C) Epo1-GFP localization in cells staged throughout the cell cycle. Arrowheads indicate sites of polarized growth.(D and K) Scs2?TM-GFP localization in wild type (D) and ?epo1 (K). Arrowheads indicate PM-associated localizations, arrows indicate altered localizations in the mutant. All scale bars, 2 ?m.(E) PCA between Scs2?TM and Epo1. Arrowheads indicate sites of interaction.(F) PCA between Epo1 and Pho88 both tagged at their C-termini. (G) Yeast expressing GFP-MSPScs2 from a plasmid. (H) PCA between Epo1 and MSPScs2 expressed from a plasmid.(I) Yeast expressing Scs2T42A?TM-GFP from a plasmid. Arrows indicate the absence of expected interactions.(J) PCA between Epo1 and Scs2T42A?TM expressed from a plasmid.(L-O) Quantification of the localizations of Scs2?TM-GFP observed in Figure 2.1 (D and K). Minimum of 25 cells per localization. Asterisks, p<0.05.(P) Serial dilutions of yeast with different alleles of SCS2 grown in the presence (1 mM) or absence of inositol at 37?C.42(Q and R) Scs2-GFP localization in wild type (E) and ?epo1 (F). Arrowheads indicate regions of the ER enriched for Scs2-GFP, arrows indicate altered localizations in the mutant.(S) Quantification of Scs2-GFP in the ER at the bud tip, as in Figure 2.1 (Q and R). Minimum of 25 cells per localization. Asterisks, p<0.05.(T) Pho88-GFP localization in the indicated strains. Arrows indicate altered localizations in the mutant. All scale bars, 2 ?m. WT, wild type.2.3.3 Epo1 interacts with the polarisome Epo1 is a soluble 944 amino acid protein with a predicted coiled-coil domain (CCD) at its C-terminus (Figures 2.2A and B). A high-throughput yeast 2-hybrid study of yeast CCDs (Newman et al., 2000) identified an interaction between the CCD of Epo1 and the CCD of Pea2 (Figure 2.2A), suggesting that Epo1 might interact with the polarisome. Therefore, we performed a proteomic analysis using Epo1-TAP to identify its binding partners. Epo1 interacted with multiple proteins with roles in cell polarity, including Pea2 (Table 2.2). Next, we determined if the Epo1 interacted directly with Pea2 by reconstituting binding in vitro using purified recombinant CCDs from these proteins. We were able to pull-down MBP-tagged Pea2 CCD using GST-tagged Epo1 CCD, whereas neither protein interacted with control proteins (Figure 2.2C). Using PCA we found that full length Epo1 interacted with Pea2 at sites of polarized growth that corresponded to the localizations of Pea2-GFP (Figures 2.2D and E). The CCDs of Epo1 and Pea2 also interacted in vivo by PCA (Figures 2.2F and G). Finally, deletion of 43the Epo1 CCD prevented its interaction with Pea2 by PCA (Figure 2.2H). Together, these data indicated that Epo1 interacted with Pea2 via their CCDs.  The Pea2-Epo1 interaction suggested that Pea2 might localize Epo1. Consistent with this, deletion of PEA2 resulted in a dramatic loss of Epo1-GFP from sites of polarized growth, although some residual localization remained (Figures 2.2I and J). In contrast, Epo1 was not required to localize Pea2-GFP (Figure 2.2K). Deletion of the Epo1 CCD also prevented its polarization (Figures 2.2L and M), indicating interaction of the Epo1 CCD with Pea2 mediated polarization of Epo1. We now tested if Epo1 contributed to the core function of the polarisome in nucleating actin cables by assessing  ?shmoo? formation. In contrast to ?pea2 cells, which showed an aberrant ?peanut? shaped shmoo (Chenevert et al., 1994), the ?epo1 mutant was indistinguishable from wild type, indicating this function of the polarisome was intact (Figure 2.2N). Lastly, we did not detect an interaction between Pea2 and Scs2 by PCA (Figure 2.2O), further supporting that Epo1 played a specific role in cortical capture of ER tubules within the polarisome.4445Figure 2.2predicted to contain alpha helicesshort CCDEpo1 (803-863)Pea2   421 aaEpo1   943 aa240-323 377-403490-510 800-920CCDPea2 (249-416)predicted to contain coiled-coilsCCDEpo1 (803-943)75503725MBP-CCDPea2GST-CCDEpo1 GSTGSTGST-CCDEpo1MBP-CCDPea2MBP-LacZ++-- ++--++-- }}BaitPreyAWindow = 14Window = 21Window = 28amino acidsProbability00. 50 100 150 200 250 300 350 400 450Coils output for Pea2Coils output for Epo1amino acidsProbability00. 100 200 300 400 500 600 700 800 900BCD Pea2 X Epo1 PCA E Pea2-GFPG1 S G2 MG1 S G2 MG1 S G2/M G1 S G2/M G1 S G2/MF Pea2 X short CCDEpo1     PCA G Epo1 X CCDPea2 PCA H3HD;(SR?&&'3&$46Figure 2.2 (continued)KO  6FV?70;3HD3&$I (SR*)3LQ?pea2FHOOVW7?pea2 W7?epo1 (SR*)3(SR?CCD*)33L[HO,QWHQVLW\J250200150100500* ***G1 S G2 M (Septum) Epo1-GFP * 6 * 0VHSWXPPea2-GFP  3L[HO,QWHQVLW\300250200150100500L (SR?&&'*)3M* * **3L[HOLQWHQVLW\200150100500G1 S G2 M (septum) :7 ?SHD ?HSRNFigure 2.2 Epo1 Is a Subunit of the Polarisome(A) Schematic of domain organizations of Epo1 and Pea2. Regions in Epo1 and Pea2 identified to bind each other by yeast two hybrid analysis are shown (CCDPea2, short CCDEpo1) (Newman et al., 2000). A longer version of the Epo1 coiled-coil domain (CCDEpo1) used for the binding studies in (C) is also shown.(B) Coiled-coil domain predictions for Pea2 and Epo1 using Coils (Lupas et al., 1991).(C) Purified recombinant fusion proteins containing GST were immobilized on glutathione beads and incubated with purified recombinant MBP fusion proteins. Bound fractions were analyzed by SDS-PAGE and Coomassie staining. Asterisk, minor contaminant band present in purified GST fraction alone.(D) PCA between Pea2 and Epo1. Arrowheads indicate localizations. All scale bars, 2 ?m.(E) Pea2-GFP localization.(F and G) PCA between Pea2 and CCDEpo1 and Epo1 and CCDPea2. CCDEpo1 and CCDPea2 were expressed from plasmids.(H) PCA between Pea2 and Epo1?CCD. Arrows indicate altered localizations relative to wild type. (I) Epo1-GFP in ?pea2 cells. (J) Quantification of Epo1-GFP at polarized sites in wild type (WT) and ?pea2 cells. A minimum of 25 cells were measured per localization. Asterisks, p<0.05 vs WT.(K) As in (J), but with Pea2-GFP in WT and ?epo1 cells. No significant changes were detected.(L)  Yeast expressing endogenous Epo1 lacking its CCD tagged with GFP.47(M) As in (K), but with Epo1-GFP and Epo1?CCD-GFP.(N) Alpha factor treatment of WT, ?pea2 and ?epo1 cells. (O) PCA between Scs2?TM and Pea2.2.3.4 ER diffusion barrier requires Epo1 and Scs2 Pea2 was previously shown to be required for formation of the ER diffusion barrier (Luedeke et al., 2005); however, its role is unknown. We reasoned that Epo1 and Scs2 might also play a role in the diffusion barrier, because Pea2 localized Epo1 to sites of polarized growth, which was required for capture of ER tubules at the bud cortex. To test for roles for Epo1 and Scs2 in the diffusion barrier we performed ER photobleaching experiments. First, we validated the use of Pho88-GFP as a general marker for diffusion of proteins within the ER. We photobleached a small region of the mother pmaER and measured fluorescence recovery within the bleached region (Figures 2.3A and B). Fluorescence recovered quickly within the bleached region, with a t1/2 of 18?4 s; and Pho88-GFP showed a substantial mobile fraction of ~72% (Figures 2.3C and D). Thus, Pho88 appeared to be highly mobile and free to diffuse within the ER membrane. In these same cells we also measured loss in fluorescence within three non-bleached regions in the mother pmaER (Figure 2.3A). Upon photobleaching, Pho88-GFP was rapidly lost from these regions with a t1/2 of ~10?3 s (Figures 2.3E and F), indicating that a diffusion barrier was not present between regions within the mother ER, as previously described (Luedeke et al., 2005). Now we examined diffusion of Pho88-GFP between mother and bud. We photobleached a region of pmaER in the bud and monitored three non-bleached regions 48in the mother, initially for a single cell (Figure 2.3G). Consistent with previous work (Luedeke et al., 2005), we found that photobleaching of the bud ER did not lead to loss of fluorescence in the mother ER (Figure 2.3H). Within the bleached region in the bud, fluorescence recovered rapidly (Figure 2.3H). Together, this indicated the existence of a diffusion barrier between the bud and mother ER. Now we performed these same experiments with ?epo1 and ?scs2 cells. In the ?epo1 and ?scs2 mutants, we observed a rapid loss in fluorescence in the mother ER upon bleaching the bud ER, whereas wild type showed no change as before (Figure 2.3I). Thus, it appeared that the diffusion barrier was compromised in the mutants. We calculated t1/2 values of 11?4s and 18?5s for ?epo1 and ?scs2 cells, respectively (Figure 2.3J). The similarity of these t1/2 values for diffusion between bud and mother ER to the t1/2 value determined for diffusion within the mother ER (10?3s, Figure 2.3F) suggested that the diffusion barrier was lost in the mutants. Analysis of diffusion of Pho88-GFP within the mother pmaER in ?epo1 and ?scs2 cells did not reveal any significant differences from wild type in t1/2 values or mobile fractions (Figure 2.3B-F), indicating that the ER membrane environment was unaltered in the mutants. Finally, the similarity between these t1/2 values and those calculated for diffusion between bud and mother further supported that the ER in the bud was no longer compartmentalized from the mother in the mutants. Thus, Epo1 and Scs2 were required for the ER diffusion barrier. We now investigated whether the Epo1 CCD was required for the diffusion barrier, since this region interacted directly with Pea2. We performed similar photobleaching experiments as before except that we now used the ER protein, Sec61, tagged with GFP. Sec61-GFP diffused rapidly within the pmaER in the bud and was 49highly mobile (Figures 2.3K-N). However, similar to Pho88-GFP, Sec61-GFP did not diffuse between mother and bud in wild type cells, and in the ?scs2 mutant, the diffusion barrier was compromised (Figure 2.3O). This was also true for the epo1-ccd mutant, (Figure 2.3O), which had a t1/2 value that was indistinguishable from ?scs2 (Figure 2.3P). Thus, the Epo1 CCD was required for the ER diffusion barrier. Lastly, we tested for a role for Sac1, since Scs2 regulates Sac1 function and hence phosphoinositide levels in the pmaER (Stefan et al., 2011), but found that loss of Sac1 did not compromise the diffusion barrier (Figure 2.3O). Given the importance of the Epo1 CCD to the ER diffusion barrier we tested whether this region also bound to Scs2. We reconstituted binding in vitro using purified recombinant Scs2 tagged with MBP (MBP-Scs2 ) and GST-tagged CCD of Epo1 (GST-CCDEpo1). MBP-Scs2 bound to GST-CCDEpo1, but not to GST alone, indicating Scs2 interacted directly with this region of Epo1 (Figure 2.3Q). Using PCA we found that deletion of the Epo1 CCD prevented interaction with Scs2 in vivo (Figure 2.3Q), consistent with Scs2 binding the CCD. Binding of Scs2 to the Epo1 CCD also suggested that this interaction was important for the ER diffusion barrier.5051Figure 2.302040608010040 60 80 1000 20Time (s)% Fluoresence at T0WT?HSR?VFVPho88-GFPBleached ROI: MotherPho88-GFPBleached ROI: Mother02040608010012040 60 80 100 1200 20Non-bleached ROI: MotherBleached ROI: Budt 1/2 (s)?HSR ?VFVWT0510152025Pho88-GFPBleached ROI: MotherNon-bleached ROI: MotherA B CMobile Fraction (%)Pre-bleach 5s post-bleachPho88-GFPBleached ROI: MotherE FDPho88-GFPBleached ROI: MotherNon-bleached ROI: MotherTime (s)% Fluoresence at T0Non-bleached ROIG H% Fluoresence at T0Time (s)Pre-bleach 5s post-bleachPho88-GFPI Pho88-GFPBleached ROI: Bud Non-bleached ROI: Mother% Fluoresence at T0Non-bleached ROITime (s)t 1/2 (s)?HSR ?VFV0510152025Pho88-GFPBleached ROI: BudNon-bleached ROI: MotherJTime (s)Pho88-GFPBleached ROINon-bleached ROI020406080100?HSR ?VFVWTt 1/2 (s)?HSR ?VFVWT05101520250 20 40 60 80 10060708090100?HSR?VFVWT0 20 40 60 80 10060708090100110 ?HSR?VFVWTFigure 2.3 Epo1 and Scs2 Are Required for the ER Diffusion Barrier(A) Confocal images of a wild type cell expressing Pho88-GFP before and 5s after photobleaching a region of pmaER in the bud (red circle). Changes in fluorescence were monitored within the bleached ROI and within three non-bleached ROIs in the mother (blue boxes). Scale bars, 2 ?m.52Figure 2.3 (continued)0 5 10 15 20 WT epo1-ccd ?VFV ?VDFSec61-GFPBleached ROI: Bud020406080100WT epo1-ccd ?VFV ?VDFt 1/2 (s)Pre-bleach 5s post-bleachMobile Fraction (%)Sec61-GFPBleached ROI: BudML NSec61-GFPBleached ROI: Bud   % Fluoresence at T0Time (s)KR% Fluoresence at T0Non-bleached ROISec61-GFPBleached ROI: Bud   Non-bleached ROI: MotherTime (s)OSec61-GFPBleached ROI: BudNon-bleached ROI: MotherPt 1/2 (s)epo1-ccd ?VFVQSec61-GFPBleached ROINon-bleached ROI20 40 60 80 100020406080100 WTepo1-ccd?VDF?VFV 6FV?70;(SR?&&'3&$0 20 40 60 80 100708090100110WTepo1-ccd?VFV?VDF0 5 10 15 20 75372575&RRPDVVLHVWDLQXQERXQGGST*67&&'(SR0%36FV?70++- ++-WB (anti-MBP): bound fractionPonceau stain: bound fraction$ B$ B$ B$ B0%36FV?70GST*67&&'(SR0%36FV?70} BaitPrey(B) Using the experimental procedure described in (A), fluorescence values were measured in the bleached ROI for a minimum of five cells in wild type (WT), ?epo1 and ?scs2 mutants. (C) t1/2 values calculated from curves in (B).(D) Mobile fractions calculated from curves in (B).(E) Similar to (A), but fluorescence was measured in the non-bleached ROI for a minimum of five cells in wild type, ?epo1 and ?scs2 mutants.(F) t1/2 values calculated from curves in (E).(G) A yeast cell expressing Pho88-GFP was bleached in a region of pmaER in the bud (red circle). Fluorescence values in three non-bleached ROIs were measured in the mother pmaER (blue boxes). (H) For the cell in (G), changes in fluorescence within the bleached and non-bleached ROIs were plotted over time as % fluorescence relative to pre-bleach (T0). Fluorescence recovery within the bleached ROI was fitted to a single exponential function.(I) Similar to (G), but for multiple cells of wild type (WT), ?epo1 and ?scs2 mutants (minimum of five cells per strain).(J) Rates of fluorescence loss were calculated for ?epo1 and ?scs2 cells in (I) and plotted as t1/2 values.(K) Yeast expressing Sec61-GFP was bleached in a region of pmaER in the bud (red circle), and fluorescence was measured in three regions of pmaER in the mother in each cell (blue boxes).(L) Fluorescence changes over time in the bleached ROI were plotted for a minimum of five cells in wild type, epo1-ccd, ?scs2 and ?sac1 mutants, as described in (K).53(M) t1/2 values calculated from curves in (L).(N) Mobile fractions calculated from curves in (L).(O and P) As in (I and J), but with cells expressing Sec61-GFP.(Q) Purified recombinant fusion proteins containing GST were immobilized on glutathione beads and incubated with purified recombinant MBP fusion proteins. Bound fractions were analyzed by western blotting and Ponceau S stain. Unbound fractions were analyzed by SDS-PAGE and Coomassie stain.(R) PCA between Scs2?TM and Epo1?CCD.542.3.5 Epo1, Scs2 and Shs1 form an ER-septin tethering complex at the bud neck The role for the septin Shs1 in creating the ER diffusion barrier suggested it might physically interact with Epo1 and Scs2 to form an ER-septin tether. Although Epo1 and Scs2 did not obviously colocalize with septins at the bud neck, we reasoned that since all three colocalized at the incipient bud site in G1 and the septum during cytokinesis, a small fraction of Scs2 and Epo1 might also localize to the neck and interact with Shs1. In support, the polarisome subunit Spa2 interacts with Shs1 by yeast two hybrid assay (Mino et al., 1998), suggesting the presence of a physical interaction between septins and the polarisome. We first tested for binding of Epo1 and Scs2 to Shs1 using PCA. Both Epo1 and Scs2 interacted with Shs1 in a similar manner, as a pair of punctae at the incipient bud site in G1, at the bud neck throughout the cell cycle and at the septum during cytokinesis (Figures 2.4A and B). These localizations closely corresponded to the localization of Shs1-GFP (Figure 2.4C) and were indicative of the septin complex (Mino et al., 1998). As a control, Pea2 did not interact with Shs1 by PCA (Figure 2.4D). Next we tested for binding between Scs2 and Shs1 by co-purification from yeast. Scs2?TM-GFP specifically co-purified with Shs1-TAP, but not in the control, indicating the presence of a stable interaction between these proteins in yeast (Figure 2.4E). Shs1 has, in addition to the GTPase domain present in all septins, a C-terminal extension (CTE) that contains a CCD (Garcia et al., 2011), raising the possibility that this region interacted with Scs2. We reconstituted binding in vitro using purified recombinant GST-tagged Shs1 CTE and MBP-Scs2. We found that the CTE specifically bound Scs2 (Figure 2.4F). Additionally, we detected an interaction between Scs2 and a longer 55version of the CTE that was extended 77 amino acids N terminally (Figure 2.4F). Using PCA we found that the CTE interacted with Scs2?TM in vivo at sites of polarized growth as well as the bud neck (Figure 2.4G). Deletion of the CTE prevented interaction between Shs1 and Scs2 at the bud neck (Figure 2.4H), even though Shs1?CTE-GFP localized normally to the neck (Figure 2.4I). The Scs2 MSP domain alone interacted with Shs1, yet the T42A mutation did not prevent binding, indicating Scs2 bound Shs1 separate from the FFAT motif binding site in Scs2 (Figures 2.4J and K).  To directly visualize tethering in vivo between the ER and septins we engineered a fusion protein, Scs2-2TMD, that in the N terminal half contained the MSP domain of Scs2, while in the C terminal half, contained the two transmembrane domains from the ER protein Sac1 followed by the Venus PCA fragment (Figure 2.4L). The topology of this fusion protein should place both the MSP domain and the PCA fragment on the cytoplasmic face of the ER membrane, accessible to Shs1 at the bud neck (Figure 2.4L). We verified that Scs2-2TMD-GFP localized throughout the ER and that the PCA version interacted with a canonical FFAT motif sequence on the ER (Figure 2.4M). We found that Scs2-2TMD interacted with Shs1 discretely at the bud neck by PCA, thus demonstrating the physical tethering of ER to septins in vivo (Figure 2.4N). We further examined binding of Epo1 to Shs1. Epo1-TAP specifically co-purified with Shs1-GFP, indicating the presence of a stable Shs1-Epo1 association in yeast (Figure 2.4O). We now tested for binding of the Shs1 CTE to the Epo1 CCD. We reconstituted binding in vitro using purified recombinant GST-tagged CTE and MBP-Epo1 CCD and found they interacted specifically (Figure 2.4P). The Epo1 CCD also interacted with the extended CTE (Figure 2.4P). By PCA, the Epo1 CCD interacted with 56full length Shs1 in vivo as did the Shs1 CTE with full length Epo1 (Figures 2.4Q and R). Deletion of the CTE prevented interaction between Shs1 and Epo1 as did deletion of the Epo1 CCD (Figures 2.4S and T). Together, these data indicated that Scs2 and Epo1 interacted with the CTE of Shs1 and formed an ER septin tether at the bud neck.5758Figure 2.4B Epo1 X Shs1 PCAA 6FV?70;6KV3&$C Shs1-GFPE FG 6FV?70;&7(3&$Strain: :%7$3WB: GFP,37$3 Lysate (10%)Yeast strains:6KV7$36FV?70*)36KV7$36FV?70*)34) ?HSR6KV7$36FV?70*)3(1) (2) (3) (4) (1) (2) (3) (4)(1) (2) (3) (4) (1) (2) (3) (4)6KV7$36FV?70*)375372575Coomassie stain: unbound fraction*67*67&7(0%36FV?70- -*67H[WHQGHG&7( - -  - -:%DQWL0%3ERXQGIUDFWLRQPonceau stain: bound fractionA B CCA BA BA B C*67*67&7(0%36FV?7050*67H[WHQGHG&7(C0%36FV?70} BaitPreyG1 S G2 0 G1 S G2 0D Pea2 X Shs1 PCA59Figure 2.4 (continued)H 6FV?70;6KV?&7(3&$*)3or9)(5(5OXPHQF\WRVROScs2 Scs26FV70';6KV3&$J 6KV;063Scs23&$M L 6FV70';))$72SL3&$6FV70'*)3K 6KV;6FV7$3&$NO PI 6KV?&7(*)350372550&RRPDVVLHVWDLQXQERXQGIUDFWLRQ*67*67&7(0%3&&'(SR++- -+*67H[WHQGHG&7( - - ++ +- -:%DQWL0%3ERXQGIUDFWLRQ3RQFHDXVWDLQERXQGIUDFWLRQ$ % &&$ %$ %$ % &*67*67&7(0%3&&'(SR0%3&&'(SR50*67H[WHQGHG&7(&} %DLW3UH\6WUDLQ:%7$3:%*)3,37$3 /\VDWH(SR7$36KV*)3(SR7$36KV*)3        7575 (SR7$36KV*)3*Figure 2.4 Epo1 and Scs2 Interact with Shs1 in Septin Filaments(A and B) PCA with the indicated proteins. Arrowheads indicate sites of interaction. All scale bars, 2 ?m.(C) Shs1-GFP localization.(D) PCA between Pea2 and Shs1. Arrows indicate the absence of expected PCA.(E) Cell lysates from yeast expressing the indicated fusion proteins were incubated with IgG beads and the bound fraction and cell lysates were analyzed by western blotting.(F) Binding of purified recombinant bait and prey fusion proteins as indicated. Asterisks, degradation products of GST-fusion proteins.(G) PCA between Scs2?TM and the CTE of Shs1 expressed from a plasmid. Arrowheads indicate strong PCA signals.60Q Shs1 X CCDEpo1 PCA R Epo1 X CTE PCAS (SR;6KV?&7(3&$ T (SR?&&';6KV3&$Figure 2.4 (continued)(H)PCA between Scs2?TM and Shs1?CTE. Arrows indicate the absence of expected PCA.(I) Yeast cells expressing Shs1?CTE-GFP. Arrowheads indicate wild type localizations.(J and K) PCA between Shs1 and the indicated proteins. Both MSPScs2 and Scs2T42A were expressed from plasmids. Arrowheads, sites of interaction.(L) Schematic of the engineering of the Scs2-2TMD construct fused to either GFP or Venus PCA fragment (VF). The single transmembrane domain (TMD) of Scs2 was replaced with the two TMDs from Sac1.(M) Scs2-2TMD chimeric fusion proteins were expressed from plasmids. Left panel: Scs2-2TMD-GFP expressed in a wild type cell. Right panel: PCA between Scs2-2TMD-VF1 and the FFAT motif from Opi1, FFATOpi1-VF2. Both plasmids were co-expressed in a wild type cell(N) PCA between Scs2-2TMD expressed from a plasmid and Shs1.(O) Cell lysates from yeast expressing the indicated fusion proteins were incubated with IgG beads and the bound fraction and cell lysates were analyzed by western blotting.(P) Binding of purified recombinant bait and prey fusion proteins.(Q) PCA between endogenous Shs1 and CCDEpo1 expressed from a plasmid. Arrowheads indicate sites of interactions. (R) PCA between endogenous Epo1 and the CTE of Shs1 expressed from a plasmid.(S) PCA between Epo1 and Shs1?CTE. Arrows indicate the absence of expected PCA.(T) PCA between Epo1?CCD and Shs1. Arrows indicate the absence of expected PCA.612.3.6 ER-septin tethering creates the ER diffusion barrier Interaction of Scs2 and Epo1 with Shs1 suggested that ER-septin tethering created the ER diffusion barrier. We now focused on a role for the Shs1 CTE since it bound directly to both Epo1 and Scs2. The Shs1 CTE has been shown to be required for the formation of septin rings in vitro, but not for incorporation into octamers (Garcia et al., 2011) and its role in vivo is unknown. Loss of Shs1 results in minor defects in septin organization and aberrant cytokinesis, which causes growth arrest under cold stress (Iwase et al., 2007). We found that the CTE was not required for Shs1 incorporation into septin filaments in vivo (Figure 2.4I) and deletion of the CTE did not cause cold sensitivity to growth (Figure 2.5A) or budding defects (Figure 2.4I), indicating the CTE was dispensable for cytokinesis. We now tested for a role for the CTE in the ER diffusion barrier by performing photobleaching experiments with Pho88-GFP. As before we bleached a region of pmaER in the bud and monitored loss in fluorescence in the mother pmaER. In contrast to wild type, the shs1-cte mutant clearly had a compromised diffusion barrier (Figure 2.5B). The t1/2 value of 8?1s. indicated that Pho88-GFP diffused between bud and mother in the shs1-cte mutant at the same rate as within the mother in wild type (t1/2 9?3s, Figure 2.3F), suggesting the barrier was completely lost. Loss of barrier function was not due to changes in the ER membrane environment (Figure 2.5C-E). Thus, the CTE of Shs1 played a specific role in the ER diffusion barrier. Minimally, ER-septin tethering could be established through the direct interaction of Scs2 with Shs1 at the bud neck, and we now examined the role for Epo1 in tethering. We found that deletion of Epo1 did not affect the ability of Scs2 to bind Shs1 in yeast 62(Figure 2.4E). However, loss of Epo1 resulted in substantial mislocalization of the Scs2-Shs1 tether in vivo to the mother distal pole in G1 and to the bud tip in S, G2 and M phase (compare Figure 2.5F to Figure 2.4A). These localizations appeared to correspond to those observed for Scs2?TM-GFP (Figure 2.1D). Thus, although Epo1 was not required for the Scs2-Shs1 interaction, Epo1 likely functioned to maintain a stable Scs2-Shs1 tether at the bud neck throughout the cell cycle. Finally, since Scs2 is highly conserved, we tested for an interaction between its human homologue, VAP-B, and Shs1 in yeast. We found that VAP-B interacted with Shs1 similarly to Scs2 (Figure 2.5G), indicating that the ER-septin tether may be conserved in humans.2.3.7 ER-septin tethering creates the nuclear envelope diffusion barrier During mitosis and nuclear migration in yeast, the intact nuclear envelope is pulled through the bud neck into the bud. An ER diffusion barrier has been identified in the nuclear membrane in M phase, which also requires Shs1 and Bud6, implying that a similar mechanism underlies this barrier (Luedeke et al., 2005; Shcheprova et al., 2008). Therefore, we tested for a role for the ER-septin tether in restricting diffusion within the nuclear envelope in M phase cells. Photobleaching analysis with Sec61-GFP revealed that a diffusion barrier indeed existed in the nuclear ER of wild type cells, as previously found (Figure 2.5H). Deletion of the Shs1 CTE, the Epo1 CCD, or Scs2 clearly compromised the diffusion barrier (Figure 2.5H).  t1/2 values determined for diffusion between mother and bud were indistinguishable from t1/2 values for diffusion within the bud nuclear ER (Figure 2.5I) indicating the nuclear membrane diffusion 63barrier was lost in the mutants. Thus the Scs2-Shs1 tether also controlled diffusion of integral proteins within the nuclear membrane during M phase.646505101520WT shs1-cte epo1-ccd ?VFVWTshs1-cte30?C 18?CA 0 20 40 60 80 100 1206080100120shs1-cte?VFVepo1-ccdWTWTshs1-cte0 20 40 60 8080100BPho88-GFPBleached ROI: BudNon-bleached ROI: Mother9070t1/2= 8.1  1.3 s+-C D Et 1/2 (s)Pho88-GFPBleached ROI: BudWT shs1-cte0510152025WT0 20 40 60 80020406080100shs1-cteTime (s)Pho88-GFPBleached ROI: Bud% Fluoresence at T0WT shs1-cte020406080100Mobile Fraction (%)Pho88-GFPBleached ROI: BudTime (s)F 6FV?70;6KV3&$LQ?HSR G 6KV;9$3%3&$Time (s)H Sec61-GFPBleached ROI: Bud NENon-bleached ROI: Mother NEBleached ROINon-bleached ROII% Fluoresence at T0Non-bleached ROI% Fluoresence at T0Non-bleached ROIG1 S G2 M G1 S G2 MSec61-GFPBleached ROI: Bud NENon-bleached ROI: Mother NEt 1/2 (s)Figure 2.5Figure 2.5 Characterizing the Role of Shs1 CTE in the ER Diffusion Barrier(A) Serial dilutions of wild type and shs1-cte mutant cells grown at 30?C and 18?C on synthetic defined media. (B) Similar to Figure 2.3H, yeast expressing Pho88-GFP were bleached in a region of pmaER in the bud and changes in fluorescence within non-bleached ROIs in the mother were plotted over time as % fluorescence relative to pre-bleach (T0), for wild type (WT) and shs1-cte cells (minimum of five cells each). Half time was calculated for shs1-cte cells.(C) Using the cells in (B), fluorescence recovery within the bleached region was measured for wild type and shs1-cte cells (minimum of five cells).(D) t1/2 values calculated from curves in (C).(E) Mobile fractions calculated from curves in (C).(F) PCA between Scs2?TM and Shs1 in ?epo1 cells. Arrows indicate mislocalized interactions in the ?epo1 mutant.(G) PCA between Shs1 and human VAP-B expressed from a plasmid.(H) A region in the bud nuclear envelope (NE) was bleached and fluorescence values in non-bleached ROIs in the mother NE were measured for the indicated strains expressing Sec61-GFP.(I) t1/2 values calculated for the NE photobleaching experiments of M phase cells expressing Sec61-GFP in (H).662.3.8 Polarization of the integral ER protein Ist2 is mediated by the ER diffusion barrier Certain mRNAs encoding ER proteins are targeted to the bud (Shepard et al., 2003), indicating that they are most likely translated in the bud, suggesting that their protein products might also be polarized within the ER. The Ist2 mRNA is polarized to the bud (Takizawa et al., 2000) and encodes a polytopic integral membrane protein that localizes to pmaER (Maass et al., 2009); although it was initially thought to be integral to the PM (Takizawa et al., 2000). The Ist2 mRNA accumulates in G2/M phase buds; consistently, the Ist2 protein is translated specifically in large buds (Takizawa et al., 2000). Newly synthesized Ist2 in the bud does not diffuse back into the mother, unless septins are disrupted (Takizawa et al., 2000), suggesting that the ER diffusion barrier might be responsible for polarizing Ist2 within the ER. Therefore, we investigated the distribution of Ist2 in wild type and in ER-septin tethering mutants. In wild type, GFP-Ist2 localized non-uniformly to the mother cell cortex, and was absent from ER tubules and the nuclear ER (Figure 2.6A). This localization was similar to Tcb3-GFP, an integral ER protein that localizes to pmaER (Figure 2.6B) (Toulmay and Prinz, 2012). However, we noticed that GFP-Ist2 was absent from small and medium sized buds (S and G2 phases) and reappeared in M phase buds (Figure 2.6A). In contrast, Tcb3-GFP was present in both buds and mothers throughout the cell cycle (Figure 2.6B). Thus, even though pmaER was present in S and G2 phase buds, GFP-Ist2 was restricted to the mother pmaER domain until M phase. Appearance of Ist2 in M phase buds was consistent with the bud-specific translation of Ist2 in G2/M phase, as previously observed (Takizawa et al., 2000).67 Next, we investigated the role of the ER diffusion barrier in Ist2 polarization. We localized GFP-Ist2 in the shs1-cte mutant, which had a compromised ER diffusion barrier. In contrast to wild type, GFP-Ist2 was present in pmaER in S and G2 phase buds in shs1-cte cells (Figure 2.6C). pmaER appeared normal in the shs1-cte mutant (Figure 2.6D), indicating that in the absence of ER-septin tethering, Ist2 diffused from the mother into the bud. We quantified these differences and in S phase wild type cells the ratio of GFP-Ist2 in mothers vs buds approached 5:1, whereas in the shs1-cte mutant it was near1:1 (Figure 2.6E). In large budded G2/M phase cells the ratio was close to 1:1 for both wild type and mutant (Figure 2.10E). We observed a similar loss of Ist2 polarization in ?epo1 and ?scs2 mutants (Figure 2.6F and G), consistent with the role for the ER-septin tether in polarizing the ER. We also observed lack of pmaER at the tips of S phase buds in these mutants, consistent with the roles of Epo1 and Scs2 in ER tubule capture and pmaER formation in the bud. Thus, Ist2 was polarized within the ER in S phase by the ER diffusion barrier at least until its expression in the bud in G2/M phase. The molecular function of Ist2 remains unknown.6869Figure 2.6Figure 2.6 The ER Diffusion Barrier Polarizes Ist2 within the ER(A and C) GFP-Ist2 localization in wild type and shs1-cte cells. Asterisks indicate absence of localization to the bud cortex. Arrows indicate mislocalization of GFP-Ist2 in the mutants. All scale bars, 2?m.(B and D) Tcb3-GFP localization in wild type and shs1-cte cells. (E) Quantification of the bud to mother fluorescence ratio of GFP-Ist2 in wild type (WT) and the shs1-cte mutant. A minimum of 36 cells per category was measured. Asterisks, p<0.0001.(F and G) GFP-Ist2 localization in ?scs2 and ?epo1 cells. Arrows indicate mislocalization of GFP-Ist2 in the mutants.702.3.9 Scs2 plays a role in S phase spindle positioning To uncover physiological functions for ER polarization we interrogated the known global genetic interaction network in yeast, which provides a functional map of the cell (Costanzo et al., 2010). This dataset contains over five million unbiased quantitative measurements of synthetic genetic interactions covering over three quarters of the yeast genome. We used this dataset and performed hierarchical clustering to generate gene clusters with similar genetic interaction profiles in order to identify genes with similar functions. We noticed that SCS2 was present in a cluster with genes that function in S phase spindle positioning, suggesting a role for ER polarization in this pathway (Figure 2.7A, ?S Phase Cluster?). In the S phase cluster were genes encoding the formin BNI1, the myosin motor MYO2, the microtubule guidance protein KAR9, the kinesins KIP3 and CIN8, several microtubule associated proteins (STU1, STU2, ASE1) and tubulin folding cofactors (CIN1, CIN2), and the transcription factor HCM1, which controls S phase expression of spindle assembly factors. Genes in the S phase cluster formed aggravating genetic interactions with a gene cluster containing M phase spindle positioning genes (Figure 2.7A, ?M Phase Cluster?), as previously observed (Tong et al., 2004). The M phase cluster included the dynein/dynactin complex, its cortical capture protein Num1, and the kinesin motor and accessory proteins required to load dynein onto the plus ends of microtubules (Huisman and Segal, 2005). Thus, the presence of SCS2 within the S phase cluster and its aggravating genetic interactions with genes in the M phase cluster implied a role for SCS2, and perhaps the ER diffusion barrier, in S phase spindle positioning.71 To evaluate the role for Scs2 in spindle positioning, we performed our own SGA analysis, which identified aggravating genetic interactions between SCS2 and many of the genes involved in M phase spindle positioning and nuclear migration (Figure 2.7B and Tables 2.3 and 2.4). We confirmed genetic interactions between SCS2 and dynein, dynactin and NUM1, and consistent with a role for Scs2 in S phase spindle positioning, these double mutants showed slow growth phenotypes (Figure 2.7C). Now we examined spindle positioning in wild type and ?scs2 cells synchronized in S-phase. In wild type cells, the spindle was predominately positioned in the mother adjacent to the neck (Figures 2.7D and E), as previously observed (Yeh et al., 1995). In contrast, in ?scs2 cells the spindle was no longer retained in the mother, but instead migrated into the neck, and in ~13% of cells, the spindle mislocalized entirely into the bud clear of the neck (Figure 2.7E). We found that spindle length was normal in ?scs2 cells, ruling out a role for defective spindle assembly (Figure 2.7F). Thus, proper S phase spindle positioning relied on Scs2, consistent with it co-clustering with S phase components and having aggravating genetic interactions with M phase components.7273Figure 2.7 -0.3  -0.2  0.0  0.2  0.3  0.5 0.5 DYN2  BIK1  YDR149C  NUM1  ARP1  LDB18  PAC11  JNM1  DYN3  DYN1  PAC1  NIP100  KIP2  SCS2  KIP3  stu1-5  HCM1  stu1-12  CIN8  stu2-12  ASE1  CLB4  KAR9  CIN1  CIN2  cmd1-8  myo2-16  BNI1  -  M Phase ClusterS Phase ClusterSPB/ Tubulin/ microtubule associatedactin associatedKinesinDynactinDynein020406080100Mother Neck Bud?VFVQ :7Q % CellsAaggravatingalleviatingno interactionEC?DUS?QLS?QXP?MQP?G\Q?OGESCS2?VFV-BFold EnrichmentmbmbmbmbWT ?VFVDQ QWT ?VFVF0 5 10 15 20 25 30 35 40CytoplasmicMicrotubule COMPONENTFUNCTIONPROCESSMicrotubule AssociatedComplexSpindle PoleBodyMicrotubuleCytoskeletonMicrotubulePlus End BindingTubulinBindingCytoskeletalProtein BindingNuclear Migrationalong MicrotubuleEstablishment of Organelle LocalizationMitotic SisterChromatid SegregationMicrotubule CytoskeletonOrganizationOrganelle FissionNuclear DivisionMitosisFigure 2.7 Scs2 Plays a Role in the Nuclear Migration Pathway in Yeast (A) Genetic interactions between genes with roles in S and M phase spindle positioning (reclustered from (Costanzo et al., 2010)). Interactions are color coded by strength and correspond to epsilon values. Gene names are color coded by function.(B) Synthetic genetic array (SGA) results for SCS2. Aggravating genetic interactions were categorized by gene ontology and fold enrichment was plotted.(C) Yeast spot assays of double mutants between SCS2 and genes required for the late stage of nuclear migration. Genes encoding components of the dynactin complex are highlighted in brown, and dynein in blue.(D) WT and ?scs2 mutant yeast expressing endogenous Tub1 tagged with GFP synchronized in S phase with hydroxyurea. b, bud. m, mother. Scale bar, 2 ?m.(E) Spindle positioning in S phase-synchronized wild type (WT) and ?scs2 cells by imaging GFP-Tub1. Scale bar = 2 ?m.(F) Spindle length measured for cells in (D).2.3.10 Scs2 recruits Num1 to pmaER To provide clues to its role in S phase spindle positioning, we now focused on protein interaction partners for Scs2. Our proteomics experiment with Scs2?TM identified an interaction with Num1 (Figure 2.1B), similar to another large-scale proteomics study (Gavin et al., 2002). Num1 is the cortical factor that captures and anchors cytoplasmic dynein at the bud cortex in M phase, thereby pulling the spindle through the neck during the process of nuclear migration (Huisman and Segal, 2005). We now investigated whether Scs2 regulated Num1on the ER.74 First, we verified the Scs2-Num1 interaction in vivo by co-IP. GFP-tagged Num1 specifically bound to Scs2?TM-TAP in yeast (Figure 2.8A). After examining the Num1 sequence, we noticed an acidic stretch of residues that contained phenylalanines which resembled a ?cryptic? FFAT motif (Mikitova and Levine, 2012), suggesting this region might interact directly with Scs2 (Figure 2.8B). We reconstituted binding in vitro using purified recombinant MBP-Scs2?TM and the Num1 FFAT motif tagged with GST and confirmed that this region of Num1 interacted directly with Scs2 (Figure 2.8C). We also verified this interaction using PCA (Figure 2.8D). Next, we tested whether the Num1 FFAT motif was necessary for binding to Scs2 in vivo using PCA. Full length Num1 interacted with Scs2-2TMD by PCA in cortical patches that were very similar to the previously observed localizations of Num1-GFP (Figure 2.8E; see also Figure 2.8G) (Heil-Chapdelaine et al., 2000). Deletion of the Num1 FFAT motif prevented this interaction (Figure 2.8F), indicating that Num1 interacted directly with Scs2 via its FFAT motif in vivo. Interaction of Num1 with Scs2 at cortical sites suggested that Scs2 might recruit Num1 to the ER, and we examined the localization of Num1-GFP in yeast. In wild type cells, Num1-GFP localized non-uniformly to the cell cortex and was enriched at the tips of M phase buds (Figure 2.8G), as was previously observed for the native Num1 protein (Farkasovsky and K?ntzel, 1995). Num1-GFP was also conspicuously absent from S phase buds, as previously observed (Heil-Chapdelaine et al., 2000). The cortical localization of Num1-GFP suggested that it might be associated with pmaER and we colocalized it with Tcb3, tagged with RFP. The localization pattern of Tcb3-RFP appeared similar to Num1-GFP suggesting Num1 was indeed associated with pmaER 75(Figure 2.8G). Using Pearson?s correlation coefficient we found very good correlation between Tcb3 and Num1 in G1 and M phases (Figure 2.8H). In S phase, this correlation decreased almost 50%, consistent with the absence of Num1-GFP from pmaER in S phase buds.  In ?scs2 cells, in contrast to wild type, Num1-GFP was no longer distributed along the cortex, but instead was concentrated in foci at the distal poles of unbudded cells and mothers, and at the tips of small and large buds; these regions did not appear to contain pmaER (Figure 2.8I). Consistently, Pearson?s correlation coefficient revealed almost no correlation between Num1-GFP and Tcb3-RFP in ?scs2 cells (Figure 2.8H). Deletion of the Num1 FFAT motif also prevented its association with pmaER (Figure 2.8J and 2.8H). Together, these data indicated that the direct interaction of Num1 with Scs2 recruited Num1 to pmaER. Failure to capture astral microtubules at the cortex of M phase buds by Num1 results in multi-nucleated mother cells (Farkasovsky and K?ntzel, 1995), and we tested if binding to Scs2 on the ER was required for this function of Num1. We measured the frequency of multinucleate cells for the ?scs2 mutant and found it was no different than wild type, whereas ?num1 cells showed the usual phenotype of ~5% multinucleated cells (Figure 2.8K). Thus, interaction with Scs2 was not required for cortical capture of microtubules by Num1, consistent with Num1-GFP localization to cortical capture sites being unaffected in ?scs2 (Figure 2.8I). Given the aggravating genetic interaction between SCS2 and NUM1, we also measured binucleate cells in the ?scs2?num1 double mutant, but found no difference compared to ?num1 alone (Figure 2.8K).767775372575Coomassie stain:          flow throughGSTGST-FFATNum10%36FV?70++- ++-IB: anti-MBPPonceau stainA B A BA BA B0%36FV?70GSTGST-FFATNum10%36FV?70}BaitPreyNum1   2748 aa 306-330101-294 2573-2683593-1384FFAT motif13X tandem repeatsPHCCDCNacidic residuesbasic residuesD Scs2-2TMD X FFATNum1 PCACBAE Scs2-2TMD X Num1 PCA F 6FV70';1XP?))$73&$G1 S G2/M G1 S G2/MStrain: WB: GFPWB: TAPIP: TAP Lysate (10%)Yeast strains:6FV?707$32) Num1-GFP6FV?707$31XP*)3(1) (2) (3) (1) (2) (3)(1) (2) (3) (1) (2) (3)Num1-GFP6FV?707$3Figure 2.878KMultinucleatedbudded cells (%)5.3 189?QXP 5.16 384?VFV?QXP?VFV 0 nWT 0 J 1XP?))$7*)37FE5)31XP?))$7*)3 MergedI 1XP*)3LQ?scs2*S*0*S*07FE5)31XP*)3 Merged7FE5)31XP*)3 Merged* *H1XP*)3#* * * ?VFV 1XP?))$7*)3*S*0* * *3HDUVRQ&RUUHODWLRQ&RHIILFLHQWG 1XP*)3Figure 2.8 (continued)*Figure 2.8 Num1 Interacts with Scs2 on the ER(A) Cell lysates from yeast expressing the indicated fusion proteins were incubated with IgG beads and the bound fraction and cell lysates were analyzed by western blotting.(B) Domain organization of Num1. Window shows acid/base composition of FFAT motif region (Scale, 10 aa). FFAT, two phenylalanines in an acidic tract. PH, pleckstrin homology domain.(C) Binding of purified recombinant bait and prey fusion proteins was performed as before.(D) PCA between Scs2-2TMD and the FFAT motif of Num1, both expressed from plasmids. All scale bars, 2 ?m.(E) PCA between Scs2-2TMD expressed from a plasmid and endogenous Num1. Arrowheads indicate PCA interactions.(F) PCA between Scs2-2TMD expressed from a plasmid and endogenous Num1?FFAT. Note the absence of cortical patches.(G) Yeast co-expressing Num1-GFP and Tcb3-RFP in wild type cells. Asterisks indicate absence of Num1-GFP localization to the bud cortex.(H) Pearson correlation coefficients were calculated for Num1-GFP and Tcb3-RFP co-expressed in wild type and ?scs2 cells and for wild type cells co-expressing Num1?FFAT-GFP and Tcb3-RFP. A minimum of 45 cells was analyzed per condition. Asterisks, p<0.0002 vs WT. #, p<10-12 vs WT G1. (I) Same as (G) but in ?scs2 cells. Arrows indicate altered Num1-GFP localizations in the mutant.79(J) Num1?FFAT-GFP co-expressed with Tcb3-RFP, quantified in Figure 2.8H.(K) DAPI-stained wild type and mutant yeast were scored for multi-nucleated mother cells.802.3.11 The ER diffusion barrier controls the localization of Num1 and the timing of nuclear migration Recruitment of Num1 to pmaER by Scs2 suggested that this interaction might be important for S phase spindle positioning. As a pmaER protein, Num1 distribution should be controlled by the ER diffusion barrier, which would prevent it from diffusing from the mother into the bud. Since Num1 is synthesized in G2/M phase (Farkasovsky and K?ntzel, 1995), the ER diffusion barrier could account for the absence of Num1 in S phase buds (Figure 2.8G). Consistent with this hypothesis, studies using photoactivatable Num1-GFP reveal that Num1 does not diffuse from mother to bud (Vorvis et al., 2008). Hence, the ER diffusion barrier would control the timing of spindle translocation by preventing premature diffusion of Num1 from the mother into the bud. Now we tested for a role for the ER diffusion barrier in localizing Num1. In contrast to wild type, in the shs1-cte mutant, Num1-GFP was no longer restricted to pmaER in the mother, but appeared to diffuse into pmaER in S phase buds (Figure 2.9A). Quantification of Num1-GFP in S phase buds revealed that in wild type, the mother to bud ratio was close to 4:1, whereas in the mutant, it approached 1:1 (Figure 2.9B), indicating the ER diffusion barrier prevented premature entry of Num1 into S phase buds. In G2/M phase the ratio was close to one for both mutant and wild type. Now we tested for S phase spindle positioning defects in the shs1-cte mutant. As before, wild type cells positioned the spindle primarily on the mother-side of the bud neck (Figure 2.9C). In contrast, the shs1-cte mutant mispositioned the spindle entirely into the bud in ~20% of cells, and in ~30% of the cells the spindle had migrated from the mother into the neck (Figure 2.9C). We deleted NUM1 in the shs1-cte mutant and found 81that the spindle no longer mispositioned into the bud or neck and remained in the mother (Figure 2.9C). Thus, it appeared that the premature migration of Num1 into S phase buds in the shs1-cte mutant resulted in mislocalization of the spindle. Defective S phase spindle positioning in the shs1-cte mutant should also result in aggravating genetic interactions with M phase spindle positioning components. Therefore, we tested for genetic interactions between shs1-cte and mutants in dynein and dynactin, which are critical components of the spindle migration apparatus. We observed slow growth phenotypes for the double mutants over the single mutant controls in both cases, confirming the presence of aggravating genetic interactions between the ER diffusion barrier mutant and the spindle translocation apparatus (Figure 2.9D). Thus, the ER diffusion barrier prevented the premature migration of Num1 into the bud, which properly positioned the spindle in the mother until nuclear migration in M phase.8283Mother Neck Bud020406080100shs1-cte (n=190)?QXPVKVFWH(n=131)WT (n=280)% CellsNucleusEERIntegral/ PeripheralER ProteinsER-Septin Tethering ComplexSeptin CollarER MembraneSeptinScs2Epo1CTEofShs1shs1-cteWT?G\QVKVFWH?G\Q?QLSVKVFWH?QLSA Num1-GFP in shs1-cte BCG1 S G2/MD**OD600Time (min) 100 200 300 400 500 600 700 800 9001000shs1-cte?QLSVKVFWH?QLS0Small buddedLarge budded00. shs1-cteBud:Mother Fluoresence Ratio**Figure 2.9Figure 2.9 The ER Diffusion Barrier Controls Spindle Positioning(A) Num1-GFP localization in shs1-cte cells. The asterisk indicates the appearance of Num1 in the bud. Scale bar, 2?m.(B) Quantification of the bud to mother fluorescence ratio of Num1-GFP in wild type (WT) and the shs1-cte mutant. A minimum of 30 cells per category was measured. Asterisks, p<0.0002.(C) S phase spindle positioning in the indicated strains, as in Figure 2.11B.(D) Growth assays of the indicated yeast strains on solid (left) or in liquid (right) media (n=6).(E) Model for polarization of the ER by ER-septin tethering. Not to scale.842.4 Discussion In this study we identify the molecular basis for polarization of the ER. We discover a subunit of the polarisome, Epo1, that interacts with Scs2 in the ER and captures ER tubules at the bud cortex. This uncovers a new role for the polarisome in ER biogenesis. Epo1 and Scs2 are part of an ER-septin tether with Shs1 at the bud neck. ER-septin tethering creates the ER diffusion barrier, which restricts the diffusion of ER proteins through ER tubules that connect the mother and bud ER, thus polarizing the ER (Figure 2.9E). We show that the integral ER protein, Ist2, is restricted to the mother ER by the ER diffusion barrier, and hence is a polarized ER protein. The ER diffusion barrier also restricts Num1 to the mother, which is required to faithfully position the spindle in the mother until nuclear migration during M phase. Thus, the ER diffusion barrier controls the timing of appearance of ER proteins in the daughter cell and fundamentally contributes to the establishment of cell polarity. Cortical capture of ER tubules in the bud and formation of the ER-septin tether at the bud neck are likely coupled processes. During bud formation in early S phase, Epo1, Scs2 and Shs1 are present at the incipient bud site, suggesting that the diffusion barrier forms at this early stage when the polarity axis is established. As the bud emerges, the majority of Epo1 remains associated with the polarisome in the bud, performing its role in ER tubule capture through binding to Scs2. A small population of Epo1 likely remains associated with Shs1 at the neck, where it maintains the Scs2-Shs1 interaction at the cell cortex, creating the ER diffusion barrier. ER polarization requires that both a new domain of ER is established in the bud, and that this domain is 85compartmentalized from the ER in the mother. Therefore, it seems practical for both of these functions to be carried out by the same protein, Epo1. How might the ER-septin tether create the ER diffusion barrier? Perhaps the Scs2-Shs1 tether positions ER tubules traversing the neck in close proximity to septin filaments such that ER proteins with domains on the cytosolic face of the ER are sterically hindered from crossing through the neck. Continuous septin structures are found in close contact with the PM, usually within a distance of 10 nm (Bertin et al., 2012) and this is sufficient to restrict the diffusion of PM proteins. Therefore, proximity of ER to septins might produce a similar effect, restricting the diffusion of ER proteins between mother and bud. Consistent with this idea, the ER diffusion barrier exists at the bud neck and Scs2 interacts with Shs1 only at the neck. In contrast, inner-leaflet proteins appear to be free from the ER diffusion barrier (Luedeke et al., 2005; Shcheprova et al., 2008). This suggests that the ER diffusion barrier exists only on the cytoplasmic leaflet of the ER membrane. Scs2 is a tail-anchored ER protein that has no lumenal domain and Scs2-Shs1 tethering occurs on the cytoplasmic face of the ER, indicating that this tether would likely be ineffective for restricting diffusion of lumenal proteins.  Shs1 could also impose ordering of Scs2 within the ER at the bud neck. Ordering of Scs2 on the cytosolic face of the ER membrane might create a mesh-like structure, which would function as a net to catch diffusing ER proteins. The MSP domain of Scs2 is highly homologous to MSP proteins in nematodes, which lack transmembrane domains, but which form membrane-associated cytoskeletal ?mesh? structures that are responsible for sperm motility (Sepsenwol et al., 1989). The structure of the Scs2 MSP 86domain shares an identical fold with nematode MSP (Kaiser et al., 2005), suggesting that Scs2 has the capacity to form related mesh-like structures on the ER. However, the MSP domain dimer interface is not perfectly conserved in Scs2, raising the possibility that interaction with Shs1 might facilitate Scs2 oligomerization and mesh formation at the bud neck. Restricting diffusion of ER proteins along ER tubules that traverse the bud neck presents a unique problem since most ER proteins will likely be distributed around the circumference of the tubule. If the ER-septin tether establishes the ER diffusion barrier by proximity to septins, then septins would need to surround ER tubules. Recent work reconstituting septin interactions in vitro shows that in addition to forming straight filaments, septins form filamentous rings and gauze-like structures (Garcia et al., 2011). The rings have diameters in the range of 400-850 nm, which are similar in size to the yeast bud neck. Remarkably, gauze-like structures form within the rings that have ~30 nm holes with regular spacing, resembling a waffle (Garcia et al., 2011). Given that the diameters of ER tubules are in the range of 20-50 nm (West et al., 2011), ER tubules could foreseeably pass through the holes in the gauze, enabling septins to surround the tubules. Interaction of Scs2 with the CTE of Shs1 within these holes in the gauze could establish a proximity-based ER diffusion barrier that surrounds the ER tubule. Gauze-like septin structures with similar-sized holes have been observed in vivo (Rodal et al., 2005); however, their proximity to the ER has not yet been investigated. ER-septin tethering maintains the ER diffusion barrier throughout the cell cycle, including within the nuclear ER during mitosis as the nucleus is pulled through the bud neck (Figure 2.7K) (Luedeke et al., 2005; Shcheprova et al., 2008). However, the 87diameter of the nucleus is much larger than the diameter of an ER tubule, which would require that the Scs2-Shs1 tether and perhaps the septin gauze be remodeled during nuclear migration. Phosphorylation of the Shs1 CTE promotes gauze formation in vitro (Garcia et al., 2011) and CTE phosphorylation in vivo is cell cycle dependent and dramatically decreases in M phase (Egelhofer et al., 2008). This suggests that dephosphorylation of Shs1 in preparation for mitosis could remodel the septin gauze and the Shs1-Scs2 tether, enabling the nuclear ER and spindle to be pulled through the bud neck, while still maintaining the ER diffusion barrier. The ER diffusion barrier provides a mechanism to control the timing of appearance of ER proteins in the bud by restricting their diffusion from the mother. This enables establishment of polarity within the ER. Ist2 is not present in buds in S phase, but appears in the ER in G2/M phase, a result of its mRNA being targeted and translated in the bud (Takizawa et al., 2000). This is also true for Num1, even though its mRNA is not known to be trafficked to the bud. However, mRNA polarization is not necessarily a prerequisite for bud specific protein translation (Shepard et al., 2003), and we propose that any and all ER proteins that are restricted by the ER diffusion barrier will be synthesized in the bud ER. In support of bud specific translation of ER proteins, ribosomes are found associated with the ER in the bud as early as S phase (West et al., 2011), and the translocon is specifically trafficked to the emerging bud through direct interaction with the exocyst tethering complex (Toikkanen et al., 2003).  882.5 TablesTable 2.1 Scs2?TM proteomics, related to Figure 2.1 Proteins quantified from the Scs2?TM-TAP SILAC proteomics. The table provides Scs2?TM binding partners and their heavy/ light isotopic peptide ratios obtained by MSQuant.NameLight/HeavyStDevFold EnrichmentSCS2 0.037 0.04 27NBA1 0.047 0 21NUM1 0.129 0.05 8VMA2 0.178 0 6IDH2 0.18 0.08 6URA2 0.187 0.11 5ESC1 0.202 0 5RIR3 0.213 0 5EPO1 0.215 0.04 5YGP1 0.225 0 4ADH1 0.228 0.04 4IDH1 0.228 0.08 4FAS1 0.23 0.01 4VMA4 0.238 0 4RHR2 0.242 0 4SEC18 0.245 0 4TUB2 0.265 0.02 4FAS2 0.281 0.01 4PMA1/2 0.289 0 3GPM1 0.315 0.04 3CHC1 0.343 0 3SSA2 0.345 0.09 3PSA1 0.35 0.08 3CCT8 0.353 0.2 3HSC82 0.355 0.09 3TDH2 0.367 0.12 3CDC19 0.374 0.15 3VMA8 0.409 0 2RPN6 0.41 0.21 2SSA1 0.426 0.1 2VPS74 0.438 0 289NameLight/HeavyStDevFold EnrichmentCDC48 0.493 0.12 2ACT1 0.546 0 2YDJ1 0.562 0.09 2LSP1 0.577 0.13 2SSB1 0.442 0.08 2PIL1 0.454 0.11 2ENO2 0.455 0.04 2HSP60 0.468 0.11 2TDH3 0.469 0 2TSA1 0.478 0.07 2VMA2 0.484 0.17 2EDE1 0.489 0 2Table 2.2 Epo1 proteomics, related to Figure 2.2Proteins identified from the Epo1-TAP proteomics. The table contains Epo1 binding partners that had at least two peptides and that were not present in an untagged control pull-down.Name protein+score# pep/des?EPO1 7274 231*SSB2 1675 45FKS1 591 26YML045W 458 20KEL1 382 18RNQ1 601 13*NEW1 333 11LYS12 266 11LYS20 279 10CHC1 176 10*TOR1 210 9DHH1 165 7RPL4A 144 7LTE1 47 7YRF158$ 135 6RPS7B 110 6PET9 90 6TFP1 87 6KEM1 77 6SEC22 92 5RPL26A 72 5RPL17B 195 4CDC39 194 4NPL3 160 4VPS1 146 4NIP1 146 4SEC2 111 4SUP45 106 4RVB2 94 4RPL36A 85 4SAC6 77 4PEA2 70 4CCT8 59 4CHA1 52 4RPS31 40 4PHO84 146 3SSD1 112 390Table 2.3 SGA results for SCS2, related to Figure 2.11Genetic interactions identified for SCS2 by SGA using a ?scs2 query strain and the nonessential haploid yeast deletion collection. Mean fitness levels with standard deviations for each double mutant with ?scs2 are provided.GeneRatio (Exp/Ctrl) Ratio SD p-value DescriptionCNM67 0.1325 0.0248 0.0244ComponentFofFtheFspindleFpoleFbodyFouterFplaque;FrequiredFforFspindleForientaTonFandFmitoTcFnuclearFmigraTonCNM67 0.1778 0.0378 0.0253ComponentFofFtheFspindleFpoleFbodyFouterFplaque;FrequiredFforFspindleForientaTonFandFmitoTcFnuclearFmigraTonCSM1 0.1799 0.0403 0.0022NucleolarFproteinFthatFformsFaFcomplexFwithFLrs4pFandFthenFMam1pFatFkinetochoresFduringFmeiosisFIFtoFmediateFaccurateFhomologFsegregaTon;FrequiredFforFcondensinFrecruitmentFtoFtheFreplicaTonFforkFbarrierFsiteFandFrDNAFrepeatFsegregaTonSWI6 0.2126 0.1040 0.0354TranscripTonFcofactor;FformsFcomplexesFwithFSwi4pFandFMbp1pFtoFregulateFtranscripTonFatFtheFG1/SFtransiTon;FinvolvedFinFmeioTcFgeneFexpression;FalsoFbindsFStb1pFtoFregulateFtranscripTonFatFSTART;FcellFwallFstressFinducesFphosphorylaTonFbyFMpk1p,FwhichFregulatesFSwi6pFlocalizaTon;FrequiredFforFtheFunfoldedFproteinFresponse,FindependentlyFofFitsFknownFtranscripTonalFcoacTvatorsCHO2 0.2250 0.0972 0.0061PhosphaTdylethanolamineFmethyltransferaseF(PEMT),FcatalyzesFtheFfirstFstepFinFtheFconversionFofFphosphaTdylethanolamineFtoFphosphaTdylcholineFduringFtheFmethylaTonFpathwayFofFphosphaTdylcholineFbiosynthesisCLA4 0.2847 0.1246 0.0080Cdc42pcacTvatedFsignalFtransducingFkinaseFofFtheFPAKF(p21cacTvatedFkinase)Ffamily,FalongFwithFSte20pFandFSkm1p;FinvolvedFinFsepTnFringFassembly,FvacuoleFinheritance,Fcytokinesis,FsterolFuptakeFregulaTon;FphosphorylatesFCdc3pFandFCdc10pCNM67 0.2958 0.1954 0.0335ComponentFofFtheFspindleFpoleFbodyFouterFplaque;FrequiredFforFspindleForientaTonFandFmitoTcFnuclearFmigraTonSPE2 0.3258 0.1912 0.0328ScadenosylmethionineFdecarboxylase,FrequiredFforFtheFbiosynthesisFofFspermidineFandFspermine;FcellsFlackingFSpe2pFrequireFspermineForFspermidineFforFgrowthFinFtheFpresenceFofFoxygenFbutFnotFwhenFgrownFanaerobicallyRPN4 0.3369 0.1486 0.0410TranscripTonFfactorFthatFsTmulatesFexpressionFofFproteasomeFgenes;FRpn4pFlevelsFareFinFturnFregulatedFbyFtheF26SFproteasomeFinFaFnegaTveFfeedbackFcontrolFmechanism;FRPN4FisFtranscripTonallyFregulatedFbyFvariousFstressFresponsesNUM1 0.3603 0.0129 0.0001Protein required for nuclear migration, localizes to the mother cell cortex and the bud tip; may mediate interactions of dynein and cytoplasmic microtubules with the cell cortexJNM1 0.4016 0.0462 0.0025Component of the yeast dynactin complex, consisting of Nip100p, Jnm1p, and Arp1p; required for proper nuclear migration and spindle partitioning during mitotic anaphase BCTK3 0.4076 0.0458 0.0078GammaFsubunitFofFCcterminalFdomainFkinaseFIF(CTDKcI),FwhichFphosphorylatesFbothFRNAFpolFIIFsubunitFRpo21pFtoFaffectFtranscripTonFandFprecmRNAF3'FendFprocessing,FandFribosomalFproteinFRps2pFtoFincreaseFtranslaTonalFfidelityNIP100 0.4269 0.0943 0.0114Large subunit of the dynactin complex, which is involved in partitioning the mitotic spindle between mother and daughter cells; putative ortholog of mammalian p150(glued)RIC1 0.4293 0.0530 0.0059ProteinFinvolvedFinFretrogradeFtransportFtoFtheFciscGolgiFnetwork;FformsFheterodimerFwithFRgp1pFthatFactsFasFaFGTPFexchangeFfactorFforFYpt6p;FinvolvedFinFtranscripTonFofFrRNAFandFribosomalFproteinFgenesPHO85 0.4619 0.0840 0.0162CyclincdependentFkinase,FwithFtenFcyclinFpartners;FinvolvedFinFregulaTngFtheFcellularFresponseFtoFnutrientFlevelsFandFenvironmentalFcondiTonsFandFprogressionFthroughFtheFcellFcycle91GeneRatio (Exp/Ctrl) Ratio SD p-value DescriptionCIK1 0.4631 0.1019 0.0171KinesincassociatedFproteinFrequiredFforFbothFkaryogamyFandFmitoTcFspindleForganizaTon,FinteractsFstablyFandFspecificallyFwithFKar3pFandFmayFfuncTonFtoFtargetFthisFkinesinFtoFaFspecificFcellularFrole;FhasFsimilarityFtoFVik1pCTK3 0.5098 0.0902 0.0249GammaFsubunitFofFCcterminalFdomainFkinaseFIF(CTDKcI),FwhichFphosphorylatesFbothFRNAFpolFIIFsubunitFRpo21pFtoFaffectFtranscripTonFandFprecmRNAF3'FendFprocessing,FandFribosomalFproteinFRps2pFtoFincreaseFtranslaTonalFfidelityDYN3 0.5237 0.0322 0.0041DyneinFlightFintermediateFchainF(LIC);FlocalizesFwithFdynein,FnullFmutantFisFdefecTveFinFnuclearFmigraTonMMS22 0.5468 0.0863 0.0196SubunitFofFanFE3FubiquiTnFligaseFcomplexFinvolvedFinFreplicaTonFrepair;FstabilizesFproteinFcomponentsFofFtheFreplicaTonFfork,FsuchFasFtheFforkcpausingFcomplexFandFleadingFstrandFpolymerase,FprevenTngFforkFcollapseFandFpromoTngFefficientFrecoveryFduringFreplicaTonFstress;FrequiredFforFaccurateFmeioTcFchromosomeFsegregaTonUBP3 0.5528 0.0473 0.0033UbiquiTncspecificFproteaseFinvolvedFinFtransportFandFosmoTcFresponse;FinteractsFwithFBre5pFtoFcocregulateFanterogradeFandFretrogradeFtransportFbetweenFtheFERFandFGolgi;FinvolvedFinFtranscripTonFelongaTonFinFresponseFtoFosmostressFthroughFphosphorylaTonFatFSer695FbyFHog1p;FinhibitorFofFgeneFsilencing;FcleavesFubiquiTnFfusionsFbutFnotFpolyubiquiTn;FalsoFhasFmRNAFbindingFacTvityVPS61 0.5598 0.0063 0.0005DubiousFopenFreadingFframe,FunlikelyFtoFencodeFaFprotein;FnotFconservedFinFcloselyFrelatedFSaccharomycesFspecies;F4%FofFORFFoverlapsFtheFverifiedFgeneFRGP1;FdeleTonFcausesFaFvacuolarFproteinFsorTngFdefectPHO85 0.5653 0.0326 0.0034CyclincdependentFkinase,FwithFtenFcyclinFpartners;FinvolvedFinFregulaTngFtheFcellularFresponseFtoFnutrientFlevelsFandFenvironmentalFcondiTonsFandFprogressionFthroughFtheFcellFcycleDIA2 0.5935 0.0865 0.0313OrigincbindingFFcboxFprotein;FformsFanFSCFFubiquiTnFligaseFcomplexFwithFSkp1pFandFCdc53p;FplaysFaFroleFinFDNAFreplicaTon;FplaysFaFroleFinFtranscripTon;FrequiredFforFcorrectFassemblyFofFRSCFcomplex,FcorrectFRSCcmediatedFtranscripTonFregulaTon,FandFcorrectFnucleosomeFposiToning;FinvolvedFinFinvasiveFandFpseudohyphalFgrowthLSM7 0.6011 0.1340 0.0466LsmF(LikeFSm)Fprotein;FpartFofFheteroheptamericFcomplexesF(Lsm2pc7pFandFeitherFLsm1pForF8p):FcytoplasmicFLsm1pFcomplexFinvolvedFinFmRNAFdecay;FnuclearFLsm8pFcomplexFpartFofFU6FsnRNPFandFpossiblyFinvolvedFinFprocessingFtRNA,FsnoRNA,FandFrRNACCR4 0.6113 0.0993 0.0337ComponentFofFtheFCCR4cNOTFtranscripTonalFcomplex,FwhichFisFinvolvedFinFregulaTonFofFgeneFexpression;FcomponentFofFtheFmajorFcytoplasmicFdeadenylase,FwhichFisFinvolvedFinFmRNAFpoly(A)FtailFshorteningVPS24 0.6142 0.0109 0.0007OneFofFfourFsubunitsFofFtheFendosomalFsorTngFcomplexFrequiredFforFtransportFIIIF(ESCRTcIII);FformsFanFESCRTcIIIFsubcomplexFwithFDid4p;FinvolvedFinFtheFsorTngFofFtransmembraneFproteinsFintoFtheFmulTvesicularFbodyF(MVB)FpathwayYJR018W 0.6161 0.0188 0.0105DubiousFopenFreadingFframeFunlikelyFtoFencodeFaFfuncTonalFprotein,FbasedFonFavailableFexperimentalFandFcomparaTveFsequenceFdataPAC1 0.6217 0.0494 0.0175ProteinFinvolvedFinFnuclearFmigraTon,FpartFofFtheFdynein/dynacTnFpathway;FtargetsFdyneinFtoFmicrotubuleFTps,FwhichFisFnecessaryFforFslidingFofFmicrotubulesFalongFbudFcortex;FsyntheTcFlethalFwithFbni1;FhomologFofFhumanFLIS1DOA4 0.6266 0.0899 0.0359UbiquiTnFisopepTdase,FrequiredFforFrecyclingFubiquiTnFfromFproteasomecboundFubiquiTnatedFintermediates,FactsFatFtheFlateFendosome/prevacuolarFcompartmentFtoFrecoverFubiquiTnFfromFubiquiTnatedFmembraneFproteinsFenFrouteFtoFtheFvacuoleDID4 0.6439 0.0802 0.0346ClassFEFVpsFproteinFofFtheFESCRTcIIIFcomplex,FrequiredFforFsorTngFofFintegralFmembraneFproteinsFintoFlumenalFvesiclesFofFmulTvesicularFbodies,FandFforFdeliveryFofFnewlyFsynthesizedFvacuolarFenzymesFtoFtheFvacuole,FinvolvedFinFendocytosisDOA1 0.6560 0.0217 0.0035WDFrepeatFproteinFrequiredFforFubiquiTncmediatedFproteinFdegradaTon,FformsFcomplexFwithFCdc48p,FplaysFaFroleFinFcontrollingFcellularFubiquiTnFconcentraTon;FalsoFpromotesFefficientFNHEJFinFpostdiauxic/staTonaryFphaseGOS1 0.6707 0.0502 0.0229vcSNAREFproteinFinvolvedFinFGolgiFtransport,FhomologFofFtheFmammalianFproteinFGOSc28/GS2892GeneRatio (Exp/Ctrl) Ratio SD p-value DescriptionYOR331C 0.6773 0.0432 0.0105DubiousFopenFreadingFframeFunlikelyFtoFencodeFaFprotein,FbasedFonFavailableFexperimentalFandFcomparaTveFsequenceFdata;FopenFreadingFframeFoverlapsFtheFverifiedFgeneFVMA4/YOR332WTHP1 0.6846 0.0192 0.0027NuclearFporecassociatedFprotein;FcomponentFofFTREXc2FcomplexF(Sac3pcThp1pcSus1pcCdc31p)FinvolvedFinFtranscripTonFelongaTonFandFmRNAFexportFfromFtheFnucleus;FinvolvedFinFpostctranscripTonalFtetheringFofFacTveFgenesFtoFtheFnuclearFperipheryFandFtoFnoncnascentFmRNP;FcontainsFaFPAMFdomainFimplicatedFinFproteincproteinFbindingSHP1 0.6881 0.0307 0.0066UBXF(ubiquiTnFregulatoryFX)FdomainccontainingFproteinFthatFregulatesFGlc7pFphosphataseFacTvityFandFinteractsFwithFCdc48p;FinteractsFwithFubiquitylatedFproteinsFin#vivoFandFisFrequiredFforFdegradaTonFofFaFubiquitylatedFmodelFsubstratePMR1 0.6902 0.0766 0.0253HighFaffinityFCa2+/Mn2+FPctypeFATPaseFrequiredFforFCa2+FandFMn2+FtransportFintoFGolgi;FinvolvedFinFCa2+FdependentFproteinFsorTngFandFprocessing;FmutaTonsFinFhumanFhomologFATP2C1FcauseFacantholyTcFskinFcondiTonFHaileycHaileyFdiseaseBIK1 0.6903 0.0840 0.0370Microtubule-associated protein, component of the interface between microtubules and kinetochore, involved in sister chromatid separation; essential in polyploid cells but not in haploid or diploid cells; ortholog of mammalian CLIP-170YJR018W 0.7057 0.0628 0.0231DubiousFopenFreadingFframeFunlikelyFtoFencodeFaFfuncTonalFprotein,FbasedFonFavailableFexperimentalFandFcomparaTveFsequenceFdataVMA16 0.7098 0.0569 0.0401SubunitFc''FofFtheFvacuolarFATPase,FwhichFfuncTonsFinFacidificaTonFofFtheFvacuole;FoneFofFthreeFproteolipidFsubunitsFofFtheFV0FdomainVMA2 0.7155 0.0417 0.0224SubunitFBFofFtheFeightcsubunitFV1FperipheralFmembraneFdomainFofFtheFvacuolarFH+cATPaseF(VcATPase),FanFelectrogenicFprotonFpumpFfoundFthroughoutFtheFendomembraneFsystem;FcontainsFnucleoTdeFbindingFsites;FalsoFdetectedFinFtheFcytoplasmYOR331C 0.7177 0.0419 0.0145DubiousFopenFreadingFframeFunlikelyFtoFencodeFaFprotein,FbasedFonFavailableFexperimentalFandFcomparaTveFsequenceFdata;FopenFreadingFframeFoverlapsFtheFverifiedFgeneFVMA4/YOR332WPFK1 0.7206 0.0554 0.0047AlphaFsubunitFofFheterooctamericFphosphofructokinaseFinvolvedFinFglycolysis,FindispensableFforFanaerobicFgrowth,FacTvatedFbyFfructosec2,6cbisphosphateFandFAMP,FmutaTonFinhibitsFglucoseFinducTonFofFcellFcyclecrelatedFgenesSPT10 0.7210 0.0468 0.0197PutaTveFhistoneFacetylaseFwithFaFroleFinFtranscripTonalFsilencing,FsequencecspecificFacTvatorFofFhistoneFgenes,FbindsFspecificallyFandFcooperaTvelyFtoFpairsFofFUASFelementsFinFcoreFhistoneFpromoters,FfuncTonsFatForFnearFtheFTATAFboxCTK2 0.7233 0.0633 0.0145BetaFsubunitFofFCcterminalFdomainFkinaseFIF(CTDKcI),FwhichFphosphorylatesFbothFRNAFpolFIIFsubunitFRpo21pFtoFaffectFtranscripTonFandFprecmRNAF3'FendFprocessing,FandFribosomalFproteinFRps2pFtoFincreaseFtranslaTonalFfidelityHUR1 0.7237 0.0667 0.0258ProteinFofFunknownFfuncTon;FreportedFnullFmutantFphenotypeFofFhydroxyureaFsensiTvityFmayFbeFdueFtoFeffectsFonFoverlappingFPMR1FgeneLSM7 0.7324 0.0428 0.0124LsmF(LikeFSm)Fprotein;FpartFofFheteroheptamericFcomplexesF(Lsm2pc7pFandFeitherFLsm1pForF8p):FcytoplasmicFLsm1pFcomplexFinvolvedFinFmRNAFdecay;FnuclearFLsm8pFcomplexFpartFofFU6FsnRNPFandFpossiblyFinvolvedFinFprocessingFtRNA,FsnoRNA,FandFrRNAPMR1 0.7340 0.0533 0.0184HighFaffinityFCa2+/Mn2+FPctypeFATPaseFrequiredFforFCa2+FandFMn2+FtransportFintoFGolgi;FinvolvedFinFCa2+FdependentFproteinFsorTngFandFprocessing;FmutaTonsFinFhumanFhomologFATP2C1FcauseFacantholyTcFskinFcondiTonFHaileycHaileyFdiseaseGAL11 0.7390 0.0405 0.0196SubunitFofFtheFRNAFpolymeraseFIIFmediatorFcomplex;FassociatesFwithFcoreFpolymeraseFsubunitsFtoFformFtheFRNAFpolymeraseFIIFholoenzyme;FaffectsFtranscripTonFbyFacTngFasFtargetFofFacTvatorsFandFrepressors;FformsFpartFofFtheFtailFdomainFofFmediator93GeneRatio (Exp/Ctrl) Ratio SD p-value DescriptionKSP1 0.7396 0.0351 0.0127Ser/thrFproteinFkinase;FassociatesFwithFTORC1FandFlikelyFinvolvedFinFTORFsignalingFcascades;FnegaTveFregulatorFofFautophagy;FnuclearFtranslocaTonFrequiredFforFhaploidFfilamentousFgrowth;FregulatesFfilamentousFgrowthFinducedFnuclearFtranslocaTonFofFBcy1p,FFus3p,FandFSks1p;FoverproducTonFcausesFallelecspecificFsuppressionFofFprp20c10AFT1 0.7421 0.0563 0.0040TranscripTonFfactorFinvolvedFinFironFuTlizaTonFandFhomeostasis;FbindsFtheFconsensusFsiteFPyPuCACCCPuFandFacTvatesFtheFexpressionFofFtargetFgenesFinFresponseFtoFchangesFinFironFavailability;FinFironcrepleteFcondiTonsFacTvityFisFnegaTvelyFregulatedFbyFGrx3p,FGrx4p,FandFFra2p,FwhichFregulateFAm1pFtranslocaTonFfromFtheFnucleusFtoFtheFcytoplasmDYN1 0.7487 0.0391 0.0180Cytoplasmic heavy chain dynein, microtubule motor protein, required for anaphase spindle elongation; involved in spindle assembly, chromosome movement, and spindle orientation during cell division, targeted to microtubule tips by Pac1pBUB3 0.7521 0.0411 0.0175KinetochoreFcheckpointFWD40FrepeatFproteinFthatFlocalizesFtoFkinetochoresFduringFprophaseFandFmetaphase,FdelaysFanaphaseFinFtheFpresenceFofFunanachedFkinetochores;FformsFcomplexesFwithFMad1pcBub1pFandFwithFCdc20p,FbindsFMad2pFandFMad3pLSM6 0.7601 0.0238 0.0061LsmF(LikeFSm)Fprotein;FpartFofFheteroheptamericFcomplexesF(Lsm2pc7pFandFeitherFLsm1pForF8p):FcytoplasmicFLsm1pFcomplexFinvolvedFinFmRNAFdecay;FnuclearFLsm8pFcomplexFpartFofFU6FsnRNPFandFpossiblyFinvolvedFinFprocessingFtRNA,FsnoRNA,FandFrRNAGET1 0.7623 0.0422 0.0205SubunitFofFtheFGETFcomplex;FinvolvedFinFinserTonFofFproteinsFintoFtheFERFmembrane;FrequiredFforFtheFretrievalFofFHDELFproteinsFfromFtheFGolgiFtoFtheFERFinFanFERD2FdependentFfashionFandFforFnormalFmitochondrialFmorphologyFandFinheritanceSSA4 0.7640 0.0634 0.0271HeatFshockFproteinFthatFisFhighlyFinducedFuponFstress;FplaysFaFroleFinFSRPcdependentFcotranslaTonalFproteincmembraneFtargeTngFandFtranslocaTon;FmemberFofFtheFHSP70Ffamily;FcytoplasmicFproteinFthatFconcentratesFinFnucleiFuponFstarvaTonSPO71 0.7643 0.0294 0.0110MeiosiscspecificFproteinFofFunknownFfuncTon,FrequiredFforFsporeFwallFformaTonFduringFsporulaTon;FdispensableFforFbothFnuclearFdivisionsFduringFmeiosisSFG1 0.7710 0.0677 0.0435NuclearFprotein,FputaTveFtranscripTonFfactorFrequiredFforFgrowthFofFsuperficialFpseudohyphaeF(whichFdoFnotFinvadeFtheFagarFsubstrate)FbutFnotFforFinvasiveFpseudohyphalFgrowth;FmayFactFtogetherFwithFPhd1p;FpotenTalFCdc28pFsubstrateKAR3 0.7714 0.0576 0.0314MinuscendcdirectedFmicrotubuleFmotorFthatFfuncTonsFinFmitosisFandFmeiosis,FlocalizesFtoFtheFspindleFpoleFbodyFandFlocalizaTonFisFdependentFonFfuncTonalFCik1p,FrequiredFforFnuclearFfusionFduringFmaTng;FpotenTalFCdc28pFsubstrateMDM32 0.7722 0.0516 0.0316MitochondrialFinnerFmembraneFproteinFwithFsimilarityFtoFMdm31p,FrequiredFforFnormalFmitochondrialFmorphologyFandFinheritance;FinteractsFgeneTcallyFwithFMMM1,FMDM10,FMDM12,FandFMDM34HUR1 0.7723 0.0226 0.0027ProteinFofFunknownFfuncTon;FreportedFnullFmutantFphenotypeFofFhydroxyureaFsensiTvityFmayFbeFdueFtoFeffectsFonFoverlappingFPMR1FgeneCTK2 0.7727 0.0378 0.0194BetaFsubunitFofFCcterminalFdomainFkinaseFIF(CTDKcI),FwhichFphosphorylatesFbothFRNAFpolFIIFsubunitFRpo21pFtoFaffectFtranscripTonFandFprecmRNAF3'FendFprocessing,FandFribosomalFproteinFRps2pFtoFincreaseFtranslaTonalFfidelitySRB2 0.7729 0.0169 0.0036SubunitFofFtheFRNAFpolymeraseFIIFmediatorFcomplex;FassociatesFwithFcoreFpolymeraseFsubunitsFtoFformFtheFRNAFpolymeraseFIIFholoenzyme;FgeneralFtranscripTonFfactorFinvolvedFinFtelomereFmaintenanceVPS1 0.7740 0.0271 0.0112DynaminclikeFGTPaseFrequiredFforFvacuolarFsorTng;FalsoFinvolvedFinFacTnFcytoskeletonForganizaTon,Fendocytosis,FlateFGolgicretenTonFofFsomeFproteins,FregulaTonFofFperoxisomeFbiogenesisLSM1 0.7740 0.0646 0.0344LsmF(LikeFSm)Fprotein;FformsFheteroheptamericFcomplexF(withFLsm2p,FLsm3p,FLsm4p,FLsm5p,FLsm6p,FandFLsm7p)FinvolvedFinFdegradaTonFofFcytoplasmicFmRNAs94GeneRatio (Exp/Ctrl) Ratio SD p-value DescriptionPML39 0.7746 0.0329 0.0120ProteinFrequiredFforFnuclearFretenTonFofFunsplicedFprecmRNAsFalongFwithFMlp1pFandFPml1p;FanchoredFtoFnuclearFporeFcomplexFviaFMlp1pFandFMlp2p;FfoundFwithFtheFsubsetFofFnuclearFporesFfarthestFfromFtheFnucleolus;FmayFinteractFwithFribosomesCTK2 0.7790 0.0415 0.0231BetaFsubunitFofFCcterminalFdomainFkinaseFIF(CTDKcI),FwhichFphosphorylatesFbothFRNAFpolFIIFsubunitFRpo21pFtoFaffectFtranscripTonFandFprecmRNAF3'FendFprocessing,FandFribosomalFproteinFRps2pFtoFincreaseFtranslaTonalFfidelityCTK2 0.7807 0.0238 0.0062BetaFsubunitFofFCcterminalFdomainFkinaseFIF(CTDKcI),FwhichFphosphorylatesFbothFRNAFpolFIIFsubunitFRpo21pFtoFaffectFtranscripTonFandFprecmRNAF3'FendFprocessing,FandFribosomalFproteinFRps2pFtoFincreaseFtranslaTonalFfidelityMXR1 0.7840 0.0184 0.0007MethioninecScsulfoxideFreductase,FinvolvedFinFtheFresponseFtoFoxidaTveFstress;FprotectsFironcsulfurFclustersFfromFoxidaTveFinacTvaTonFalongFwithFMXR2;FinvolvedFinFtheFregulaTonFofFlifespanCTK2 0.7866 0.0069 0.0001BetaFsubunitFofFCcterminalFdomainFkinaseFIF(CTDKcI),FwhichFphosphorylatesFbothFRNAFpolFIIFsubunitFRpo21pFtoFaffectFtranscripTonFandFprecmRNAF3'FendFprocessing,FandFribosomalFproteinFRps2pFtoFincreaseFtranslaTonalFfidelityYJL175W 0.7888 0.0615 0.0486DubiousFopenFreadingFframeFunlikelyFtoFencodeFaFfuncTonalFprotein;FdeleTonFconfersFresistanceFtoFcisplaTn,FhypersensiTvityFtoF5cfluorouracil,FandFgrowthFdefectFatFhighFpHFwithFhighFcalcium;FoverlapsFgeneFforFSWI3FtranscripTonFfactorLAA1 0.7901 0.0276 0.0060APc1FaccessoryFprotein;FcolocalizesFwithFclathrinFtoFtheFlatecGolgiFapparatus;FinvolvedFinFTGNcendosomeFtransport;FphysicallyFinteractsFwithFAPc1;FsimilarFtoFtheFmammalianFp200;FmayFinteractFwithFribosomes;FYJL207CFisFaFnoncessenTalFgeneHKR1 0.7937 0.0288 0.0163MucinFfamilyFmemberFthatFfuncTonsFasFanFosmosensorFinFtheFSho1pcmediatedFHOGFpathwayFwithFMsb2p;FproposedFtoFbeFaFnegaTveFregulatorFofFfilamentousFgrowth;FmutantFdisplaysFdefectsFinFbetac1,3FglucanFsynthesisFandFbudFsiteFselecTonUMP1 0.7938 0.0098 0.0027ShortclivedFchaperoneFrequiredFforFcorrectFmaturaTonFofFtheF20SFproteasome;FmayFinhibitFprematureFdimerizaTonFofFproteasomeFhalfcmers;FdegradedFbyFproteasomeFuponFcompleTonFofFitsFassemblySPA2 0.7954 0.0361 0.0156ComponentFofFtheFpolarisome,FwhichFfuncTonsFinFacTnFcytoskeletalForganizaTonFduringFpolarizedFgrowth;FactsFasFaFscaffoldFforFMkk1pFandFMpk1pFcellFwallFintegrityFsignalingFcomponents;FpotenTalFCdc28pFsubstrateACA1 0.7960 0.0328 0.0113BasicFleucineFzipperF(bZIP)FtranscripTonFfactorFofFtheFATF/CREBFfamily,FmayFregulateFtranscripTonFofFgenesFinvolvedFinFuTlizaTonFofFnoncopTmalFcarbonFsourcesYOR314W 0.7977 0.0400 0.0274DubiousFopenFreadingFframeFunlikelyFtoFencodeFaFprotein,FbasedFonFavailableFexperimentalFandFcomparaTveFsequenceFdataCTF4 0.8036 0.0244 0.0139ChromaTncassociatedFprotein,FrequiredFforFsisterFchromaTdFcohesion;FinteractsFwithFDNAFpolymeraseFalphaF(Pol1p)FandFmayFlinkFDNAFsynthesisFtoFsisterFchromaTdFcohesionMSS11 0.8038 0.0541 0.0324TranscripTonFfactorFinvolvedFinFregulaTonFofFinvasiveFgrowthFandFstarchFdegradaTon;FcontrolsFtheFacTvaTonFofFMUC1FandFSTA2FinFresponseFtoFnutriTonalFsignalsSPC2 0.8065 0.0368 0.0148SubunitFofFsignalFpepTdaseFcomplexF(Spc1p,FSpc2p,FSpc3p,FSec11p),FwhichFcatalyzesFcleavageFofFNcterminalFsignalFsequencesFofFproteinsFtargetedFtoFtheFsecretoryFpathway;FhomologousFtoFmammalianFSPC25GEP4 0.8077 0.0357 0.0234MitochondrialFphosphaTdylglycerophosphataseF(PGPFphosphatase),FdephosphorylatesFphosphaTdylglycerolphosphateFtoFgenerateFphosphaTdylglycerol,FanFessenTalFstepFduringFcardiolipinFbiosynthesis;FnullFmutantFisFsensiTveFtoFtunicamycin,FDTTVPH1 0.8103 0.0399 0.0266SubunitFaFofFvacuolarcATPaseFV0Fdomain,FoneFofFtwoFisoformsF(Vph1pFandFStv1p);FVph1pFisFlocatedFinFVcATPaseFcomplexesFofFtheFvacuoleFwhileFStv1pFisFlocatedFinFVcATPaseFcomplexesFofFtheFGolgiFandFendosomesYGL109W 0.8135 0.0215 0.0069DubiousFopenFreadingFframeFunlikelyFtoFencodeFaFprotein,FbasedFonFavailableFexperimentalFandFcomparaTveFsequenceFdata;FoverlapsFtheFuncharacterizedFgeneFYGL108C95GeneRatio (Exp/Ctrl) Ratio SD p-value DescriptionTRS65 0.8152 0.0478 0.0357SubunitFofFTRAPPII,FaFmulTmericFguanineFnucleoTdecexchangeFfactorFforFYpt1p;FinvolvedFinFintracGolgiFtrafficFandFtheFretrogradeFpathwayFfromFtheFendosomeFtoFGolgi;FroleFinFcellFwallFbetacglucanFbiosynthesisFandFtheFstressFresponseBIM1 0.8155 0.0546 0.0469Microtubule-binding protein that together with Kar9p makes up the cortical microtubule capture site and delays the exit from mitosis when the spindle is oriented abnormallyCTK2 0.8182 0.0399 0.0246BetaFsubunitFofFCcterminalFdomainFkinaseFIF(CTDKcI),FwhichFphosphorylatesFbothFRNAFpolFIIFsubunitFRpo21pFtoFaffectFtranscripTonFandFprecmRNAF3'FendFprocessing,FandFribosomalFproteinFRps2pFtoFincreaseFtranslaTonalFfidelityWSC4 0.8186 0.0499 0.0415ERFmembraneFproteinFinvolvedFinFtheFtranslocaTonFofFsolubleFsecretoryFproteinsFandFinserTonFofFmembraneFproteinsFintoFtheFERFmembrane;FmayFalsoFhaveFaFroleFinFtheFstressFresponseFbutFhasFonlyFparTalFfuncTonalFoverlapFwithFWSC1c3ASF1 0.8200 0.0312 0.0221NucleosomeFassemblyFfactor,FinvolvedFinFchromaTnFassemblyFandFdisassembly,FanTcsilencingFproteinFthatFcausesFderepressionFofFsilentFlociFwhenFoverexpressed;FplaysFaFroleFinFregulaTngFTy1FtransposiTonSEC66 0.8218 0.0294 0.0176NoncessenTalFsubunitFofFSec63FcomplexF(Sec63p,FSec62p,FSec66pFandFSec72p);FwithFSec61Fcomplex,FKar2p/BiPFandFLhs1pFformsFaFchannelFcompetentFforFSRPcdependentFandFpostctranslaTonalFSRPcindependentFproteinFtargeTngFandFimportFintoFtheFERCRH1 0.8270 0.0387 0.0152ChiTnFtransglycosylaseFthatFfuncTonsFinFtheFtransferFofFchiTnFtoFbeta(1c6)FandFbeta(1c3)FglucansFinFtheFcellFwall;FsimilarFandFfuncTonallyFredundantFtoFUtr2;FlocalizesFtoFsitesFofFpolarizedFgrowth;FexpressionFinducedFbyFcellFwallFstressERV14 0.8273 0.0581 0.0486ProteinFlocalizedFtoFCOPIIccoatedFvesicles,FinvolvedFinFvesicleFformaTonFandFincorporaTonFofFspecificFsecretoryFcargo;FrequiredFforFtheFdeliveryFofFbudcsiteFselecTonFproteinFAxl2pFtoFcellFsurface;FrelatedFtoFDrosophilaFcornichonMEI4 0.8290 0.0418 0.0246MeiosiscspecificFproteinFinvolvedFinFdoublecstrandFbreakFformaTonFduringFmeioTcFrecombinaTon;FrequiredFforFchromosomeFsynapsisFandFproducTonFofFviableFsporesAIF1 0.8299 0.0279 0.0156MitochondrialFcellFdeathFeffectorFthatFtranslocatesFtoFtheFnucleusFinFresponseFtoFapoptoTcFsTmuli,FhomologFofFmammalianFApoptosiscInducingFFactor,FputaTveFreductaseRAD1 0.8314 0.0121 0.0017SinglecstrandedFDNAFendonucleaseF(withFRad10p),FcleavesFsinglecstrandedFDNAFduringFnucleoTdeFexcisionFrepairFandFdoublecstrandFbreakFrepair;FsubunitFofFNucleoTdeFExcisionFRepairFFactorF1F(NEF1);FhomologFofFhumanFXPFFproteinECM21 0.8360 0.0536 0.0489ProteinFinvolvedFinFregulaTngFtheFendocytosisFofFplasmaFmembraneFproteins;FidenTfiedFasFaFsubstrateFforFubiquiTnaTonFbyFRsp5pFandFdeubiquiTnaTonFbyFUbp2p;FpromoterFcontainsFseveralFGcn4pFbindingFelementsTOP1 0.8361 0.0393 0.0331TopoisomeraseFI,FnuclearFenzymeFthatFrelievesFtorsionalFstrainFinFDNAFbyFcleavingFandFrecsealingFtheFphosphodiesterFbackbone;FrelaxesFbothFposiTvelyFandFnegaTvelyFsupercoiledFDNA;FfuncTonsFinFreplicaTon,FtranscripTon,FandFrecombinaTonPSD1 0.8371 0.0040 0.0001PhosphaTdylserineFdecarboxylaseFofFtheFmitochondrialFinnerFmembrane,FconvertsFphosphaTdylserineFtoFphosphaTdylethanolamineYOR008CcA 0.8402 0.0517 0.0454PutaTveFproteinFofFunknownFfuncTon,FincludesFaFpotenTalFtransmembraneFdomain;FdeleTonFresultsFinFslightlyFlengthenedFtelomeresVPS4 0.8412 0.0040 0.0001AAAcATPaseFinvolvedFinFmulTvesicularFbodyF(MVB)FproteinFsorTng,FATPcboundFVps4pFlocalizesFtoFendosomesFandFcatalyzesFESCRTcIIIFdisassemblyFandFmembraneFrelease;FATPaseFacTvityFisFacTvatedFbyFVta1p;FregulatesFcellularFsterolFmetabolism96Table 2.4 Aggravating genetic interactions identified in the ?scs2 SGA grouped according to gene ontology term, related to Figure 2.11 and Figure 2.12.COMPONENTGO Term Enrichment P-value Gene(s) annotated to the termcytoplasmic microtubule9/197.08E-08BIM1/YER016W, DYN1/YKR054C, NDL1/YLR254C, CIK1/YMR198W, JNM1/YMR294W, DYN3/YMR299C, PAC1/YOR269W, NIP100/YPL174C, KAR3/YPR141Cmicrotubule associated complex5/150.00529DYN1/YKR054C, CIK1/YMR198W, JNM1/YMR294W, DYN3/YMR299C, NIP100/YPL174Cspindle pole body 7/180.00076BIK1/YCL029C, BIM1/YER016W, DYN1/YKR054C, CIK1/YMR198W, JNM1/YMR294W, CNM67/YNL225C, NIP100/YPL174C, KAR3/YPR141Cmicrotubule cytoskeleton11/550.0000265BIK1/YCL029C, BIM1/YER016W, DYN1/YKR054C, NDL1/YLR254C, CIK1/YMR198W, JNM1/YMR294W, DYN3/YMR299C, CNM67/YNL225C, PAC1/YOR269W, NIP100/YPL174C, KAR3/YPR141CFUNCTIONGO Term Enrichment P-value Gene(s) annotated to the termMicrotuble Plus End Binding3/3 0.00217BIM1/YER016W, NDL1/YLR254C, PAC1/YOR269WTubulin Binding 6/19 0.00080 BIK1/YCL029C, NUM1/YDR150W, BIM1/YER016W, NDL1/YLR254C, PAC1/YOR269W, NIP100/YPL174CCytoskeletal Protein Binding8/45 0.00287 BIK1/YCL029C, NUM1/YDR150W, BIM1/YER016W, PEA2/YER149C, SPA2/YLL021W, NDL1/YLR254C, PAC1/YOR269W, NIP100/YPL174CPROCESSGO Term Enrichment P-value Gene(s) annotated to the termNuclear Migration along Microtubule9/16 3.45E-08 BIK1/YCL029C, NUM1/YDR150W, BIM1/YER016W, DYN1/YKR054C, NDL1/YLR254C, CIK1/YMR198W, DYN3/YMR299C, PAC1/YOR269W, KAR3/YPR141CEstablishment of Organelle Localization13/408.87E-09SHP1/YBL058W, BIK1/YCL029C, NUM1/YDR150W, BIM1/YER016W, DYN1/YKR054C, NDL1/YLR254C, CIK1/YMR198W, JNM1/YMR294W, DYN3/YMR299C, TOP1/YOL006C, PAC1/YOR269W, NIP100/YPL174C, KAR3/YPR141CMitotic Sister Chromatid Segregation10/450.00013SHP1/YBL058W, CSM1/YCR086W, BIM1/YER016W, AFT1/YGL071W, CTF8/YHR191C, DYN1/YKR054C, CIK1/YMR198W, TOP1/YOL006C, CTF4/YPR135W, KAR3/YPR141CMicrotubule Cytoskeleton Organization10/480.00024SHP1/YBL058W, BIK1/YCL029C, NUM1/YDR150W, BIM1/YER016W, SWE1/YJL187C, DYN1/YKR054C, CIK1/YMR198W, JNM1/YMR294W, CNM67/YNL225C, NIP100/YPL174COrganelle Fission 14/810.0000131SHP1/YBL058W, BIK1/YCL029C, CSM1/YCR086W, NUM1/YDR150W, BIM1/YER016W, AFT1/YGL071W, CTF8/YHR191C, VPS1/YKR001C, DYN1/YKR054C, CIK1/YMR198W, TOP1/YOL006C, BUB3/YOR026W, CTF4/YPR135W, KAR3/YPR141CNuclear Division 12/730.00025SHP1/YBL058W, BIK1/YCL029C, CSM1/YCR086W, BIM1/YER016W, AFT1/YGL071W, CTF8/YHR191C, DYN1/YKR054C, CIK1/YMR198W, TOP1/YOL006C, BUB3/YOR026W, CTF4/YPR135W, KAR3/YPR141CMitosis 12/720.00021SHP1/YBL058W, BIK1/YCL029C, CSM1/YCR086W, BIM1/YER016W, AFT1/YGL071W, CTF8/YHR191C, DYN1/YKR054C, CIK1/YMR198W, TOP1/YOL006C, BUB3/YOR026W, CTF4/YPR135W, KAR3/YPR141C973 Chapter 3: Plasma Membrane-Endoplasmic Reticulum Contact Sites Regulate Phosphatidylcholine Synthesis3.1 IntroductionThe structure of the ER of Saccharomyces cerevisiae is somewhat unique amongst eukaryotes, in that its reticular network, a characteristic of all eukaryotic cells, lies just beneath the PM. Reconstruction of total yeast ER in individual cells by 3D electron tomography (West et al., 2011) has revealed that PM-associated ER (pmaER) consists of ER tubules and flattened fenestrated ER sheets that are in close apposition to the cytosolic leaflet of the PM. Regions of pmaER that are apposed to the PM are devoid of ribosomes (West et al., 2011), consistent with these being sites of physical contact between ER and PM (Pichler et al., 2001). PM-ER contacts likely play important roles in all eukaryotic cells (Levine, 2004) and in yeast, pmaER is enriched in lipid synthesizing enzymes (Pichler et al., 2001), suggesting PM-ER contacts are important for lipid metabolism. Protein families with lipid related functions have been found specifically localized at PM-ER contact sites (Loewen et al., 2003; Schulz et al., 2009; Toulmay and Prinz, 2012), where they are thought to play roles in non-vesicular lipid transport between PM and ER, although the role for the contacts themselves has not been directly demonstrated. Recently, it has been found that localization of the integral ER phosphatidylinositol phosphate phosphatase, Sac1, to PM-ER contacts regulates phosphatidylinositol 4-phosphate (PI4P) levels in the PM in trans (Stefan et al., 2011). In trans catalysis by Sac1 requires interaction with Osh3, a soluble lipid binding protein of the oxysterol binding protein (OSBP) family that localizes to PM-ER contacts (Stefan et 98al., 2011). Consistent with a role for contacts in regulating Sac1, a mutant with reduced pmaER has increased levels of PI4P (Manford et al., 2012). We previously found that two genes with roles in ER biogenesis in yeast, SCS2 and ICE2, exhibit an aggravating genetic interaction, and ?scs2?ice2 double mutants have reduced pmaER, suggesting that pmaER performs an essential function required for cell growth (Loewen et al., 2007). Ice2 is an integral ER protein of unknown function that is required for inheritance of pmaER (Estrada de Martin et al., 2005). Scs2 is a highly conserved tail-anchored protein of the VAP family that localizes proteins containing FFAT motifs to the ER (Loewen et al., 2003) and regulates yeast phospholipid synthesis (Loewen et al., 2004). Scs2 localizes the OSBP homologues Osh2 and Osh3 to pmaER through interaction with the FFAT motifs in these proteins (Loewen et al., 2003; Schulz et al., 2009), which is required for regulation of in trans Sac1 activity (Stefan et al., 2011).3.2 Methods3.2.1 Plasmids and yeast strainsThe pGAL-OSH1, OSH2 and OSH3 multicopy plasmids (pOSH1, -2, -3) used in this study were a kind gift from C. Beh (Kozminski et al., 2006) and express the native untagged protein under control of the galactose promoter (GAL1-10). pGST-PAH1 was isolated from the GST-ORF yeast array (Sopko et al., 2006) and confirmed by sequencing. It expresses Pah1 tagged at the N-terminus under control of the galactose promoter. HA- tagged Pah1 (pPAH1) and the catalytically dead D398E variant (pPAH1 99D398E; containing the D398E mutation in the Pah1 coding sequence) were a kind gift from G. Carman (Han et al., 2007). The pOPI3 plasmid is based on pRS416 (CEN, URA3) (Sikorski and Hieter, 1989) and expresses the Opi3 protein with an N-terminal myc tag (MEQKLISEEDL) under control of the constitutive portion of the PHO5 promoter and was constructed by amplifying the OPI3 coding sequence from yeast genomic DNA and cloning the PCR product into the Bgl2 (5?) and SacI (3?) restriction sites. pSCS2 was constructed as pOPI3 except that the SCS2 gene was inserted. pCHO2 was obtained from the MoBY-ORF collection (Ho et al., 2009) and contains the CHO2 gene with its endogenous promoter and terminator cloned into a centromere-based vector. RFP-ER contains the C- terminal transmembrane domain of Scs2 fused to monomeric DsRed in pRS416 (CEN, URA) under control of the PHO5 promoter. All yeast strains are based on S288C. Deletion strains were obtained from freezer stocks of the haploid yeast deletion collection (BY4741, MAT a, KanMX), a gift from C. Boone. All deletion strains were confirmed by PCR. GFP-tagging of endogenous proteins was done by homologous recombination of PCR-generated fragments in haploids at the C-terminus of the endogenous protein using the pKT128 (SpHIS5) plasmid (Sheff and Thorn, 2004) in the BY7043 background (Tong and Boone, 2006). Gene deletion strains were constructed in BY4741 using PCR-generated fragments from pKT127 (KanMX) (Sheff and Thorn, 2004) or in BY7092 using p4339 (NatR) (Tong and Boone, 2006). Double and triple deletion strains were generated by standard yeast genetic techniques.1003.2.2 Yeast growth assays10- fold serial dilutions of log phase cells were spotted using a pin-frogger (Sigma) onto agar plates containing synthetic defined (SD) media with the appropriate amino acid dropouts and either 2% glucose or 2% galactose as indicated. Ethanolamine, choline or monomethylethanolamine were added to SD media to a final concentration of 1 mM.All growth assays were performed at 30?C for at 24-48 hours.3.2.3 Array-based genome-wide suppressor screenThe array-based genome-wide suppressor screen for the ?scs2?ice2 mutant was performed by modifying the synthetic genetic array (SGA) method (Tong and Boone, 2006) and using the GST-ORF collection (Open Biosystems) in place of the deletion mutant array (Sopko et al., 2006). GST-ORFs are expressed under control of the GAL1/10 promoter and tagged at the N-terminus with GST.The GST-ORF collection was  arrayed using a RoToR HDA robot (Singer Instruments) at a density of 1536 spots per plate onto SD media containing glucose. This array was crossed with a ?scs2::NatMX ?ice2::KanMX mutant query strain (Y7092 background) grown on SD media containing choline and diploids were selected on medium also containing choline prior to sporulation for 5 days. Haploid ?scs2?ice2 double mutant progeny containing the GST-ORF plasmids were recovered on media lacking His, Arg, Lys, and containing canavanine, thialysine, G418, and Nat. The final pinning step was to media containing 2% galactose and lacking choline. Suppressors were identified by visual inspection of plates and GST-ORF plasmids were recovered and sequenced to confirm the identity of the suppressors.1013.2.4 In vivo methylation assayTo monitor conversion of PE to PC, 100 ml of cells were grown in SD media containing either 2% raffinose and 2% galactose or 2% dextrose to an OD600 of 0.5. For the experiments shown in Fig. 3.1 G and Fig. 3.2 D, SD media was supplemented with 1 mM choline to support growth of the ?cho2 and ?scs2?ice2 mutants, otherwise yeast were grown in the absence of choline. Cells were collected by centrifugation, washed once in 0.67% Yeast Nitrogen Base (YNB) then resuspended in 12 ml YNB + 2% raffinose, 2% galactose (YNB-RG). After addition of 24 ?Ci of [3H]-ethanolamine, cells were shaken at 37 ?C for 0.5 - 1 h. Cells were then collected by centrifugation, washed once in 1.6 ml YNB-RG, resuspended in 120 mL SD media and shaken at 30 ?C. Aliquots of 20 ml were removed every hour, collected by centrifugation, washed once in 1 ml water, and then frozen for subsequent analysis. After thawing cell pellets and washing once in 1 ml water, cells were resuspended in 200 ?l water and glass beads added to the top of the meniscus. Cells were lysed by three 30 s cycles of agitation in a Precellys 24 tissue homogenizer. The lysate was collected by washing the beads twice with 800 ?l water and transferred to a glass tube. Lipids were extracted by the addition of 6 ml 2:1 methanol:chloroform, and vortexed once, followed by addition of 2 ml of 0.9% w/v NaCl and vortexing once more (Voss et al., 2012). Phase separation was achieved by centrifugation at 1500 g for 1 min. The upper aqueous phase was discarded and the lower organic phase transferred to a clean tube. The solvent was evaporated under a stream of nitrogen and the resultant lipid film resuspended in 60 ?l chloroform containing 2 mg/ml PS/PE/PC standards. Following addition of 140 ?l of HPLC buffer (90:3:1 102acetonitrile:methanol:phosphoric acid), the entire sample was injected into a GE Healthcare AKTA FPLC system and lipids separated on a LiChrospher 60 Si column. Fractions corresponding to the PE and PC peaks were collected and activity determined by scintillation counting.A background reading corresponding to column flow-through between the PE and PC fractions was subtracted from the counts in the PE and PC fractions. PE to PC conversion rates were calculated from the initial linear portion of the assays (minimum of three time points, beginning at t=0 h) and regression analysis was performed using GraphPad Prism software to determine the slopes and the associated error. PC synthesis rates are presented as [PC/(PC+PE)]/h.3.2.5 In vitro Opi3 methylation assayMicrosomes containing Opi3 were isolated from wild type strain D273-10B as described (de Kroon et al., 2003), and pelleted through 20% (w/v) sucrose in buffer (0.6 M sorbitol, 50 mM Tris-HCl pH 7.5) at 130,000 g for 1 h (SW41 rotor, Beckman) before use. [3H]-PME-loaded mitochondria from a ?opi3 strain SH414 prepared as described (de Kroon et al., 2003) were subjected to lipid extraction.The dried mitochondrial lipid extract was hydrated in buffer, and large unilamellar vesicles (liposomes) were obtained by extrusion through 200 nm pore size filters after 10 freeze-thaw cycles. Microsomes at 0.5 mg protein/ml were incubated with [3H]-PME-loaded liposomes at 0.4 mM phospholipid-phosphorous in buffer supplemented with 5 mM S- adenosyl-L-methionine at 30?C for 2 h. Lipids were extracted before or after separating liposomes from microsomes on a 20% (w/v) sucrose cushion in buffer also containing 1 mM EDTA by centrifugation at 130,000 g for 1 h. Recovery of [3H]-labeled lipid was determined by liquid scintillation counting, and the conversion of [3H]-PME was quantitated after thin 103layer chromatography as described (Janssen et al., 2002). Lipid extraction was performed and phospholipids were quantitated (according to (Rouser et al., 1970)). Protein concentrations were determined using the BCA method (Pierce) with 0.1% (w/v) SDS added and BSA as a standard.3.2.6 NBD-PC, NBD-PE and FM4-64 cellular uptake assays The assays were performed essentially as described in (Pomorski et al., 2003). Briefly, stock solutions of NBD-PE and NBD-PC (10 mM; Avanti Polar Lipids) were prepared in DMSO. Log phase cells were pre-incubated at 4?C and NBD-lipids were added to a final concentration of 100 ?M, vortexed thoroughly, and incubated at either 4?C or room temperature for the indicated length of time. After incubation, cells were washed in ice-cold media without glucose, containing 2% sorbitol and 20 mM NaN3. Cells were then mounted on a microscope slide for confocal microscopy. For FM4-64 labeling, log phase cells were incubated either at 4?C or room temperature with 40 ?M FM 4-64 for 15 and 60 minutes. Cells were then washed twice in ice-cold media and mounted on a microscope slide for confocal microscopy.3.3 Results3.3.1 PM-ER contacts play roles in the methylation pathway of phospholipid biosynthesis.To uncover clues about PM-ER contact site function we examined the yeast global genetic interaction network (Costanzo et al., 2010), which is a comprehensive map of pairwise genetic interactions in which genes with similar functions form coherent clusters. We noticed that SCS2 and ICE2 were in a cluster enriched for genes involved 104in phospholipid metabolism (Figure 3.1A). These included genes required to make phosphatidylcholine (PC) by the Kennedy pathway, a salvage pathway that synthesizes PC and PE from the lipid precursors choline, ethanolamine and diacylglycerol (DAG) (Figure 3.1B) (Carman and Han, 2011). This cluster showed predominately aggravating genetic interactions with a cluster containing both PEMT enzymes, encoded by CHO2 and OPI3, which synthesize PC by the methylation pathway (Figures 3.1A & B). Also in this cluster was the Ino2-Ino4 transcription factor, which activates expression of Kennedy and methylation pathway genes (Carman and Han, 2011), as well as ICE2, further suggesting a function for ER-PM contacts in the methylation pathway. This clustering suggested that PM-ER contacts might regulate PC synthesis. We found that choline but not ethanolamine rescued the ?scs2?ice2 slow growth phenotype (Figure 3.1C), indicating that the mutant likely had a PC synthesis defect in the methylation pathway. Overexpression of OPI3 in the ?scs2?ice2 mutant rescued its growth defect (Figure 3.1D), whereas overexpression of CHO2 did not (Figure 3.2), suggesting Opi3 function was compromised. Addition of monomethylethanolamine, which is converted into phosphatidylmonomethylethanolamine (PME) by the Kennedy pathway and bypasses the requirement for Cho2, did not rescue growth of ?scs2?ice2 cells (Figure 3.21) and was consistent with loss of Opi3 function in the mutant.Cho2 performs the first PE methylation, whereas Opi3 is primarily responsible for the second and third methylations (Figure 3.1B). Opi3 can also methylate first, but at a less efficient rate (Kodaki and Yamashita, 1987). Due of this redundancy, ?cho2 and ?opi3 cells grow poorly on media lacking choline, but they are not obligate choline auxotrophs. In contrast, ?cho2?opi3 double mutants are obligate auxotrophs and are 105rescued by overexpression of Opi3, but not Cho2 (Kodaki and Yamashita, 1987). The aggravating genetic interaction between Scs2 and Cho2 and the alleviating interaction between Scs2 and Opi3 observed in the gene cluster (Figure 3.1A), suggested that Scs2 might modify Opi3 function directly. Consistent with Scs2 regulating Opi3, we found that ?scs2?cho2 cells were obligate choline auxotrophs (Figure 3.1E), and overexpression of Scs2 rescued the choline auxotrophy of the ?cho2 mutant (Figure 3.1F). For ICE2, we uncovered a genetic interaction with PSD1 that was rescued by ethanolamine (Figure 3.2), further supporting a role for Ice2 in the methylation pathway as implied by the gene cluster.  To measure Opi3 function in the ?scs2?ice2 mutant, we pulse-labelled cells with [3H]-ethanolamine and monitored conversion of radiolabelled PE into PC. In both wild type and mutant cells, the rate of incorporation of label into PC was linear for at least the first three hours of the assay (Figure 3.2). As expected, we found that the rate of PC synthesis in a ?cho2 mutant was significantly reduced (Figure 3.1G). Consistent with decreased Opi3 function, PC synthesis was also reduced in ?scs2?ice2 (Figure 3.1G). Overexpression of Opi3 increased the rate of PC synthesis back to levels similar to wild type (Figure 3.1H & Figure 3.2). Thus, reduced synthesis of PC in the ?scs2?ice2 mutant likely accounted for the choline auxotrophy of the mutant. Finally, the ?scs2?ice2 mutant was sensitive to dithiothreitol (Figure 3.1I), indicating increased ER stress, also consistent with decreased Opi3 function (Thibault et al., 2012).106107Figure 3.1 RER1  SCS3  INO2  SCS2  YET1  ICE2  DGK1  SEY1  HNM1  PCT1  SPT23  CKI1  SSP2  YOR246C  CHO2  OPI3  ICE2  INO2  INO4 -0.25-0.17-0.08 0.00 0.08 0.17 0.25A?LFH?VFV?VFV?LFH- + Cho + EtnWTCBDWT?VFV?LFH- + pOPI3PS PE PME PDE PCPSD136'OPI3 OPI3&+2Etn(in) Cho(in)PCT1G HCKI1CPT1EKI1EPT1ECT1DAGPAPAH1Kennedy PathwayDGK1Methylation PathwayHNM1Etn(out) Cho(out)HNM1alleviatingno interactionaggravatingWT?FKR- S6&6WT?FKR?VFV?FKR?VFVE FFigure 1Figure 1. Opi3 function is compromised in ?scs2?ice2 cells. (A) Gene clusters containing SCS2 and ICE2 revealed a potential role for PM-ER contacts in phospholipid metabolism. Genes are colour-coded according to pathways in (B). Data was clustered from {Costanzo:2010jl}. (B) Major pathways of phospholipid synthesis in S. cerevisiae. Genes encoding enzymes required for each step are shown in italics. Cho, choline; Etn, ethanolamine; PA, phosphatidic acid; DAG, diacylglycerol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PME, phosphatidylmonomethylethanolamine; PDE, phosphatidyldimethylethanolamine; PC, phosphatidylcholine. (C) Ten-fold serial dilutions of the indicated mutant yeast strains grown on SD media (-) or SD supplemented with either choline (+Cho) or ethanolamine (+Etn). (D) Wild type (WT) and ?scs2?ice2 yeast grown on SD media transformed with either a plasmid expressing Opi3 (+ pOPI3) or a control plasmid (-). (E) Mutant yeast grown on SD media without choline. (F) Yeast grown on SD media transformed with either a plasmid expressing Scs2 (+ pSCS2) or a control plasmid (-). (G) In vivo PE methylation assay. Log phase yeast were pulse-labelled with [3H]-Etn and the rate of PC synthesis was determined by measuring conversion of PE into PC over time. Error bars, SEM. Asterisks, P < 0.005 vs WT. (H) PE methylation assay for indicated strains expressing Opi3 from a plasmid (n.s., not significant vs WT). (I) Yeast grown on SD media containing choline with 1 mM diothiothreitol (+DTT) or without (-).WT?VFV?LFH?KDF- + DTTI00. of PC synth. (/h)Rate of PC synth. (/h)n.s.* **Rate$=$PC/$(PE+PC)$per$* *Figure 3.1 Opi3 Function Is Compromised in ?scs2?ice2 Cells. (A) Gene clusters containing SCS2 and ICE2 revealed a potential role for PM-ER contacts in phospholipid metabolism. Genes are colour-coded according to pathways in Figure 3.1B. Data were clustered from (Costanzo et al., 2010).(B) Major pathways of phospholipid synthesis in S. cerevisiae. Genes encoding enzymes required for each step are shown in italics. Cho, choline; Etn, ethanolamine; PA, phosphatidic acid; DAG, diacylglycerol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PME, phosphatidylmonomethylethanolamine; PDE, phosphatidyldimethylethanolamine; PC, phosphatidylcholine.(C) Ten-fold serial dilutions of the indicated mutant yeast strains grown on SD media (-) or SD supplemented with either choline (+Cho) or ethanolamine (+Etn).(D) Wild type (WT) and ?scs2?ice2 yeast grown on SD media transformed with either a plasmid expressing OPI3 (+ pOPI3) or a control plasmid (-).(E) Mutant yeast grown on SD media without choline.(F) Yeast grown on SD media transformed with either a plasmid expressing SCS2 (+ pSCS2) or a control plasmid (-).(G) In vivo PE methylation assay. Log phase yeast were pulse-labelled with [3H]-Etn and the rate of PC synthesis was determined by measuring conversion of PE into PC over time. Error bars, SEM. Asterisks, p < 0.005 vs WT.(H) PE methylation assay for indicated strains expressing OPI3 from a plasmid (n.s., not significant vs WT). (I) Yeast grown on SD media containing choline with 1 mM diothiothreitol (+DTT) or without (-).108Figure 3.2 (A) Yeast growth assays of mutants containing either a plasmid expressing CHO2 (+pCHO2) or empty vector (-) on SD media lacking choline.(B) Yeast growth assays of mutants on SD media in the absence (-) or presence of 1 mM monomethylethanolamine (+MME).109Figure 3.2(C) Yeast growth assays of indicated mutants on SD media without choline or ethanolamine (-), with choline (+Cho) or with ethanolamine (+Etn).(D) Incorporation of [3H]-ethanolamine into PC over time in wild type, ?cho2 and ?scs2?ice2 yeast grown in the presence of choline. Plotted is the fraction of counts in PC out of total PE+PC counts at each time point.(E) Incorporation of [3H]-ethanolamine into PC over time in wild type and ?scs2?ice2 cells expressing Opi3 from a plasmid grown in the absence of choline. Plotted is the fraction of counts in PC out of total PE+PC counts at each time point. Error bars = SD.3.3.2 The PC methyltransferase, Opi3, functions at ER-PM contactsLoss of Opi3 function in the ?scs2?ice2 mutant suggested that Opi3 functioned at PM-ER contacts. Using an endogenous type I integral ER protein Pho88 tagged with GFP we verified our previous findings that ?scs2?ice2 cells had normal ER tubules and nuclear ER, but greatly diminished ER at the cell cortex (Figure 3.3A). To characterize PM-ER contacts, we used a specific marker that is an integral ER membrane protein that localizes to PM-ER contacts by interacting directly with the PM (Toulmay and Prinz, 2012). As previously found, Tcb3-GFP localized only to the pmaER domain at the cell cortex, but not the nuclear ER or ER tubules (Figure 3.3B). Consistent with 3D electron tomography showing that pmaER forms distinct domains in the bud and mother and is absent from the bud neck (West et al., 2011), Tcb3-GFP was discontinuous through the neck and otherwise localized to pmaER throughout the cell cycle (Figure 3.3C). In the ?scs2?ice2 mutant, Tcb3-GFP localization was clearly disrupted. pmaER defects were pronounced in small and medium-sized buds, in which Tcb3-GFP often failed to localize 110to the bud periphery and instead localized to ER tubules near the centre of the bud (Figure 3.3D). In mothers, Tcb3-GFP also mislocalized to the nuclear ER. Now we examined the localization of the endogenous Opi3 enzyme tagged with GFP, which appeared functional (Figure 3.4B). In wild type cells Opi3-GFP localized throughout the ER (Figure 3.3E). We observed some additional diffuse staining in the vacuole that was likely a result of turnover of Opi3-GFP. In the ?scs2?ice2 mutant, localization of Opi3-GFP to the nuclear ER remained intact, however, it was almost completely absent from pmaER (Figure 3.3E). Quantification of Opi3-GFP in the nuclear ER revealed no change in its expression level (Figure 3.4), indicating that loss of Opi3 function likely resulted from the defect in pmaER in the mutant. Consistent with this, Opi3 overexpression did not appear to rescue pmaER (Figure 3.4C). Scs2 localizes both Osh2 and Osh3 to PM-ER contacts (Loewen et al., 2003), and we tested for their roles in regulating Opi3. Overexpression of Osh2 partially restored growth of the ?scs2?ice2 mutant, whereas Osh3 fully rescued (Figure 3.3F). In contrast, Osh1, which is localized to the nucleus-vacuole junction by Scs2 (Loewen et al., 2003) did not rescue, and in fact impeded growth (Figure 3.3F). Osh3 overexpression restored PC synthesis in the ?scs2?ice2 mutant (Figure 3.3G), consistent with rescue of growth of the mutant. However, Osh3 did not rescue pmaER in ?scs2?ice2 cells (Figure 3.4), but it did restore growth of the ?cho2 mutant (Figure 3.3H), suggesting that Osh3 regulated Opi3 function at contacts.111Figure 3.3 PM-ER Contacts Regulate Opi3. (A) Images of yeast cells expressing endogenous Pho88-GFP. Arrows indicate nuclear ER, arrowheads indicate pmaER, and asterisks indicate absence of pmaER. All scale bars = 2 ?m.112Figure 3.3(B) Yeast expressing endogenous Tcb3-GFP (green) and RFP-ER (red) expressed from a plasmid.(C and D) Yeast expressing Tcb3-GFP staged throughout the cell cycle. Double arrowheads indicate mislocalization of Tcb3-GFP to ER tubules, asterisks indicate absence of pmaER.(E) Yeast expressing endogenous Opi3-GFP. Asterisks indicate vacuoles. (F) Growth of WT and ?scs2?ice2 yeast overexpressing Osh proteins from plasmids grown on SD media containing galactose in the absence (-) or presence (+Cho) of choline.(G) PE methylation assay for indicated strains expressing Osh3 from a plasmid.(H) Growth assays of WT and ?cho2 mutant yeast overexpressing Osh3 from a plasmid grown on SD media containing galactose in the absence of choline.113114Figure 3.4Figure 3.4 (A) Growth assays of WT and Opi3-GFP yeast on SD media lacking choline.(B) Quantification of Opi3-GFP in the nuclear ER of WT and ?scs2?ice2 mutant yeast (n = number of cells; error bars = SD).(C) Images of WT and ?scs2?ice2 yeast cells (transformed with the indicated plasmids) expressing endogenous Tcb3-GFP grown on SD media containing galactose. Asterisks indicate regions with disorganized pmaER in the mutants. Scale bars = 2 ?m.(D) Incorporation of [3H]-ethanolamine into PC over time in wild type and ?scs2?ice2 cells expressing Osh3 from a plasmid grown in the absence of choline. Plotted is the fraction of counts in PC out of total PE+PC counts at each time point. Error bars = SD.3.3.3 Pah1 is a multi-copy suppressor for PC synthesis defects and restores PM-ER contacts in ?scs2?ice2 cellsWe now exploited the choline auxotrophy phenotype of the ?scs2?ice2 mutant in a screen to identify regulators of PM-ER contact structure. We identified a suppressor plasmid carrying the PAH1 gene (Figure 3.5A), which encodes a highly conserved phosphatidic acid (PA) phosphatase enzyme of the lipin family that catalyzes the conversion of PA into DAG in the Kennedy pathway (Figure 3.1B) (Han et al., 2006). Surprisingly, a catalytically inactive mutant of Pah1, D398E (Han et al., 2007), also rescued the choline auxotrophy of the ?scs2?ice2 mutant (Figure 3.5B), arguing against rescue via the Kennedy pathway. Consistent with this, Pah1 still rescued a ?pct1?scs2?ice2 triple mutant with an inactivated Kennedy pathway (Figure 3.5C). Overexpression of Pah1 rescued PC synthesis in the ?scs2?ice2 mutant (Figures 3.5D 115& 3.6A), indicating Pah1 rescued Opi3 function. However, it did not rescue the ?cho2 mutant (Figure 3.6B), suggesting Pah1 did not regulate Opi3 directly. These results suggested that Pah1 might rescue pmaER in the ?scs2?ice2 mutant. In contrast to Opi3 and Osh3, overexpression of GST-Pah1 appeared to restore Tcb3-GFP localization to pmaER (Figure 3.5E), and Pah1 also restored Opi3-GFP to pmaER (Figure 3.6D). Therefore, we performed an ultrastructural analysis of PM-ER contacts using transmission electron microscopy. We quantified ER segment length, frequency, and the overall ratio of PM-ER contacts to PM perimeter (Figures 3.5F and 3.7). In the ?scs2?ice2 mutant we found that contacts were decreased to ~7% in buds and ~ 13% in mothers, compared to ~25% of the cell periphery in wild type (Figure 3.5G). In buds, the decrease resulted from both a decrease in contact site length (Figure 3.5H) and frequency (Figure 3.5I). In mothers, the decrease resulted only from reduced frequency since contact site length was unaffected. Overexpression of both Pah1 and D398E Pah1 in the ?scs2?ice2 mutant restored contacts to near wild type levels in both buds and mothers (Figure 3.5G). Rescue resulted from increased contact length and frequency in buds (Figures 3.5H & I), indicating Pah1 rescued the defect in formation of pmaER in the bud. In wild type cells, Pah1 increased contacts to ~40% of the cell periphery, a result of increased frequency, supporting a physiological role for Pah1 in initiating contacts. Finally, Pah1 rescued the dithiothreitol sensitivity of the ?scs2?ice2 mutant, which did not require activation of the unfolded protein response (Figure 3.6), further supporting that Pah1 directly rescued the defect in PM-ER contacts by a mechanism that was independent of activation of general stress response pathways.116Figure 3.5 Pah1 Regulates PM-ER Contacts. (A & C) Serial dilutions of (A) ?scs2?ice2 and (C) ?scs2?ice2?pct1 yeast overexpressing GST-Pah1 from a plasmid under control of the galactose promoter (+pGST-PAH1) on SD media containing either glucose (Dex) or galactose (Gal).117Figure 3.5(B) Yeast expressing either HA-PAH1 or the catalytically inactive D398E mutant from a plasmid grown on SD media lacking choline.(D) Opi3 methylation assay for wild type and ?scs2?ice2 cells overexpressing GST-PAH1 (n.s., not significant vs WT).(E) Images of Tcb3-GFP in ?scs2?ice2 yeast overexpressing GST-Pah1. Scale bar = 2 ?m.(F) Schematic of ultrastructural assay and representative TEM images illustrating PM-ER contacts (arrows).(G) Ratio of PM-ER contacts to PM perimeter. **, P <10-4 vs WT; *, P <0.001 vs WT; ##, P <0.01 vs ?scs2?ice2, #, P <0.05 vs ?scs2?ice2.(H) PM-ER contact length. *, P < 0.005 vs WT; #, P < 0.005 vs ?scs2?ice2.(I) Frequency of PM-ER contacts. **, P < 0.005 vs WT; *, P <0.05 vs WT; ##, P <0.005 vs ?scs2?ice2; #, P <0.05 vs ?scs2?ice2. Error bars, SEM.118Figure 3.6 (A) Incorporation of [3H]-ethanolamine into PC over time in wild type and ?scs2?ice2 cells expressing GST-Pah1 from a plasmid grown in the absence of choline. Plotted is 119Figure 3.6the fraction of counts in PC out of total PE+PC counts at each time point. Error bars = SD.(B) Growth assays of wild type and ?cho2 yeast expressing GST-Pah1 from a plasmid grown on SD media containing galactose and lacking choline.(C) Yeast grown on SD media with Cho (-) and with 1 mM diothiothreitol (+DTT).(D) Images of ?scs2?ice2 yeast expressing endogenous Opi3-GFP and transformed with a plasmid expressing GST-Pah1 grown on SD medium containing galactose and lacking choline. Arrowheads, Opi3-GFP localizing to the pmaER. Arrows, Opi3-GFP localizing to the nuclear ER. Scale bar = 2 ?m.120Figure 3.7 Representative transmission electron microscopy images of wild type and ?scs2?ice2 yeast expressing GST-Pah1 from a plasmid (+PAH1) grown on SD media containing galactose and choline. pmaER has been indicated on the images by tracing in red and nuclear ER has been traced in blue. n, nucleus; v, vacuole. Scale bars = 500 nm.121Figure Opi3 may function in trans at PM-ER contactsOur results suggest that PM-ER contacts provide a spatial mechanism to regulate synthesis of PC by the yeast PEMT enzyme, Opi3. A consequence of altered Opi3 activity at contacts could be altered PM stability, as has been found in the livers of PEMT -/- mice, which have increased PE:PC in their PM (Li et al., 2006). Yeast mutants with increased PE:PC in the PM are sensitive to nonionic detergents, which destabilize the bilayer (Sch?ller et al., 2007). We found that ?lem3/ros3 cells, which have a buildup of PE in the PM due to decreased PE flippase activity (Kato et al., 2002; Saito et al., 2007), were sensitive to NP-40 (Figure 3.8A). We also found that ?opi3 cells, which have increased PME (Bilgin et al., 2011), were NP-40 sensitive (Figure 3.8B), suggesting that PME accumulated in the PM. The ?scs2?ice2 mutant was similarly NP-40 sensitive, which was suppressed by Opi3 (Figure 3.8C). Importantly, D398E Pah1 also suppressed, consistent with rescue of PM-ER contacts and reconstitution of Opi3 function by Pah1 (Figure 3.8D). Loss of Osh3 also caused NP-40 sensitivity, further supporting its role in regulating Opi3 at contacts (Figure 3.8E). This function for Osh3 was independent of its role in regulating PI4P levels in the PM (Stefan et al., 2011), because ?sac1 cells were insensitive to NP-40 (Figure 3.8E). NP-40 sensitivity was not due to decreased PE flippase activity, because ?scs2?ice2 cells were insensitive to cinnamycin (Ro09-0198), which binds PE located in the outer leaflet of the PM and lyses cells (Kato et al., 2002; Saito et al., 2007), whereas ?lem3/ros3 cells were highly sensitive (Figure 3.8F). Previous work suggested that Opi3 could act in trans in catalyzing the methylation of PME located in a juxtaposed membrane (Janssen et al., 2002). Hence, 122the role for PM-ER contacts might be to provide Opi3 access to PE/PME located in the PM for in trans methylation. We tested this possibility using an in vitro assay. We incubated liposomes containing tritiated PME with ER microsomes containing Opi3 and monitored synthesis of PC (Figure 3.8G). Mixing of liposomes and microsomes resulted in conversion of ~35% of the PME into PDE and PC (Figure 3.8H). Recovery of the liposomal fraction, which contained the majority of the radiolabel (Figure 3.9), revealed a similar lipid distribution with ~30% of the PME converted to PDE and PC (Figure 3.8I). Thus, accumulation of PDE and PC in the liposomal fraction was consistent with in trans Opi3 activity. We did not detect defects in endocytosis or uptake of NBD-PE/PC in the ?scs2?ice2 mutant (Figure 3.9), indicating lipid transport between PM and ER was likely not disrupted, further supporting that Opi3 functioned in trans at contacts.123Figure 3.8 PM-ER Contacts Affect PM Stability in trans. (A-E) Yeast growth assays done in the absence (-) or presence of 0.1 % NP-40 (+NP-40) on SD media containing choline.(F) Yeast growth assays done in the absence (-) or presence of cinnaymcin (+) on SD media containing choline.(G) Schematic of in vitro trans-methylation assay. Opi3 topology is based on mammalian PEMT and boxes indicate regions containing catalytic sites (Shields et al., 2005).(H) Distribution of label before and 120 min after mixing.(I) Distribution of label in the liposomal fraction 120 min after mixing. Error bars, SD.124Figure 3.8Figure 3.9(A) Lipid radiolabel recovered after separation of liposomes and microsomes in the in vitro Opi3 assay. Radiolabel recovered from the top and bottom of the sucrose cushion after sedimentation is plotted as a percentage of total label before sedimentation. Note that ~30% of total label was not recovered after sedimentation.125Figure 3.9(B) Endocytosis assay. Uptake of FM 4-64 dye at 4?C (cold) or room temperature (RT ) for 15 min in the indicated strains. Note that at 4?C the FM 4-64 dye remained in the plasma membrane whereas at RT it was endocytosed and labelled internal membranes.(C) Lipid transport assay.The indicated strains were labelled with either NBD-PC or NBD-PE for the indicated times at 4?C and imaged. Cells were additionally labeled with FM 4-64 under the same conditions to ensure endocytosis was blocked for the duration of the assay. ?lem3 cells were used as a control to show specificity of uptake of NBD-labelled lipids by the non-endocytic lysolipid transport pathway. (Riekhof et al., 2007). Asterisks mark unlabeled cells in the ?lem3 control. Note that during the time course of the assay, FM 4-64 remained in the plasma membrane whereas NBD lipids were transported in a Lem3-dependent manner to internal membranes that included pmaER (arrows) and the nuclear ER (arrowheads). No differences in transport of NBD-PC or NBD-PE to the ER in the ?scs2?ice2 mutant were observed. Scale bars = 2 ?m.3.4 DiscussionIn this study, we demonstrated for the first time that membrane contact sites between the ER and PM are required for lipid biosynthesis. We propose that PM-ER contacts provide a structural mechanism to regulate PC synthesis in the PM by physically coupling the ER-localized PEMT to its PE in the PM in trans.Contact sites between the ER and other organelles are present in all eukaryotic cells, and have only recently begun to be characterized in molecular detail. Their roles in calcium signalling and transport between ER and PM, and ER and mitochondria are quite well understood (Carrasco and Meyer, 2011; Hayashi et al., 2009), but the 126molecular mechanisms underlying their roles in lipid synthesis and transport remain ill-defined (Toulmay and Prinz, 2011). Of particular importance are roles for lipid transport proteins (LTPs) that localize to contact sites and are hypothesized to facilitate non-vesicular trafficking of lipids at contacts (Im et al., 2005; Levine, 2004). Whether LTPs truly perform this function remains the subject of considerable debate. Recently, a non-transport role for LTPs in regulating the activity of Sac1 at PM-ER contacts has also been reported (Stefan et al., 2011), indicating that contacts may also have functions in regulating the activity of key lipid metabolic enzymes. However, in no instances have contacts been dissected genetically to demonstrate their direct involvement in lipid synthesis or transport. Our present study demonstrates a direct role for PM-ER contacts in lipid synthesis in yeast we show that loss of PM-ER contacts in the ?scs2?ice2 mutant disrupts de novo phosphatidylcholine synthesis by the phosphatidylethanolamine N-methyltransferase (PEMT) enzyme, Opi3. In a screen using the ?scs2?ice2 mutant to uncover novel regulators of PM-ER contacts, we identified a phosphatidic acid phosphatase of the lipin family, Pah1, which both restored contact structure as well as Opi3 function when overexpressed. We show that Opi3 activity requires the oxysterol-binding protein homologue Osh3, which localizes to PM-ER contacts (Loewen et al., 2004). Osh3 has previously been shown to activate in trans Sac1 activity at PM-ER contacts (Stefan et al., 2011); however, we now show a Sac1-independent function for Osh3 in regulating Opi3.We propose that by restricting the localization of Osh3 to PM-ER contact sites and by providing the lipid substrate in trans, Opi3 activity can be restricted to PM-ER 127contacts and PC can be synthesized directly in the PM. Similar to its proposed role in regulation of Sac1 (Stefan et al., 2011), Osh3 may present PME or PE in the PM to Opi3 located at PM-ER contacts. In trans methylation by Opi3 may enable cells to rapidly adjust the PME/PE:PC ratio of the PM, affecting the physical properties of the bilayer. In trans methylation by Opi3 requires that PME is available in the PM. Lipidomic analysis of subcellular fractions of yeast organelles identified PME in the Golgi, but not in any other compartments, including ER microsomes and PM (Schneiter et al., 1999). This indicates that PME is a constituent of the secretory pathway and is likely trafficked to the PM where it is rapidly converted into PC. Finally, the active site of all eukaryotic PEMTs resides on the cytoplasmic face of the ER membrane, accessible to the PM (Shields et al., 2003), and prokaryotic PEMTs are soluble enzymes that lack transmembrane domains (Kl?sener et al., 2009) and hence, by their nature associate with the PM in trans. Thus, PM-ER contacts may regulate PC synthesis by providing PEMT enzymes located in the ER access to their lipid substrates located in trans in the PM.This study may be pertinent to human health since over-activity of the mammalian PEMT enzyme has now been shown to cause ER stress leading to obesity and diabetes (Fu et al., 2011). Thus, our work in yeast identifying PM-ER contacts and Osh3 as regulators of PEMT may contribute to our understanding of the molecular mechanisms underlying these and related metabolic diseases.1284 Chapter 4: A Conserved ER-Membrane Complex Facilitates Phospholipid Exchange Between the ER and Mitochondria4.1 IntroductionMitochondria are critical cellular components that are needed for energy production, lipid metabolism, calcium regulation, and apoptosis.  Most proteins and lipids necessary for mitochondrial biogenesis are not synthesized in mitochondria and must be imported. Although protein import into mitochondria is relatively well understood, much less is knowN about phospholipid transfer to mitochondria.  Phospholipid synthesis occurs largely in the endoplasmic reticulum (ER) and mitochondria acquire phospholipids from the ER at regions of close contact between these organelles (Prinz, 2010).  Zones of close contact between organelles, often called membrane contact sites, are regions where lipids, small molecules, and other signals are transferred between organelles (Elbaz and Schuldiner, 2011; Toulmay and Prinz, 2011).Protein complexes proposed to mediate ER-mitochondria contacts have been identified in mammalian cells and in S. cerevisiae (de Brito and Scorrano, 2008; Kornmann et al., 2009; Szabadkai et al., 2006).  The only such complex that has been identified in yeast to date is called the ER-mitochondria encounter structure (ERMES), which contains an integral ER glycoprotein (Mmm1), a cytosolic protein (Mdm12) and two proteins in the outer mitochondrial membrane (Mdm10 and Mdm34) (Kornmann et al., 2009).  The ERMES complex may play a role in phospholipid exchange between the 129ER and mitochondria.  The study that identified this complex found that phospholipid exchange between the ER and mitochondria decreases 2-5 fold in cells missing this complex. However, two subsequent studies found that the transfer of phosphatidylserine (PS) from the ER, where it is produced (Zinser et al., 1991), to mitochondria did not significantly slow in cells lacking the ERMES complex (Nguyen et al., 2012; Voss et al., 2012). These findings suggest that protein complexes in addition to ERMES mediate ER-mitochondria tethering since lipid exchange between these organelles probably occurs only at contacts and may be required for cell viability.  Consistent with this, cells lacking ERMES proteins are viable (Kornmann and Walter, 2010).  The mechanism of phospholipid exchange between the ER and mitochondria at sites of contact between these organelles is not well understood, but is thought to be nonvesicular in nature (Osman et al., 2011). PS transport from the ER to mitochondria has been found to be reduced ~ 2-fold in certain yeast mutants, but the proteins involved do not seem to directly mediate transfer.  Met30 is a subunit of an ubiquitin ligase complex (Schumacher et al., 2002) that ubiquitinates the transcription factor Met4p, which, in turn, regulates PS transport to mitochondria (Voelker, 2009).  Cells missing the ERMES complex and proteins of the reticulon family needed to shape the reticular ER have a similar ~ 2-fold defect in ER to mitochondria PS transfer (Voss et al., 2012).  Why lipid exchange slows in these mutants, however, is not understood. An important function of PS transport to mitochondria is in the synthesis of phosphatidylethanolamine (PE) (Osman et al., 2011).  PE is critical for mitochondrial function (Gohil and Greenberg, 2009) and although PE can be made outside mitochondria, for unknown reasons this PE is not efficiently transported to mitochondria 130(Burgermeister et al., 2004).  Failure to import PE may explain why all cells from yeast to humans have an enzyme that converts PS to PE in the mitochondrial matrix.  In yeast, this protein is called PS decarboxylase 1 or Psd1 (Figure 4.1A) (Trotter and Voelker, 1995).  PE produced in mitochondria by Psd1p can be transferred back to the ER and converted to phosphatidylcholine (PC) by the methyltransferases Cho2 and Opi3 (Figure 4.1A).  There is a second PS decarboxylase in yeast, called Psd2, which resides in the Golgi complex, endosomal system or vacuole (Trotter and Voelker, 1995).  PE can also be synthesized from diacylglycerol (DAG) and ethanolamine, a metabolic pathway known as the Kennedy pathway (Figure 4.1A) (Carman and Han, 2011).  The Kennedy pathway can also produce PC from DAG and choline (Figure 4.1A).In this study, we used a novel genetic screen to identify genes required for phospholipid exchange between the ER and mitochondria.  We found that mutants missing multiple proteins of the ER-membrane protein complex (EMC) have dramatic defects in PS transport to mitochondria.  This complex is thought to contain six conserved proteins, called Emc1-6 (Jonikas et al., 2009).  The EMC has previously been suggested to play roles in the cellular response to ER stress, in membrane protein folding, or the unfolded protein response in the ER (Christianson et al., 2011; Jonikas et al., 2009); nontheless, its function is not known. Our findings indicate that the EMC mediates lipid transfer from the ER to mitochondria, perhaps by facilitating close contacts between these organelles.   1314.2 Methods4.2.1 Yeast strains, plasmids, and genetic methodsStrains and plasmids used in this study are listed in the Supplemental Table 4.  Media used were:  YPD (1% yeast extract, 2% peptone, 2% glucose), YPGly (1% yeast extract, 2% peptone, 3% glycerol), and SC (2% glucose, 0.67% yeast nitrogen base without amino acids, and amino acid dropout mix from BIO101). Where indicated, ethanolamine or choline was added to a concentration of 5 mM. 5-FOA was added at a concentration of 1mg/ml. Single deletion strains were obtained from freezer stocks of the haploid yeast deletion collection (BY4741, Mat a, KanMX, a gift from C. Boone) unless otherwise stated. Other gene deletions were constructed using the PCR method with the heterologous markers S. pombe HIS5 (pKT128), K. lactis URA3 (pKT209) or NatR (p4339). Double deletion strains were derived from the meiotic products of heterozygous diploids with at least three spores of each genotype being compared. All yeast cells expressing GFP fusion proteins were tagged endogenously in haploids unless otherwise indicated. C-terminally tagged GFP strains were constructed by standard methods involving single-step gene replacement using the pKT128 (SpHIS5) plasmid (Sheff and Thorn, 2004) in the wild type Y7043 background and crossed to the indicated single deletion mutants (BY4741; kanMX). Mdm12-RFP was created similarly to C-terminal GFP fusions except using pMRFP-NAT in BY4741. For PCA, the plasmids used for C-terminal genomic tagging were created as follows: Venus-YFP fragments F1 and F2 were amplified by PCR from p413-TEF-Zip-linker-Venus YFP-F1 and p415-TEF-Zip linker-Venus YFP-F2 (gift of S. Michnick (Tarassov et al., 2008), and cloned into 132plasmids pKT128 and pKT209 (Sheff and Thorn, 2004), respectively, replacing yEGFP and including a myc tag (MEQKLISEEDL) in the linker region to give pHVF1CT (pFA6a-myc-VF1-HIS5) and pUVF2CT (pFA6a-myc-VF2-URA3). For N-terminal integrations, plasmid pHVF1NT (HIS5-PHO5-VF1) was created by replacing eGFP in plasmid pTLHPG (HIS5-PHO5-GFP; gift of T. Levine) with the Venus F1 PCR fragment. To create the Tom5?TM-VF1 strain the C-terminal 21 amino acids of endogenous Tom5 were replaced in-frame by VF1.4.2.2 Synthetic Genetic Array (SGA) Analysis for CHO2 and EMC6 SGA analysis was performed according to established protocols (Tong and Boone, 2006) essentially as previously described (Young et al., 2010) using a Singer RoToR Colony Arraying robot (Singer Instruments). ?cho2::URA3 and ?emc6::URA3 query strains were constructed using standard techniques in strain background Y7092 and crossed to the yeast haploid deletion mutant array (DMA) using a Singer RoToR HDA robot. Following diploid selection, spots were replicated three times and sporulated for 5 days. Haploids were germinated on SD-media lacking histidine, arginine and lysine supplemented with thialysine and canavanine (both at 100 ?g/ml). Control sets of single deletion strains were generated by plating on media containing 5-fluoroorotic acid to counter-select for the ?cho2::URA3 or ?emc6::URA3 alleles and G418 sulfate (200 ?g/ml) to select for the DMA strain; while double mutants were selected for by plating on media lacking uracil and containing G418 sulfate. A further round of selection was performed on the same media. For the CHO2 SGA screen in the presence of choline, all plates additionally contained 1 mM choline. Arrays were imaged using a flatbed scanner. Balony software ( was used to measure spot 133sizes, determine cut-off values for genetic interactions and define strains the showed statistically significant changes in growth rate. Cut-off values for genetic interactions were defined for each screen by determining three standard deviations from the mean of the ratios of the double mutant to single mutant growth rates. Double mutant strains that met the cut-off and showed significant changes in growth relative to the corresponding single mutant control (one-tailed students? t-test; p < 0.05; n=3) were considered as genetic interactions. For the CHO2 SGA screen, aggravating genetic interactions identified in the screen done in the absence of choline were considered rescued if they were no longer identified as genetic interactions according to the above criteria in the screen done in the presence of choline. Gene ontology analysis was performed using Funspec ( and Cytoscape ( {Robinson et al., 2002, BMC Bioinformatics, 3, 35; Cline et al., 2007, Nat Protoc, 2, 2366-82}.4.2.3 Protein Subcellular Localization by Confocal Microscopy Log phase live yeast cells were imaged using a Zeiss LSM-5 Pascal confocal microscope and Zeiss Pascal software. Unless otherwise stated, all proteins were tagged at the C-terminus of the endogenous protein. Optical slices were taken through the centre of each cell and images being directly compared were captured with identical microscope settings on the same day.4.2.4 In vivo labeling with [3H]serine. Cells were labeled with L-[3-3H]serine (American Radiolabeled Chemicals) as described (Raychaudhuri and Prinz, 2008) with the following modifications.   About 2 134OD600 units of cells from a saturated culture were added to 25mL of SC medium and incubated at 30oC. When the cultures reached an OD600 of about 0.3, 10 ?g/mL myriocin (SigmaAldrich, stock = 500 ?g/mL in methanol) was added to the medium, the cells were grown for 30 minutes, and cerulenin (SigmaAldrich, stock = 5 mg/mL in dimethyl sulfoxide) was added to 10 ?g/mL in the medium.  About 5 minutes later, 50 ?Ci of [3H]serine was added to the medium and the cells were grown for an additional 30 minutes.  The culture was then added to an equal volume of ice-cold water and it was washed once with ice- cold water. Cells were lysed in a Mini-BeadBeater-8 (BioSpec). Lipids were extracted as described (Parks et al., 1985), separated by HPLC and the fractions containing PS, PE, and PC were collected and analyzed by liquid scintillation counting.4.2.5 Mitochondrial extracts and in vitro [3H]serine labeling Crude mitochondria were prepared as described (Voss et al., 2012). Briefly cells were grown in YPD medium to an OD600 of ~0.3, washed once with water, and incubated in 1mL 0.1 M Tris-SO4 (pH 9.4) containing 10mM DTT for 10 minutes at 30?C. Cells were washed once with spheroplast buffer (1.2 M sorbitol, 20 mM Tris pH 7.4) and resuspended in 1.5 mL of the same buffer containing 1mg/mL zymolyase  20T (Seikagaku Biobusiness, Japan). After incubation for 60 min at 30?C, cells were pelleted (5 min, 500 x g) and washed twice with spheroplast buffer. Cells were resuspended in ice-cold lysis buffer (0.6 M mannitol, 2 0mM Tris pH 7.4, 1 mM EDTA, 1mM  PMSF and protease inhibitors, [Roche]) and lysed with a dounce using a B-pestle. The extract was centrifuged twice for 5 min at 3000 x g to remove unlysed cells and debris. The 135supernatant was centrifuged at 9,600 x g for 10 min and the pellet containing crude mitochondria was resuspended in lysis buffer using a dounce (B-pestle). To label crude mitochondria with [3H], 1-2 mg of crude mitochondria in 1mL of lysis buffer were heated to 30oC and 10 ?Ci of L-[3-3H]serine (American Radiolabeled Chemicals) was added and MnCl2 was added to 0.6 ?M.  After 20 minutes, serine and EDTA were added to 0.5 mM and 5 mM, respectively. Samples of 200 ?L were taken after 0, 5, 10, and 15 minutes and added to 6 mL of chloroform:methanol (1:2). Lipids were extracted, separated by HPLC, and extracted as described in the previous section. 4.2.6 Mitochondrial purification and determination of mitochondrial steady state lipid About 2 OD600 units of cells from a saturated culture were washed with water, resuspended in 50 mL of fresh SC medium containing 200 ?Ci of [3H]acetate (American Radiolabeled Chemicals), and grown at 30oC for at least 3-4 generations. Crude mitochondria were purified as described in the previous section and further purfied by equilibrium centrifugation using density gradients made from OptiPrep (Axis-Shield, Oslo, Norway) as described {Nunnari et al., 2002, Methods Enzymol, 351, 381-93}. Lipids were extracted and separated by one-dimensional TLC as described by {Vaden et al., 2005, Anal Biochem, 338, 162-4}.  TLC plates were scanned on a RITA Star Thin Layer Analyzer (Raytest). 4.2.7 Psd assay Psd assay was performed as described {Raychaudhuri and Prinz, 2008, Proc Natl Acad Sci U S A, 105, 15785-90} except that the concentration of the substrate, 1-136oleoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphoserine (Avanti Polar Lipids), was 500 ?M.4.1 Results4.1.1 Genetic screen for components that mediate transport of phospholipids between ER and mitochondria In a prior synthetic genetic array (SGA) screen for the PSD1 gene we uncovered an aggravating genetic interaction with the CHO2 gene.  We found that the growth defect of cells missing Psd1 and Cho2 was rescued by the addition of ethanolamine or choline to the medium (Figure 4.1B), which allows cells to make PE and PC via the Kennedy pathway (Figure 4.1A). We reasoned that genes required for transport of PS from ER to mitochondria and PE from mitochondria to ER would similarly have negative genetic interactions with CHO2 that would be rescued by ethanolamine or choline. Therefore, we performed an SGA screen for the CHO2 gene in the absence and presence of choline to identify genes that functioned in lipid transport between ER and mitochondria. The results of this genome-wide screen are shown in Figure 4.1C, in which we the plotted growth of double mutants in the absence of choline versus their growth in its presence. We identified 191 double mutants that exhibited slow growth phenotypes whose growth was significantly improved by choline addition (Figure 4.1C). According to gene ontology classifications, membrane-associated functions were highly represented among these groups and included ER, vacuole and endosomal compartments (Figure 4.1D). In the Cellular Component category, we noticed almost thirty-fold enrichment in EMC proteins, a conserved, uncharacterized ER membrane 137protein complex (Figure 4.1D) (Jonikas et al., 2009). In our screen, genes encoding the six subunits of the EMC showed strong aggravating genetic interactions with CHO2 that were rescued by choline addition (Figure 4.3A). We verified these genetic interactions by spot assay and found that these interactions were rescued by choline but not ethanolamine, suggesting that the EMC has a function distinct from PE production by Psd1 (Figure 4.2).138139Figure 4.1Figure 4.1 Genome-Wide Screen for Regulators of Phospholipid Synthesis.(A)  Phospholipid synthesis in the methylation pathway is compartmentalized between ER and mitochondria. PS synthesized in the ER is transported to mitochondria for conversion into PE and transported back to the ER for conversion to PC. The Kennedy pathway synthesizes PE and PC from ethanolamine (etn) and choline (cho) independent of lipid transport between ER and mitochondria.(B)  Yeast growth assays for the indicated mutants in the absence (nil) or presence of ethanolamine (+ etn) or choline (+ cho).(C)  Results of SGA screen for CHO2 in the absence (-) or presence (+) of choline. Genetic interactions are plotted as the log2 of the ratio of growth of single versus double mutants with ?cho2 in the absence and presence of choline. Interactions rescued by choline (green triangles) predominately clustered on the X axis, whereas interactions not rescued (red squares) were present on the diagonal.(D)  Enrichment of functional groups for the genes that showed interactions and were rescued by choline in (C). Fold enrichment represents the frequency of a given term in our dataset relative to the frequency of that term in the whole genome.140Figure 4.2 Yeast growth assays of mutants identified in the CHO2 SGA screen, related to Figure 4.1.141Figure 4.2Serial dilutions of the indicate strains were spotted onto agar plates containing synthetic complete media (SC) or with or without ethanolamine or choline.4.1.2 Genetic interactions reveal that the EMC functions in the phospholipid synthesisTo uncover additional functional information about the EMC we examined the global genetic interaction network, which is a comprehensive map of pairwise genetic interactions in which genes with similar functions form coherent clusters (Costanzo et al., 2010). We noticed that all EMC genes except for EMC5, which was not present in the global network, formed a discrete cluster, suggesting EMC genes share similar functions (Figure 4.3B). Interestingly, also in this cluster were CHO2 and OPI3, which encode the two methyltransferases that convert PE to PC (Figure 4.1A), further supporting a role for EMC genes in phospholipid metabolism. EMC genes showed primarily aggravating genetic interactions with a cluster of genes that contained INO2 and INO4, which encode both subunits of the Ino2/4 transcriptional activator complex required for expression of gene involved in phospholipid synthesis, including CHO2 and OPI3.Next, we performed an SGA screen for one of the EMC genes, EMC6, to further define functions for the EMC complex. We identified 36 aggravating and 41 alleviating genetic interactions with EMC6 (Table 4.1). Genetic interactions with EMC6 revealed enrichment for functions associated with lipid metabolism, mitochondria, membrane traffic, cell polarity and morphogenesis, cell signaling and chromatin (Figure 4.3C). Although EMC genes has been found to have links to the ER unfolded protein response 142(UPR) (Christianson et al., 2011; Jonikas et al., 2009), we did not find significant enrichment for ER stress response functions and EMC6 did not interact genetically with either HAC1 or IRE1, two key factors required for induction of the UPR. Our screen did uncover genetic interactions with key regulators of phospholipid metabolism, INO2, INO4 and SCS2, as well as CHO2 (Figure 4.3C), further supporting a role for EMC proteins in phospholipid synthesis. We also did not observe aggravating genetic interactions between EMC6 and any of the remaining EMC genes (Table 4.1).143144Figure 4.3Figure 4.3 The EMC genes function in phospholipid metabolism.(A)  Genetic interactions identified between EMC genes and CHO2. Plotted is the ratio of spot size of the single EMC mutants versus the corresponding double mutants with ?cho2 in the absence and presence of choline.(B)  EMC gene cluster identified in the global genetic interaction map (Costanzo et al., 2010) and aggravating genetic interactions with a cluster of genes that function in the methylation pathway of phospholipid synthesis. Aggravating interactions have negative values and alleviating interactions have positive values. Trees identify isolated clusters identified in the global genetic interaction map.(C)  Functional map for EMC6 derived using genetic interactions identified in the EMC6 SGA screen. Colored nodes represent functional groups and edges define associations between groups. Node size and edge thickness indicate their level of significance within the network. Genes (grey nodes) identified in the screen that are associated with each functional group are shown (blue edges).4.1.3 EMC proteins form a complex in the ER We now examined the individual localizations of EMC proteins by creating endogenous GFP fusion proteins and imaging by confocal microscopy. We found that each of Emc1-6 localized throughout the yeast ER and were expressed at similar levels (Figure 4.4A). Next we verified interactions between each EMC protein by Protein-Fragment Complementation Assay (PCA) in which we tagged each endogenous Emc protein with one half of the Venus fluorescent protein. Shown in Figure 4.4B is the 145matrix containing all pair-wise interactions between the EMC proteins. We observed PCA interactions in the ER between most EMC proteins and there was no single EMC protein that failed to interact with any other EMC protein, suggesting that all six EMC proteins did indeed form a complex within the ER. Interestingly, we did not detect interactions between Emc1 and Emc3 or Emc 1 and Emc4, whereas as all other EMC proteins interacted, suggesting that Emc1 was organized distinctly within the complex. To test the functional interdependence of the EMC proteins we examined the localization of GFP-tagged EMC proteins in various ?emc deletion mutant strains. We found that deletion of EMC6 resulted in a dramatic reduction of Emc2-5 in the ER, whereas the localization of Emc1 was unaffected (Figure 4.4C). This indicated an important role for Emc6 in complex organization and further suggested that Emc1 was distinct within the complex. All EMC proteins except Emc2 contain predicted transmembrane domains, therefore Emc2 must be peripherally associated with the ER. Consistent with a central role for Emc6 in complex organization, Emc6 was primarily responsible for localizing Emc2 to the ER (Figure 4.4D). Together, the PCA interaction data between EMC proteins and their functional interdependence for ER localization indicated that EMC proteins formed a complex within the ER.146147Figure 4.4Figure 4.4 EMC proteins interact in a complex in ER(A)  Yeast expressing EMC proteins endogenously tagged with GFP imaged by confocal microscopy, top panel = fluorescence image, bottom = DIC.(B)  Interactions between EMC proteins in the ER imaged using Venus PCA. Images of cells expressing proteins fused to either of the two haves of the Venus proteins (VF1 or VF2).(C)  Localization of EMC proteins tagged with GFP in emc6? cells. (D)  Emc2 tagged with GFP imaged in EMC mutants. All scale bars 2 ?m.4.1.4 ER to mitochondria PS transport decreases in cells missing multiple Emc proteins.  To determine if the EMC plays a role in phospholipids exchange between the ER and mitochondria we used an in vivo assay to measure PS import into mitochondria from the ER.  After synthesis in the ER, PS can be transported to mitochondria and converted to PE by Psd1p (Carman and Henry, 1999).  Thus, the conversion of newly synthesized PS to PE has been used to estimate the amount of PS transfer from the ER to mitochondria (Voelker, 2009). We metabolically labeled cells with [3H]serine, which is used for PS production in the ER (Zinser et al., 1991). Previously, we have shown that, using the labeling conditions described in Materials and Methods, cells produce PS and PE at linear rates and that little of the radiolabeled PE is converted to PC (Raychaudhuri and Prinz, 2008).  Strains were labeled with [3H]serine for 30 minutes and the ratio of  [3H]PS to [3H]PE calculated.  In a wild-type strain, this ratio was 2.5.  Cells missing 148either Psd1p or Psd2p had a significant decrease in the [3H]PS to [3H]PE ratio and this ratio was close to zero in a strain missing both proteins (Figure 4.5A).  Using this assay we determined the amount of PS converted to PE in cells missing any one of the Emc proteins.  Surprisingly, we found that the amount of PS converted PE did not decrease significantly in these strains (Figure 4.5A and data not shown).  We speculated that the Emc proteins might have redundant functions and therefore measured ER to mitochondria PS transport in cells missing multiple Emc proteins.  A strain lacking Emc2p and Emc6p had a ~25% decrease in the ratio of [3H]PS to [3H]PE compared to wild-type.  Cells missing additional Emc proteins had more substantial transport defects; a strain missing Emc1, Emc2, Emc3, and Emc6 (4x-emc) or one missing these proteins and Emc5 (5x-emc) had ~50% reduction in the ratio of [3H]PS to [3H]PE (Figure 4.5A). These strains contain Psd2, which is outside mitochondria and converts a significant fraction of newly synthesized PS to PE (Figure 4.5A).  Since the amount of PS to PE conversion in 4x-emc and 5-emc cells is about the same as that of cells lacking Psd1, our findings suggest that very little PS to PE conversion occurs in the mitochondria of these strains. To test this, we sought to make a 5x-emc psd2? strain to measure PS to PE conversion only in mitochondria.  However, we found that 5x-emc psd2? cells were not viable.  We transformed 5x-emc cells with a plasmid containing PSD2 and URA3 and then deleted PSD2 on the chromosome.  The resulting strain was not able to grow on medium with 5-fluoroorotic acid (5-FOA), which is toxic to URA3 strains and selects against the plasmid with URA3 and PSD2 (Figure 4.5B).  Therefore, 5x-emc psd2? cells were not viable.  Together, these findings suggest 149that ER to mitochondria PS transport is dramatically reduced in cells missing multiple EMC proteins. It is possible that 5x-emc psd2? was not viable because it was unable to produce sufficient PE.  However, adding ethanolamine to the medium, which can be used to make PE by the Kennedy pathway (Figure 4.1A), did not restore viability to 5x-emc psd2? cells (Figure 4.5B). This strain probably has defects in lipid metabolism in addition to a significant reduction in PS transport to mitochondria. To rule out that the decrease in PS to PE conversion in 5x-emc cells was caused by a reduction in Psd1p activity or mislocalization of Psd1p, we determined the amount of Psd activity in mitochondria derived from 5x-emc cells. An in vitro Psd assay was performed using a fluorescent PS analog [7-nitro-2?1,3-benzoxadiazol-4-yl]-PS (NBD-PS). We found mitochondrial Psd activity was not reduced in mitochondria from 5x-emc cells compared to those from wild-type cells but rather was significantly increased for unknown reasons (Fig 4.5C).  These findings suggest that 5x-emc cells have a significant decrease in the transfer of PS from the ER to mitochondria.150Figure 4.5 Cells missing multiple EMC proteins have defects in PS transfer from the ER to mitochondria(A)  Cells with the indicated genotypes were labeled with [3H]serine for 30 minutes and the ratio of [3H]PS converted to [3H]PE determined (mean + s.d, n = 3-5 independent experiments). The dashed red line indicates the amount of conversion that occurred in psd1? cells. * = p < 0.05 compared to wild-type, two-tailed t-test.151Figure 4.5(B)  PSD activity of crude mitochondria incubated with NBD-PS for 1 hour at 30?C. PSD activity was normalized to that of wild-type crude-mitochondria (mean + s.d., n = 2-3 independent experiments). * = p < 0.05 compared to wild-type, two-tailed t-test.(C)  10-fold serial dilutions of cultures of the indicated strains were spotted onto SC medium with or without 5-FOA and ethanolamine. The plates were incubated at 30?C for four days.4.1.5 Mitochondria in 5x-emc cells are nonfunctional and have abnormal phospholipid levels.  Because 5x-emc cells have reduced PS transport from ER to mitochondria, we wondered if mitochondria from 5x-emc cells have reduced amounts of PS and PE.  To measure phospholipid levels, wild-type and 5x-emc cells were labeled with [3H]acetate for at least 3-4 generations and the relative abundance of the major phospholipids in purified mitochondria was determined. We found that PS levels in the mitochondria of 5x-emc cells were about 50% lower than those in wild-type mitochondria (Figure 4.6A).  Therefore, reduced ER to mitochondria PS transport in 5x-emc cells results in decreased steady-state PS levels in mitochondria. Notably, PE levels were also reduced about 50%. This reduction is probably caused by the defect in ER to mitochondria PS transport in 5x-emc cells since most PE in mitochondria is generated from PS by Psd1p (Burgermeister et al., 2004).  Thus, a reduction in PS levels in mitochondria probably results in lower levels of PE in mitochondria.  Interestingly, the relative abundance of other phospholipids was increased in 5x-emc mitochondria, particularly phosphatidic acid (PA) and cardiolipin (CL) (Figure 4.6A).  These changes may reflect a mechanism 152by which cells compensate for low levels of mitochondrial PE, which is thought to be critical for proper mitochondrial function (Gohil and Greenberg, 2009).  Because the lipid profile of mitochondria in 5x-emc cells was dramatically altered, we wondered if the mitochondria were functional. Yeast strains with nonfunctional mitochondria cannot grow on media containing nonfermentable carbon sources such as glycerol.  We found that 5x-emc cells and other strains missing multiple EMC proteins cannot grow on the glycerol-containing medium YPGly, indicating that cells missing multiple EMC proteins do not have functional mitochondria, probably because of the abnormal levels of phospholipids in the mitochondria of these strains.  Interestingly, 5x-emc cells had a substantial growth defect even on the glucose containing medium YPD (Figure 4.6B) and had abnormal morphology with many cells have multiple buds (Figure 4.7). 153Figure 4.6 Mitochondria from cells missing emc proteins have reduced levels of PS and PE and are not functional(A)  Wild-type and 5x-emc cells were grown for at least three generations in medium containing [3H]acetate and the amount of the six major phospholipids in purified mitochondria was determined (mean + s.d., n = 3 independent experiments). * = p < 0.05, two-tailed t-test.(B)  10-fold serial dilutions of the indicated strains on YPD and YPGly plates. The plates were incubated at 30?C for three days.154Figure 4.6Figure 4.7 5x-emc cells have abnormal shapes, related to Figure 4.6.DIC images of wild-type and 5x-emc cells in mid-logarithmic growth phase. Bar = 5 ?m.155Figure 5x-emc cells have a reduced rate of ER to mitochondria PS transfer in vitro Since we found that 5x-emc cells have reduced ER to mitochondria PS transport in vivo, we wondered if a similar defect could be detected in vitro.  We used a previously established two-step assay to monitor the transfer of newly synthesized PS from the ER to mitochondria (Voss et al., 2012).  In the first step, crude mitochondria are incubated for 20 minutes with  [3H]serine and Mn2+. The presence of Mn2+ is required by PS synthase but inhibits the conversion of [3H]PS to [3H]PE by Psd1. Thus, in the second step of the reaction, Mn2+ is chelated by EDTA and the PS to PE conversion rate was determined.  Since Psd2 is not present in this assay , all PS to PE conversion in this assay is mediated by Psd1 and indicates that PS synthesized in ER-derived membranes has been transferred to mitochondria.  Using mitochondria derived from wild-type cells, we found that about 1 % [3H]PS synthesized was converted to PE per minute (Figure 4.8A).  When mitochondria from 3x-emc (missing Emc2, Emc5, and Emc6) and 5x-emc cells were used, this rate decreased about 2-fold and 3-fold respectively (Figure 4.8A). It should be noted that for all the strains tested the rate of PS to PE conversion was linear (R2 > 0.9). Since mitochondria derived from 5x-emc cells have Psd activity that is not lower than those from wild-type (Figure 4.5C), these finding indicate that the rate of ER to mitochondria PS transfer is significantly reduced in crude mitochondria derived from 5x-emc cells.4.1.7 The EMC and ERMES complex are required for viability and ER to mitochondria PS transport Because the ERMES complex tethers the ER and mitochondria, we wondered whether PS transfer would be slower in crude mitochondria missing Emc proteins and 156the ERMES complex. As disruption of a single ERMES component causes disassembly of the whole complex (Kornmann et al., 2009), we sought to delete one of the four genes encoding the ERMES proteins in 5x-emc cells.  However, we were unable to delete MMM1 in 5x-emc cells, probably because the resulting strain inviable.  Therefore we introduced the conditional mmm1-1 allele into 5x-emc cells.  This strain was not viable at non-permissive temperature of 37oC (Figure 4.8B).  We found that when 5x-emc mmm1-1 cells were shifted to restrictive temperature (37oC), they stopped growing after 4 hours.  Therefore, we isolated crude mitochondria from 5x-emc mmm1-1 cells 3 hours after shift to 37oC.  In these mitochondria, the rate of PS to PE conversion was reduced ~5-fold (Figure 4.8A).  Because mitochondria derived from 5x-emc mmm1-1 cells do not have less Psd activity than those from wild-type (Figure 4.5C), these findings indicate that PS transfer from ER to mitochondria is almost abolished in 5x-emc mmm1-1 cells at non-permissive temperature.  It should be noted that PS to PE transfer slowed only modestly in mitochondria derived from mmm1-1 cells (Figure 4.8A), indicating that the effects of mmm1-1 and 5x-emc mutations on ER to mitochondria PS transfer were additive.4.1.8 An artificial ER-mitochondria tether restores PS transfer in cells missing multiple Emc proteins and ERMES proteins. The decrease in ER to mitochondria PS transfer in cells missing EMC proteins could be caused by a decreased ability to transport PS or by inefficient tethering of the ER and mitochondria.  To distinguish between these possibilities, we determined if artificially tethering the ER and mitochondria corrects the PS transport defect in mitochondria derived from 5x-emc and 5x-emc mmm1-1 cells.  For these studies we 157used a fusion protein called ChiMERA, which has previously been shown to tether the ER and mitochondria {Kornmann et al., 2009, Science, 325, 477-81}.  When this fusion protein was expressed in 5x-emc and 5x-emc mmm1-1 cells, it corrected the ER to mitochondria PS transport defect in mitochondria derived from these strains (Figure 4.8C).  It also restored the ability of 5x-emc mmm1-1 cells to grow at elevated temperature (Figure 4.8B).  These findings suggest that the defect in ER to mitochondria PS transfer in 5x-emc and 5x-emc mmm1-1 cells is probably caused by inefficient tethering of the ER and mitochondria. 158Figure 4.8Figure 4.8 5x-emc mmm1-1 cells are not viable and have a dramatic reduction in ER to mitochondria PS transfer at nonpermissive temperature.(A)  Crude mitochondria were incubated with [3H]serine and Mn2+. After 20 minutes at 30?C, EDTA and an excess of unlabeled serine were added; chelation of Mn2+ by EDTA inhibits PS synthase and allows Psd1 to function. The samples were collected over 15 minutes and the rate of [3H]PS to [3H]PE conversion per minute was calculated (mean + s.d., n = 3-5 independent experiments). * = p < 0.05 compared to wild-type, two-tailed t- test.(B)  Cultures of strains with the indicated genotypes were grown at 23?C and 10-fold serial dilutions were spotted on to YPD plates and incubated at 23?C or 37?C for 4 days. (C) The rate of PS to PE conversion of strains expressing ChiMERA was determined as in (A) (mean + s.d., n = 3 independent experiments).4.1.9 The EMC interacts with Tom5 at ER-mitochondria contact sites  Our findings suggested that EMC may play a role in ER-mitochondrial tethering, a conclusion that was further supported by the genetic interactions we uncovered between EMC6 and genes with mitochondria-related functions. Therefore, we searched for genes identified in the CHO2 SGA screen that encoded mitochondrial outer membrane proteins. We identified TOM5, which encodes a small subunit of the TOM complex that is an integral protein of the mitochondrial outer membrane that showed a strong genetic interaction with CHO2 that was rescued by choline (Figure 4.2). Thus, Tom5 was an ideal candidate for interacting with the EMC and we tested for a physical interaction using PCA. We found that both Emc1 and Emc2 interacted with Tom5 in 159punctae that were suggestive of the localization of the ERMES complex (Figure 4.9A and B)  (Kornmann et al., 2009). Interestingly, when we deleted the transmembrane domain of Tom5 to make Tom5?TM, the soluble form interacted with Emc2 on the ER (Figure 4.9C), indicating that localization of Tom5 to the outer mitochondrial membrane was not required for its interaction with the EMC. This also suggested that proximity of ER to mitochondria would regulate binding of the EMC to Tom5 located in the mitochondrial outer membrane. To verify that the EMC interacted with Tom5 at ER-mitochondria contacts, we colocalized the PCA interaction between Emc1 and Tom5 with the ERMES subunit Mdm12, tagged endogenously with RFP. We found that in 100% of the cells we examined, the Emc1-Tom5 PCA punctae colocalized with ERMES foci (Figure 4.9D).  This indicated that the EMC-Tom5 interaction likely formed a bridge between ER and mitochondria at the same contact sites as defined by ERMES. We did not detect an interaction between Tom5 and another integral ER protein, Ale1, using PCA, even though Ale1-GFP localized throughout the ER similar to EMC proteins (Figure 4.10).  Ale1 has a cytoplasmically oriented C-terminus, which should be accessible to Tom5 on mitochondria (Pagac et al., 2011). Deletion of individual EMC genes also had no affect on the localization of Mdm34-RFP indicating the EMC likely did not regulate ERMES complex assembly (Figure 4.11). Thus, this data further supported a specific role for the EMC at contacts between ER and mitochondria. Next we examined the functional relevance of the EMC-Tom5 interaction. We did not observe growth defects of the Emc2-Tom5 PCA strain grown on fermentable or nonfermentable carbon sources (Figure 4.9E), indicating that mitochondrial function was likely normal. However, we found that the PCA between Emc2 and Tom5?TM caused 160this strain to grow poorly on both media types (Figure 4.9E), suggesting that recruitment of Tom5?TM to the ER by the EMC interfered with normal mitochondrial function. Since loss of EMC function in the 5x-emc mutant lead to decreased PS transfer and impaired mitochondrial function, this suggested that the interaction between the EMC and full length Tom5 at contacts might be important for PS transfer. Therefore we measured PS transfer between ER and mitochondria in cells expressing Tom5?TM-VF1, Emc2-VF2, or both proteins.  Expression of both proteins in the same cell caused a dramatic decrease in the amount of [3H]PS converted to [3H]PE (Figure 4.9F) suggesting that PS transfer to mitochondria was compromised. Together, these findings indicated that Tom5 and the EMC interacted at sites of close contact of the ER with mitochondria and that blocking this interaction dramatically reduced PS transfer between these organelles.161Figure 4.9 The EMC interacts with Tom5 at ER-mitochondria contacts(A&B) Interactions between Tom5 and Emc1 (A) and Emc2 (B) proteins imaged by Venus PCA.(C)  Interaction between Tom5?TM and Emc2 by PCA.(D)  Colocalization of the Tom5-Emc interaction and Mdm12-RFP of the ERMES complex.162Figure 4.9(E)  Yeast growth assays for the indicated strains on media containing glucose (YPD) or glycerol (YPGly). Tom5 x Emc2 and Tom5?TM x Emc2 indicate haploid strains used for PCA containing Tom5 tagged with vF1 and Emc2 tagged with vF2.(F)  Cells with the indicated genotypes were labeled with [3H]serine as in Figure 4.5 A. The ratio of [3H]PS converted to [3H]PE was determined and expressed as a percent of wild- type cells (mean + s.d, n = 3 independent experiments). * = p < 0.05 compared to wild- type, two-tailed t-test. All scale bars = 2 ?m.163Figure 4.10 Ale1 control for PCA between the EMC and Tom5, related to Figure 4.9.(A)  Emc1 x Tom5 PCA in diploids.(B)  Ale1 x Tom5 PCA in diploids captured with identical microscope settings as in (A).(C) Ale1 tagged at the endogenous gene locus with GFP. All scale bars; 2?m.164Figure 4.10ABCFigure 4.11 Localization of ERMES subunit Mdm34 is not disrupted in EMC mutants, related to Figure 4.9. Mdm34-RFP tagged at the endogenous locus was imaged in various EMC deletion mutants using confocal microscopy. Scale bars = 2 ?m.165Figure 4.114.2 DiscussionLipid exchange between the ER and mitochondria is critical for mitochondrial membrane biogenesis and lipid metabolism. Here we used a novel genetic screen to identify mutants with defects in these processes. We found that cells missing multiple components of a conserved protein complex of the ER, called EMC, had dramatic reductions in the amount of PS transferred from the ER to mitochondria both in vitro and in intact cells. This lead to a reduction in the amount of PS and PE in mitochondria. A role for the EMC in phospholipid metabolism was further supported by our unbiased global genetic analyses. We found that the EMC proteins localize to the ER where they form a complex, consistent with previous findings (Jonikas et al., 2009). The EMC interacts with the mitochondrial outer membrane protein Tom5 at sites of close contact between the ER and mitochondria. The ERMES complex is also found at these sites, which are thought to be regions of lipid exchange between these organelles. A PCA interaction between Emc2 and the cytosolic portion of Tom5 (Tom5?TM) reduced PS transfer to mitochondria, suggesting that the EMC interaction with full length Tom5 is important for lipid transfer at contact sites. Together, these findings indicate that the EMC localizes to sites of ER to mitochondria contact where it facilitates PS transfer from the ER to mitochondria.Our data suggest that the EMC may perform a tethering function at ER- mitochondria contact sites. Using our in vitro PS transport assay we found that the rate of transport was decreased almost 70% in the 5x-emc mutant and was almost completely abolished in combination with the ERMES mutation, mmm1-1. Strikingly, this  transport defect was entirely ameliorated in the 5x-emc mmm1-1 mutant that expressed 166ChiMERA. We previously showed that synthesis of PE in this assay results from transport of PS from ER that remains associated with mitochondria, since transport cannot occur between donor membranes from psd1? cells, which cannot synthesize PE and wild type acceptor membranes (Voss et al., 2012). PE synthesis by mitochondria with associated ER was also unaffected by dilution (Voss et al., 2012), indicating that tethering rather than the presence of soluble transport factors is critical for in vitro transport, consistent with previous studies (Schumacher et al., 2002). Thus, the decreased PS transfer to mitochondria in vitro in the EMC mutants and rescue by an artificial tethering protein strongly support a role for the EMC in tethering. Although unlikely, it is also possible that expression of ChiMERA artificially bypasses the requirement for membrane-associated lipid transporters or that it enhances the transport activity of mutated, but partially functional EMC complex by increasing ER- mitochondria contacts. However, a direct role for the EMC in lipid transport is unlikely since EMC proteins do not contain known lipid binding or transport domains. Further support for a tethering function for the EMC comes from genetic interactions between the EMC and ERMES. Similar to ERMES mutants, we found that disruption of the EMC complex interfered with mitochondrial respiration, indicating that mitochondrial function was compromised in the absence a functional EMC. This may be a result of decreased mitochondrial PE in these mutants, similar to psd1? cells. However, a small amount of growth of EMC mutants was observed on fermentable carbon sources, suggesting that contacts between ER and mitochondria were not completely lost. Consistent with this, we did not observe defects in ERMES in EMC mutants. Strikingly, additional inactivation of ERMES in the EMC defective strain 167resulted in synthetic lethality, even on fermentable carbon sources, suggesting that contacts were completely lost in this mutant. Consistent with lethality arising from loss of ER-mitochondrial tethering, expression of ChiMERA completely rescued growth of the 5x-emc mmm1-1 mutant. Thus, these data indicate that the EMC and ERMES, together, are likely responsible for tethering of ER to mitochondria, a process which we now show is essential for cell growth.Although our initial genetic screen was designed to identify proteins with specific functions in mediating lipid transport between ER and mitochondria, our data supports a more general role for the EMC in ER-mitochondrial tethering. We found that 5x-emc psd2? cells are not viable and do not grow even when supplemented with ethanolamine. Since psd1? psd2? cells can grow if ethanolamine is in the medium, 5x-emc psd2? cells must have other defects in lipid metabolism or other essential processes in addition to altered PS transport to Psd1 in mitochondria. Genetic interactions with EMC6 revealed functional links to vesicular transport and the Golgi complex, consistent with a role for Psd2 in Golgi/vacuole compartments. Additionally, Emc6 contains a putative Rab5-interacting protein domain that may also explain its functions in the secretory pathway. Therefore, the EMC may play a role in secretions, although the precise nature awaits further studies. Similarly, the ERMES tethering complex has functions in addition to tethering (Kornmann and Walter, 2010). Finally, the synthetic lethality observed between the EMC and ERMES mutants indicates that abolished ER-mitochondrial tethering may result in other physiological defects. These could include defective calcium homeostasis, mitochondrial protein import and inheritance of mitochondrial DNA. The nature of this synthetic lethality will need to be 168investigated further to better understand additional functions for ER-mitochondrial tethering beyond phospholipid synthesis.Even though the EMC appears to be a complex in the ER, loss of individual Emc proteins had little effect on PS transfer. This suggests that proteins in the EMC have overlapping tethering functions and that assembly of tethering complexes containing EMC proteins may not require all of the proteins. However, we did detect a modest, but significant 25% decrease in PS transport in cells expressing a nonmitochondrial version of Tom5 lacking its C-terminal transmembrane domain. Additionally, in Emc2 x Tom5?TM PCA cells transport was reduced even further, suggesting that this interaction interfered with tethering. We also found that all six EMC proteins interacted with full length Tom5 at ER-mitochondria contacts by PCA (data not shown), providing a possible explanation for the lack of transfer defects in individual EMC mutants. It therefore seems likely that multiple EMC proteins interact with Tom5 to tether ER to mitochondria and that loss of individual EMC components is not sufficient to disrupt this tethering.Tom5 is a tail-anchored membrane protein that is one of three small subunits of the translocase of the outer mitochondrial membrane (TOM) complex that is responsible for import of most, if not all mitochondrial proteins across the outer membrane (Neupert and Herrmann, 2007). The two other small subunits, Tom6 and Tom7 are similar in length and topology, but share no sequence homology with each other or Tom5. However, all these proteins perform an overlapping essential function since the triple deletion mutant interferes with import and is lethal, whereas the individual deletions have only minor effects (Alconada et al., 1995; H?nlinger et al., 1996). It may be that 169Tom6 and Tom7 can compensate for loss of Tom5 function in ER-mitochondria tethering, explaining why we only see a partial defect in PS transfer in Tom5?TM cells.In summary, we have shown that the EMC plays an important role in mediating tethering and lipid trafficking between the ER and mitochondria, which we demonstrate for the first time are essential processes. Understanding how the EMC facilitates tethering and how tethering makes possible lipid exchange and signaling between the ER and mitochondria are important topics for future studies.4.3 TablesTable 4.1 Genetic interactions with EMC6, related to Figure 4.3.Aggravating and alleviating interactions are listed along with the strength of the interactions plotted as the ratio of the growth of the double mutant with ?emc6 versus the single mutant.Query gene Target gene Type Strength (Ratio ??/?)EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6AAH1 aggravating 0.63ASF1 aggravating 0.80ATP1 aggravating 0.19ATP25 aggravating 0.76BUR2 aggravating 0.60CHO2 aggravating 0.09CIK1 aggravating 0.77CKA2 aggravating 0.80CLA4 aggravating 0.70ECM3 aggravating 0.25ELM1 aggravating 0.64GEP4 aggravating 0.56GET1 aggravating 0.27HTD2 aggravating 0.69INO2 aggravating 0.55KAP123 aggravating 0.22LPD1 aggravating 0.67170Query gene Target gene Type Strength (Ratio ??/?)EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6MCT1 aggravating 0.70MDY2 aggravating 0.75MHR1 aggravating 0.75MRPL15 aggravating 0.57MRPL25 aggravating 0.68MRPL49 aggravating 0.75MSM1 aggravating 0.69MSO1 aggravating 0.37NPL3 aggravating 0.67PET123 aggravating 0.71PML39 aggravating 0.55PPA2 aggravating 0.71SCS2 aggravating 0.81SIP3 aggravating 0.65SLM5 aggravating 0.69SRV2 aggravating 0.78UBR2 aggravating 0.72VPS33 aggravating 0.55VPS41 aggravating 0.75AAT2 alleviating 1.43ADA2 alleviating 1.76AFT1 alleviating 1.25ASE1 alleviating 1.26ATP11 alleviating 1.23AVO2 alleviating 1.41BEM4 alleviating 1.24BRO1 alleviating 1.82BTS1 alleviating 1.22BUB3 alleviating 1.24CTK2 alleviating 1.77EAP1 alleviating 1.38GCR2 alleviating 1.22ILM1 alleviating 1.49INO4 alleviating 1.29LDB16 alleviating 1.44LEO1 alleviating 1.22MON2 alleviating 1.64MTM1 alleviating 1.48NOP16 alleviating 1.18OCA6 alleviating 1.43OPI11 alleviating 1.24PHO80 alleviating 1.55PHO85 alleviating 2.11RAD6 alleviating 2.18RPB9 alleviating 1.37RPL43A alleviating 1.29RPS10A alleviating 2.16171Query gene Target gene Type Strength (Ratio ??/?)EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6EMC6RPS19B alleviating 2.74SAN1 alleviating 1.34SER3 alleviating 1.34SIN3 alleviating 1.42SKY1 alleviating 1.21SNF6 alleviating 1.73SSO2 alleviating 1.45SUB1 alleviating 1.22SUR1 alleviating 2.55TIM18 alleviating 1.34VPS1 alleviating 1.25VRP1 alleviating 1.62ZAP1 alleviating 1.241725 Conclusions5.1 Chapter summaryIn Chapter 2, we show that the ER diffusion barrier is created by an ER-septin tether that results from the direct interaction between the septin Shs1 and the integral ER protein Scs2. We have mapped the domains responsible for this interaction and reconstituted binding in vitro with purified recombinant proteins. We then show that this binding is required to create the ER diffusion barrier in vivo. We also purify Shs1-Scs2 tethering complex from yeast, demonstrating the stable association of these proteins in vivo. Additionally, we demonstrate that a physiologically important function of the ER diffusion barrier is to restrict diffusion of the spindle from mother to bud until M phase, which affects cell growth when lost. We demonstrate that Scs2 interacts directly with the spindle capture protein Num1 via its FFAT motif (Scs2 binding element), which recruits Num1 to the peripheral ER. As an ER-associated protein, Num1 is restricted by the ER diffusion barrier, which prevents it from diffusing from the mother into the bud during S and G2 phases. Loss of the diffusion barrier (in shs1-cte cells) results in Num1 mislocalization to the bud and Num1-dependent spindle mispositioning to the bud. Finally, the aggravating genetic interactions between the shs1-cte allele and genes in the nuclear migration illustrate the importance of the ER diffusion barrier to maintaining normal cell physiology. These findings demonstrate that the ER diffusion barrier is critical for establishment of cell polarity and for cell growth.In Chapter 3, we defined a new role for PM-ER junctions in the regulation of PC synthesis in the de novo methylation pathway. We found that Opi3, the key enzyme in 173methylating PE to yeld PC, localizes to PM-ERJs and is dependent on Scs2 and Ice2. Further, we identified a non-catalytic role for the PA phosphatase Pah1 in this process. Overexpression of Pah1 rescues both PM-ERJ formation and the PC synthesis defects in ?scs2?ice2 double mutants. The lipid binding protein Osh3 also rescues the ?scs2?ice2 double mutant, perhaps by facilitating lipid transfer to Opi3 at PM-ERJs. However, it did not restore PM-ER junctions. Lastly, we found that Opi3 could act in trans in vitro, consistent with its role at PM-ERJs. Overall, this study demonstrates the possibility of localized phospholipid synthesis at PM-ER junctions. Since PM-ERJ formation occurs concurrently with cell growth, this process may efficiently couple lipid synthesis to membrane expansion during cell growth.In Chapter 4, we uncovered new components for mitochondria-ER tethering and defined the role for mito-ERJs in non-vesicular lipid traffic and phospholipid synthesis. First, we discovered a highly conserved protein complex of the ER called EMC interacts with Tom5 on the mitochondria at mito-ERJs. Mechanistically, we propose that their interactions facilitated tethering of ER and mitochondria at mito-ERJs because a 5x-EMC deletion is synthetic lethal with mutations in the ERMES complex, but is rescued by expression of the artificial ER-mitochondria tether CHiMERA. Specifically, they were required for proper transfer of PS from the ER to mitochondria. These experiments suggest that the EMC complex binding to Tom5 at the mitochondrial surface forms a membrane tether between ER and the outer membrane of mitochondria which ensures efficient PS transfer.1745.2 Common discussions5.2.1 The significance of ER polarization.Polarization of the ER likely plays important roles in many cell types. The ER travels on actin cables in squid axons (Tabb et al., 1998) and insect photoreceptors, where it rapidly translocates in response to light (Sturmer et al., 1995). Myosin V motors  are involved in these processes (Tabb et al., 1998), similar to their role in ER inheritance in yeast (Estrada et al., 2003). Natural myosin V mutations in mouse and rat result in neurological disorders where a key feature is lack of smooth ER and IP3 signalling in Purkinje cell spines (Dekker-Ohno et al., 1996; Futaki et al., 2000; Takagishi et al., 1996). Our work in yeast has shown that the ER protein Scs2 is critical for polarizing the ER (Chapters 2 and 3). The human homolog of Scs2, called VAP, has been linked to the motorneuron disease amyotrophic lateral sclerosis (ALS) (Nishimura et al., 2004). Although the pathophysiology of ALS is still not well understood, our work indicates that loss of ER polarization may be a contributing cause. Supporting evidence comes from transgenic mice displaying ALS-like motorneuron dysfunctions; the neurons in these mice not only have defects in mitochondria and microtubules but also show a loss of smooth ER in axons (Collard et al., 1995). Additionally, in another mouse model of convulsive limb movements and opisthotonic seizures, smooth ER is missing in the dendritic spine of Purkinje cells in their cerebella (Takagishi et al., 1996). Thus, polarization of ER is important for critical functions in neurons.1755.2.2 ER Polarization by the ER diffusion barrierDiffusion barriers contribute to the polarization of membranes in many well-differentiated cells. For instance, epithelial cells are polarized into apical and basolateral domains by tight junctions which prevent the lateral diffusion of plasma membrane proteins (Caudron and Barral, 2009). In the dendritic ER of neurons, membrane proteins  diffuse rapidly, but their diffusion becomes restricted at dendritic branch points by an unknown diffusion barrier (Cui-Wang et al., 2012). Mammalian septins localize to the base of dendritic spines and at dendritic branch points, and they regulate dendritic branching and spine morphogenesis (Tada et al., 2007; Xie et al., 2007), suggesting that ER-septin tethering might polarize the ER in dendrites. Polarization of the IP3 receptor in dendritic spines of mouse Purkinjie-cells is required for calcium signaling, and mutations that prevent IP3 receptor polarization cause severe ataxia in mice that mimics amyotrophic lateral sclerosis (ALS) in humans (Wagner et al., 2011). This suggests an important role for the ER diffusion barrier in nerve transmission. Lastly, septins are found at the base of many other ER-containing polarized structures including filopodia, pseudopodia, cilia, and the cytokinetic cleavage furrow (Saarikangas and Barral, 2011), suggesting that ER polarization by diffusion barriers may have widespread implications in biology.5.2.3 The significance of ER junctionsThe physiological functions of ER junctions are only beginning to be understood. pmaER, the ER component of PM-ERJs were discovered to contain an enrichment of lipid synthesizing enzymes over 10 years ago (Pichler et al., 2001), but it is only recent that the molecular details are becoming unveiled, such as in Ca2+ signaling (Carrasco 176and Meyer, 2011), in phosphoinositide metabolism (Manford et al., 2012; Stefan et al., 2011) and in phospholipid synthesis (Tavassoli et al., 2013). The first two roles suggest that PM-ERJs may be a hub for intracellular signaling that transduces signals in the PM (Ca2+, PIP ) to effector proteins in the ER network (Stefan et al., 2013). Since the gaps of PM-ERJs are narrow (<10 nm), PM-ERJs have an ideal architechture for relaying these signals efficiently (Pichler et al., 2001; West et al., 2011). Consistently, STIM-Orai interaction occurs across the junctional space, and lipid transfer proteins of the Osh family localize to PM-ERJs. In addition, lipid synthesis at ER junctions may have broader implications. For example, PC synthesis is important for human health, as it is the major phospholipid that constitutes biological membranes. Mice lacking PE methyl transferases (PEMT -/-) develop acute liver failure when fed a choline deficient diet, because hepatocytes lacking PEMTs cannot produce adequate amounts of PC, offsetting the balance of PE to PC ratios in the PM, which compromises PM membrane integrity and ultimately leads to leakage of liver enzymes, cell damage and inflammation (Koteish and Diehl, 2002). Similarly, patients with nonalcoholic steatohepatitis also have decreased PC in their hepatocytes (Li et al., 2006). Therefore, localized synthesis of phospholipids and their non-vesicular trafficking may be a viable mechanism for fine-tuning lipid composition in membranes. Moreover, mito-ERJs are important for mitochondrial dynamics. ER tubules are found to mark sites of mitochondrial fission (Friedman et al., 2011). Normal mitochondrial function and dynamics requires the delivery of amino phospholipids, such as PS synthesized in the ER, to mitochondria by non-vesicular transport pathways. 177Therefore, mito-ERJs may regulate mitochondrial biogenesis. Moreover, mito-ERJs have also been proposed to play a role in calcium signaling, suggesting diverse implications in general cellular metabolism. For example, mito-ERJs are found to be involved in apoptosis, Alzheimer's disease pathology and insulin signaling (Area-Gomez et al., 2012; Raturi and Simmen, 2013; Sebasti?n et al., 2012). Additionally, the mito-ERJ localized ubiquitin ligase Gp78 suggests a role for these contacts in protein quality control (Wang et al., 2000). All in all, studying mito-ERJs has emerged as an important area of research.178Bibliography1. Achleitner, G., Gaigg, B., Krasser, A., Kainersdorfer, E., Kohlwein, S.D., Perktold, A., Zellnig, G., and Daum, G. (1999). 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