Medicine, Faculty of
DSpace
UBCV
Sridhar, Vaishnavi
Doctor of Philosophy - PhD
University of British Columbia
Eukaryotic organelle membranes are composed of lipids and proteins. Most lipid precursors are synthesized in the endoplasmic reticulum (ER) and are transported to other membranes. Transport can proceed either via the vesicular or the non-vesicular route, primarily via lipid transport proteins at membrane contact sites (MCSs). MCSs are sites of close apposition between organelle membranes, which are present in eukaryotes including plants, fungi and animals. These sites are maintained by protein tethers, which function in lipid transport, maintenance of membrane integrity, membrane biogenesis and ion regulation. One such protein tether, Vps13, is highly conserved and functions as a bulk lipid transporter at many MCSs. Bulk transport of lipids is required to confer rapid changes in lipid composition or for membrane elongation, such as in case of generation of prospore membranes or for autophagosome formation or during endocytosis. At ER-PM MCSs, Vps13-like proteins could function in membrane elongation during endocytosis, thus making it important to identify Vps13-like proteins at these sites. I identified an uncharacterized protein Fmp27 and its paralog Ypr117w, which have secondary structure similarities to Vps13, with a potential role in lipid homeostasis at ER-PM MCSs. Fmp27 is a well conserved protein, and its mammalian ortholog is overexpressed in breast cancer, whereas its fly and plant orthologs are implicated in organismal development. I found that Fmp27 is anchored to the ER and localizes to ER-PM cortical sites. Furthermore, I identified an interacting partner, Ybl086c, which binds to Fmp27 and recruits it to these cortical sites via a conserved region in its C-terminus. I found that Fmp27/Ypr117w and Ybl086c are not required for the growth of cells in response to various stresses such as temperature, salt, osmolarity and detergents. Using fluorescent lipid probes, I found that these proteins do not have a clear role in the distribution of the glycerophospholipids phosphatidylserine (PS), phosphatidylinositol-4-phosphate (PI4P) and phosphatidylinositol-4,5-bisphosphate PI(4,5)P₂ in cells. Instead, I found that the deletion of Fmp27/Ypr117w and its binding partner confer sensitivity to the drug amphotericin B, indicating elevated levels of accessible sterols at the plasma membrane. This indicates a role for these proteins in lipid homeostasis.
1 Investigating the role of the putative lipid transport protein Fmp27 at ER-PM membrane contact sites by Vaishnavi Sridhar BS-MS (Indian Institute of Science Education and Research, Mohali) 2017 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Cell and Developmental Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2023 ©Vaishnavi Sridhar, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Investigating the role of the putative lipid transport protein Fmp27 at ER-PM membrane contact sites. submitted by Vaishnavi Sridhar in partial fulfilment of the requirements for the degree of Doctor of Philosophy In Cell and Developmental Biology Examining Committee: Additional Supervisory Committee Members: Dr. Abel Rosado, Associate Professor, Botany, UBC Supervisory Committee Member Dr. Elizabeth Conibear, Professor, Medical Genetics, UBC Supervisor Dr. Christopher Loewen, Professor, Cell and Developmental Biology, UBC Supervisory Committee Member Dr. Ivan Robert Nabi, Professor, Cell and Developmental Biology, UBC Supervisory Committee Member Dr. Franck Duong, Professor, Biochemistry and Molecular Biology, UBC University Examiner Dr. Calvin Yip, Associate Professor, Biochemistry and Molecular Biology, UBC University Examiner iii Abstract Eukaryotic organelle membranes are composed of lipids and proteins. Most lipid precursors are synthesized in the endoplasmic reticulum (ER) and are transported to other membranes. Transport can proceed either via the vesicular or the non-vesicular route, primarily via lipid transport proteins at membrane contact sites (MCSs). MCSs are sites of close apposition between organelle membranes, which are present in eukaryotes including plants, fungi and animals. These sites are maintained by protein tethers, which function in lipid transport, maintenance of membrane integrity, membrane biogenesis and ion regulation. One such protein tether, Vps13, is highly conserved and functions as a bulk lipid transporter at many MCSs. Bulk transport of lipids is required to confer rapid changes in lipid composition or for membrane elongation, such as in case of generation of prospore membranes or for autophagosome formation or during endocytosis. At ER-PM MCSs, Vps13-like proteins could function in membrane elongation during endocytosis, thus making it important to identify Vps13-like proteins at these sites. I identified an uncharacterized protein Fmp27 and its paralog Ypr117w, which have secondary structure similarities to Vps13, with a potential role in lipid homeostasis at ER-PM MCSs. Fmp27 is a well conserved protein, and its mammalian ortholog is overexpressed in breast cancer, whereas its fly and plant orthologs are implicated in organismal development. I found that Fmp27 is anchored to the ER and localizes to ER-PM cortical sites. Furthermore, I identified an interacting partner, Ybl086c, which binds to Fmp27 and recruits it to these cortical sites via a conserved region in its C-terminus. I found that Fmp27/Ypr117w and Ybl086c are not required for the growth of cells in response to various stresses such as temperature, salt, osmolarity and detergents. Using fluorescent lipid probes, I found that these proteins do not have a clear role in the distribution of the glycerophospholipids phosphatidylserine (PS), phosphatidylinositol-4-phosphate (PI4P) and phosphatidylinositol-4,5-bisphosphate PI(4,5)P2 in cells. Instead, I found that the deletion of Fmp27/Ypr117w and its binding partner confer sensitivity to the drug amphotericin B, indicating elevated levels of accessible sterols at the plasma membrane. This indicates a role for these proteins in lipid homeostasis. iv Lay Summary In eukaryotes such as humans, cells contain highly specialized compartments called organelles. Cellular organelles are constantly communicating with one another for various cellular functions. One way that organelles can communicate is via membrane contact sites (MCSs), which are sites where organelles are near one another. Proteins bridge the apposing membranes at MCSs, and mutations in these proteins prevents efficient communication between organelles. This impairs the normal functioning of cells and leads to diseases. In this study, I investigated the role of previously uncharacterized proteins at the endoplasmic reticulum (ER) and plasma membrane (PM) contact sites. I determined how these proteins localize to the ER-PM MCSs and found that they have a role in lipid homeostasis. I conducted these studies in budding yeast, since they possess organelles, pathways, and proteins similar to humans, and are easy to manipulate, making them an ideal system to use. v Preface This thesis includes unpublished data from a manuscript in preparation. Author contributions are listed below. For chapter 2 and 3, I performed all the experiments, and designed and constructed most of the strains, plasmids and oligos. Some oligos were designed by our lab manager Michael Davey, while some strains were constructed by fellow graduate students Samantha Dziurdzik and Shawn Shortill or were obtained from the yeast knock out collection. Dr. Elizabeth Conibear assisted in performing the bioinformatic analysis in Figure 2.1 and 2.5. Some of the plasmids used in chapter 2 and 3 were gifts from other labs. Manuscript details: A conserved interactor targets Fmp27/Hob1 to ER-PM contact sites for a shared role in lipid homeostasis Samantha K. Dziurdzik 1,2* , Vaishnavi Sridhar 1,3* , Sarah D. Neuman 4 , Judith Yan 1,3 , Michael Davey 1 , Stefan Taubert 1,2,3 , Arash Bashirullah 4 , Elizabeth Conibear 1,2,3¥ *Contributed equally 1 Centre for Molecular Medicine and Therapeutics, British Columbia Children’s Hospital Research Institute, University of British Columbia, Vancouver, Canada V5Z 4H4 2 Department of Medical Genetics, University of British Columbia, Vancouver, Canada V6H 3N1 3 Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada V6H 3N1 4 Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, WI 53705-2222, USA. vi Table of Contents Abstract……………………………………………………………………………………...iii Lay Summary……………………………………………………………………………….iv Preface……………………………………………………………………………………….v Table of Contents…………………………………………………………………………..vi List of Tables...…………………………………………………………………………......x List of Figures...……………………………………………………………………...….....xi List of abbreviations...………………………………………………………………........xiii Acknowledgements...…………………………………………………………………....xviii Dedication.…...…………………………………………………………………………....xix Chapter 1: Introduction……………………………………………………………………..1 1.1 Membrane lipids and lipid biosynthetic pathways………………………………...1 1.1.1 Membrane lipid composition……………………………………………………1 1.1.1.1 Techniques used to study lipid distribution in cells.................................8 1.1.2 Lipid biosynthesis is compartmentalized................................................…10 1.2 Lipid transport in cells………………………………………................................14 1.2.1 Vesicular transport……………………………………………………………..15 1.2.2 Non-vesicular transport by translocases…………………………………….16 1.2.3 Non-vesicular transport at membrane contact sites………………………..16 1.3 Lipid transport proteins are important for membrane contact site function…... 17 1.3.1 LTPs as shuttles………………………………………………………………..18 1.3.2 LTPs as lipid transfer bridges………………………………………………...19 1.4 Tethers are required for MCS formation………………………………………….21 1.5 ER structure, organization, and function in protein synthesis..........................23 1.5.1 ER structure and organization...................................................................23 1.5.2 ER function in protein synthesis................................................................24 1.6 The ER makes membrane contact sites with different organelles…………….25 1.6.1 ER-Golgi MCSs.………………………………………………………………..27 1.6.2 ER-Mitochondria MCSs...……………………………………………………..27 vii 1.6.3 ER-peroxisome MCSs…………………………………………………….......28 1.6.4 ER-endosomes/lysosomes/vacuole MCSs.…..…………………………….28 1.6.5 ER-LD MCSs…………………………………………………………………...29 1.6.6 ER-PM MCSs…………………………………………………………………..29 1.7 ER-PM MCS tether proteins.....................................……...…….….................30 1.8 Other ER-PM MCS proteins……………………………………………………….32 1.9 Vps13 is a LTP that transports lipids at various MCSs………………………..34 1.9.1 Vps13 localizes to different MCSs …………………………………………..35 1.9.2 Vps13 has glycerophospholipid transfer activity…………………………...38 1.10 Atg2 transports the lipids required for autophagosome expansion………….40 1.11 Vps13-like proteins at ER-PM MCSs…………………………………………...42 1.11.1 Csf1 is a Vps13-like protein with a conserved role in cold stress response....................................................................................................................42 1.11.2 Fmp27 and Hob2 (Ypr117w) are putative lipid transport proteins at ER-PM MCSs………………………………………………………………………………….........43 1.12 Summary and Overview………………………….……………………………...44 Chapter 2: Characterization of the interaction of Fmp27, a Vps13-like protein with its binding partner………………………………………………………………………..47 2.1 Synopsis…………………………………………………………………………....47 2.2 Introduction…………………………………………………………………………47 2.3 Results……….…………………………………………………………………......48 2.3.1 Fmp27 is a protein with structural similarities to Vps13 and localizes to ER-PM MCSs……………………………………………………………………………….48 2.3.2 Fmp27 is an ER protein that localizes to ER-PM contact sites….….........54 2.3.3 Fmp27 is recruited to cortical sites by Ybl086c…………………………….56 2.3.4 Ybl086c interacts with and recruits Fmp27 via its C-terminus……………62 2.3.5 Ybl086c interacts with and recruits Fmp27 via a conserved region in its C-terminus……………………………………………………………………………………..68 2.4 Conclusion and Discussion……………………………………………………....72 2.4.1 Fmp27 and Ypr117w are Vps13-like proteins……………………………...72 viii 2.4.2 Fmp27 localizes to the ER via its N-terminus and is recruited to ER-PM MCSs by Ybl086c………………………………………………………………………….72 2.4.3 How is Ybl086c recruited to the PM?.......................................................74 2.4.4 Do Fmp27 and Ybl086c form a complex?................................................74 2.5 Materials and Methods..……………………………………………………….......75 2.5.1 Yeast media……………………………………………………………….......75 2.5.2 Yeast strains and plasmids…………………………………………………..75 2.5.3 Bioinformatics………………………………………………………………….75 2.5.4 Fluorescence microscopy…………………………………………………….77 2.5.5 Image analysis………………………………………………………………...77 2.5.6 Co-immunoprecipitation and Western Blotting………..…………………...78 Chapter 3: Investigating the function of Fmp27/Ypr117w and their binding partner Ybl086c......................................................................................................................80 3.1 Synopsis…………………………………..………………………………………...80 3.2 Introduction………………..………………………………………………………..80 3.3 Results……………………..………………………………………………….........82 3.3.1 Fmp27/Ypr117w and Ybl086c do not have a role in the growth of cells in response to different stress conditions…………………………………………………..82 3.3.2 Fmp27/Ypr117w and Ybl086c do not have a clear role in phospholipid distribution…………………………………………………………………………………..87 3.3.3 Fmp27/Ypr117w and Ybl086c have a role in the retention of sterols in a bound state at the PM………………………………………………………................92 3.4 Conclusion and Discussion………………………………………………………101 3.4.1 Fmp27/Ypr117w and Ybl086c retain sterols in a bound state at the PM.101 3.4.2 Which glycerophospholipids are transported by Fmp27/Ypr117w?.........103 3.5 Materials and Methods.……….……………….………………………………….104 3.5.1 Yeast strains and plasmids………………...……………………………......104 3.5.2 Chemicals………...…………………………………………………………...104 3.5.3 Growth Assays………...……………………………………………………...104 3.5.4 Fluorescence microscopy…………………………………………………....105 3.5.5 Image analysis…………..............................................…………………....106 ix Chapter 4: Discussion and Future Directions………………………………………..107 4.1 Summary of key findings…………………………………………………………107 4.2 Characterizing the recruitment of Ybl086c and Fmp27 to ER-PM MCSs…..107 4.3 Investigating the role of Fmp27/Ypr117w and Ybl086c in stress response...109 4.4 Investigating the role of Fmp27/Ypr117w and Ybl086c in lipid transport…...110 4.5 Investigating the role of Fmp27/Ypr117w and Ybl086c in retaining sterols at the PM…………………………………………………………………..............................111 4.6 Characterization of the interaction and the function of Fmp27/Ypr117w and Ybl086c in other model organsims…………………………………………………...113 4.7 Limitations of the study..................................................................................116 4.8 Conclusion………………………………………………………………………....118 Appendices…………...………………………………………………………………....120 Appendix A: Supplementary material for Chapter 1……………………………….120 Appendix B: Supplementary material for Chapter 2............................................122 Appendix B1: HHpred results for the query Vps13 650-1390………………....122 Appendix B2: HHpred results for the query Vps13 APT1 domain…………….123 References…………….………………………………………………………………...139 x List of Tables Supplementary Table 1: List of yeast strains used in Chapter 2 and 3……............124 Supplementary Table 2: List of plasmids used in Chapter 2 and 3…..…................128 Supplementary Table 3: List of oligos used in Chapter 2 and 3…....……...............131 Supplementary Table 4: List of antibodies used in Chapter 2………...……............138 xi List of Figures Figure 1.1 Generic chemical structures of glycerophospholipid species in eukaryotic cells ……..…………...….…….....................................................................3 Figure 1.2 Chemical structures of glycosphingolipid species in fungal cells............5 Figure 1.3 Chemical structures of cholesterol and ergosterol .................................6 Figure 1.4 Ergosterol biosynthesis pathway in S. cerevisiae.................................11 Figure 1.5 Sphingolipid biosynthesis pathway in S. cerevisiae..............................13 Figure 1.6 Mechanisms of lipid transport between membranes and leaflets......…15 Figure 1.7 The ER makes contacts with different organelles….…..…..…..……….26 Figure 1.8 ER-PM MCS proteins……………………..………....……………………..30 Figure 1.9 Vps13 and Atg2 are LTPs that localize to different MCSs…….………..36 Figure 2.1 HHpred identifies Fmp27 and Ypr117w as Vps13-like proteins in S. cerevisiae................................................................................................................50 Figure 2.2 Predicted structures of HHpred hits and Vps13-like proteins……..…...52 Figure 2.3 Predicted structures of Atg2, Fmp27 and Ypr117w and degree of confidence of the predictions.....................................................................................53 Figure 2.4 Fmp27 localizes to cortical sites and contains a hydrophobic helix for ER localization………………………………………………………………………..........55 Figure 2.5 Ybl086c is a well-conserved protein with functional correlation to Fmp27.....................................................................................................................57 Figure 2.6 Ybl086c predicted structure and degree of confidence of the predicted structure.....................................................................................................................60 Figure 2.7 Ybl086c localizes to cortical sites and recruits Fmp27………...………..61 Figure 2.8 Interaction between Fmp27 and Ybl086c increases upon overexpression……………………………………………………………………………..63 Figure 2.9 Ybl086c interacts with Fmp27 and Ypr117w via its C-terminus….........64 Figure 2.10 The C-terminus of Ybl086c is sufficient to recruit overexpressed Fmp27 to cortical sites……………………………………............................................66 Figure 2.11 Mapping the region of Ybl086c C-terminus sufficient for recruiting Fmp27 to the PM ……………………………………………………………………….....68 Figure 2.12 Ybl086c recruits overexpressed Fmp27 to cortical sites via a conserved region in its C-terminus…………………………………………….…….......69 Figure 2.13 Ybl086c interacts with Fmp27 via a conserved region its C-terminus.71 Figure 3.1 fmp27Δ, ybl086cΔ and ypr117wΔ do not show any growth defects at different temperatures……………………………………………………………………..83 Figure 3.2 fmp27Δ, ybl086cΔ and ypr117wΔ do not show any specific phenotypes in response to NaCl and LiCl…….…………………………………………………….….85 xii Figure 3.3 fmp27Δ, ybl086cΔ and ypr117wΔ do not show any specific phenotypes in response to alkaline pH, high osmolarity conditions and an ionic detergent SDS.86 Figure 3.4 fmp27Δ, ybl086cΔ and ypr117wΔ do not show differences in PI4P localization compared to WT, BY4741…………………………………………………..89 Figure 3.5 fmp27Δ, ybl086cΔ and ypr117wΔ do not show differences in PS localization compared to WT, BY4741…………………………………………………..90 Figure 3.6 fmp27Δ, ybl086cΔ, ypr117wΔ do not show differences in PI(4,5)P2 localization compared to WT, BY4741…………………………………………………..91 Figure 3.7 fmp27Δ, ybl086cΔ and ypr117wΔ do not show sensitivity in response to fluconazole……………………………………………………………………………....93 Figure 3.8: fmp27Δypr117wΔ, ybl086cΔ and fmp27Δybl086cΔ show sensitivity in response to valinomycin……….………………………………………………………….94 Figure 3.9: fmp27Δypr117wΔ, ybl086cΔ and fmp27Δybl086cΔ show slight resistance in response to myriocin………………..……….…………………………….96 Figure 3.10: fmp27Δypr117wΔ, ybl086cΔ and fmp27Δybl086cΔ show sensitivity in response to amphotericin B………………………………………………………………97 Figure 3.11: laf1Δ, fmp27Δypr117wΔ, ybl086cΔ and fmp27Δybl086cΔ show sensitivity in response to amphotericin B………………………………………………..99 Figure 3.12: Ybl086c 15A mutant cannot rescue the amphotericin B sensitive phenotype of ybl086cΔ............................................................................................100 xiii List of abbreviations A.U.: Arbitrary units ACBD: Acyl-CoA binding domain ANOVA: Analysis of variance APT: Aberrant Pollen Transmission ATG: Autophagy-related ATP: Adenosine triphosphate BLTP: Bridge-like lipid transfer protein cER: Cortical Endoplasmic Reticulum CERS: Ceramide synthase Cers: Ceramides CoA: Coenzyme A Co-IP: Co-immunoprecipitation CSF: Cold Sensitive for Fermentation DAG: Diacylglycerol DIC: Differential interference contrast DMSO: Dimethyl sulfoxide EEIG1: Early estrogen-induced gene 1 ELO: Fatty Acid Elongation EM: Electron microscopy ER: Endoplasmic reticulum ERMES : ER-mitochondria encounter structure complex E-Syts: Extended Synaptotagmins FAM: Family FFAT: Phenylalanines in an acidic tract FL: Full Length FLARES: Fluorescent lipid-associated reporters FMP: Found in Mitochondrial Proteome FRET: Forster resonance energy transfer FYVE: Fab1, YOTB, Vac1, and EEA1 GCS: Glucosylceramide synthase xiv GFP: Green fluorescent protein GPI: Glycosylphosphatidylinositol GRAM: Glucosyltransferases, Myotubularins and other membrane associated proteins HA: Hemagglutinin HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIS: Histidine HMG-CoA reductase: 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase HMM: Hidden Markov Model HOG: High osmolarity glycerol HPH: Hygromycin resistance gene Hyg B: Hygromycin B IgG: Immunoglobulin G IM: Isolation membrane IP: Immunoprecipitate IST: Increased sodium tolerance LAM: Lipid transfer protein Anchored at Membrane contact sites LCBs: Long chain bases LD: Lipid droplets LE: Late endosome LEU: Leucine Lpt: Lipopolysaccahride transport protein LTP: Lipid transport proteins M(IP)2C: Mannosyl di-inositol phosphorylceramide. mCh: m-Cherry MCSs: Membrane contact sites MIPC: Mannosyl inositol phosphorylceramide MORN: Membrane occupation and recognition nexus MUSCLE: Multiple sequence comparison by log-expectation N.S.: No significance NAT: Nourseothricin nER: Nuclear Endoplasmic Reticulum xv NVJ: Nuclear-vacuolar junctions OD600: Optical density at 600nm OE: Over-expressed ORF: Open reading frame ORP: Oxysterol binding protein-related protein OSBP: Oxysterol binding protein OSH: Oxysterol binding protein homolog PA: Phosphatidic acid PAGE: Polyacrylamide gel electrophoresis PC: Phosphatidylcholine PCR: Polymerase chain reaction PE: Phosphatidylethanolamine PH: Pleckstrin homology PI(3)P: Phosphatidylinositol 3-phosphate PI(3,4)P2: Phosphatidylinositol 3,4-bisphosphate PI(3,4,5)P3: Phosphatidylinositol 3,4,5-triphosphate PI(3,5)P2: Phosphatidylinositol 3,5-bisphosphate PI(4)P: Phosphatidylinositol 4-phosphate PI(4,5)P2: Phosphatidylinositol 4,5-bisphosphate PI(5)P: Phosphatidylinositol 5-phosphate PI: Phosphoinositide PLC: Phospholipase C PM: Plasma membrane PMN: Piecemeal microautophagy of nucleus PMSF: Phenylmethylsulfonyl fluoride PRELI: Protein of Relevant Evolutionary and Lymphoid Interest PS: Phosphatidylserine PtdIns: Phosphatidylinositol PX: Phox homology RANK: Receptor activator of the nuclear factor-κB RANKL: Receptor activator of the nuclear factor-κB ligand xvi RBG: Repeating Beta Groove RFP: Red fluorescent protein S.E.M.: Standard error of the mean SAM: Sterile α motif SCS: Suppressor of Choline Sensitivity SD: Synthetic dextrose SDS: Sodium dodecyl sulphate SEC: Secretion defective SIP: SNF1-Interacting Protein SMP: Synaptotagmin-like Mitochondrial lipid-binding Protein SMS: Sphingomyelin synthase SOAR: STIM1 Orai activating region SOCE: Store operated calcium entry SPT: Serine-Palmitoyl Transferase StAR: Steroidogenic Acute Regulatory protein STIM: Stromal interacting molecule TCB: Tricalbin proteins TGN: Trans Golgi Network TMD: Transmembrane domain TRIAP: TP53-regulated inhibitor of apoptosis TULIP: Tubular lipid binding proteins URA: Uracil UV: Ultraviolet VAB: Vps13 adaptor binding Vac: Vacuole partitioning VAP: Vesicle-associated membrane protein-associated protein vCLAMPs: Vacuole-mitochondrial patches VPS: Vacuolar protein sorting WB: Western blot WCL: Whole Cell Lysate WT: Wild type xvii YPD: Yeast extract, peptone, dextrose YSP: Yeast Suicide Protein xviii Acknowledgements I would like to thank my supervisor Dr. Elizabeth Conibear for her support, guidance, insights, and fruitful discussions towards the completion of this work. I am grateful for the support received from my lab members, both past and present. My committee members and the CELL Program were instrumental in providing much needed support to complete my PhD. I am grateful to SERB (DST, India) for funding my studies and giving me an opportunity to pursue my studies. It would not have been possible for me to pursue my PhD otherwise. I am also grateful to St. John’s College, UBC for providing me with a great place to live and develop. I would also like to acknowledge that all my work was done on the traditional, unceded and ancestral lands of the Musqueam First Nations, wherein I was able to work, learn and play. I would like to thank all my friends for helping me through this journey and for their immense support. My parents, brother and Karan have helped and guided me through each step of this process, and I feel blessed. Last but not the least, I am grateful to the almighty, the supreme being. xix Dedicated to my parents, brother and Karan Sarve Krishnaarpanam astu 1 Chapter 1 1. Introduction Eukaryotic cells are compartmentalized into organelles, which are enclosed by membranes. These membranes are made of lipids and proteins. Organelles have specialized functions and need to communicate for the exchange of ions, lipids, and proteins (Scorrano et al., 2019). The communication between organelles takes place by different mechanisms that involve specific cellular machinery. Defects in the machinery involved in organelle communication result in dysfunction and disease. In the following sections, I highlight the different membrane lipids, their synthesis and transport mechanisms. I further describe how specific organelles contact each other. I conclude by explaining specific examples of proteins involved in inter-organelle communication, their importance, and my research objectives. 1.1 Membrane lipids and lipid biosynthetic pathways Organelle membranes are made up of different classes of lipids (Hankins et al., 2015), and many lipid precursors are primarily synthesized in the endoplasmic reticulum (ER) by enzymes (Fagone and Jackowski, 2009; Holthuis and Menon, 2014). The differential lipid composition of membranes is important for various membrane properties and for the association of proteins with membranes. In section 1.1.1, I describe the different membrane lipids and their distribution in eukaryotic cells. This section additionally describes the contribution of lipids to different membrane properties. Subsection 1.1.1.1 describes some commonly used methods to study lipid distribution in cells. In section 1.1.2, I describe lipid biosynthetic pathways and the enzymes involved in these pathways. 1.1.1 Membrane lipid composition Most eukaryotic membranes are fluid lipid bilayers composed of different proportions of the three major classes of lipids, which are glycerophospholipids, sphingolipids and sterols (van Meer et al., 2008). These lipids contribute to various membrane functions, such as maintaining membrane curvature, membrane thickness, membrane fluidity, membrane permeability, membrane integrity and interaction with proteins. Most lipids 2 contain hydrophobic tails and a polar head group, and hence are amphipathic. The hydrophobic tails of lipids form the interior of the fluid lipid bilayers, while the polar head groups face the cytosol or extracellular space. Most organelle membranes are made up of two leaflets, an inner leaflet, and an outer leaflet. The two leaflets in membrane bilayers have different compositions of lipids, which is more pronounced in case of some membranes (van Meer et al., 2008). Glycerophospholipids make up the majority of all organelle membranes and contain two fatty acid chains and a phosphate head group ester attached to a glycerol backbone. Fatty acid chains can differ in their length, the number of double bonds, the position of double bonds and hydroxylation (Holthuis and Menon, 2014). Additionally, a variety of phospho-head groups, which are either uncharged or charged are incorporated into glycerophospholipids, such as phosphate, choline, serine, inositol, glycerol and ethanolamine, to respectively give rise to phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidyl glycerol (PG) and phosphatidylethanolamine (PE) (Holthuis and Menon, 2014) (Figure 1.1). PC is the most abundant lipid in organelle membranes, while PE and PG are mainly found in mitochondrial membranes. The inositol group in PI can be further phosphorylated at different positions to generate PI(3)P, PI(4)P, PI(4,5)P2, PI(3,5)P2, PI(3,4)P2 and PI(3,4,5)P3. PA, PS, PE, PI and its derivatives are negatively charged and hence, termed anionic glycerophospholipids. Organelle membranes are enriched in PI derivatives, which contribute to specific membrane signatures, such as PI4P at the PM and Golgi, PI(4,5)P2 at the PM, PI3P at endosomes and PI(3,5)P2 at lysosomes/vacuoles (Di Paolo and De Camilli, 2006; Holthuis and Menon, 2014). The inner leaflet of the PM is enriched in charged glycerophospholipids such as PA, PE, PS, PI(4)P and PI(4,5)P2 (van Meer, 2008). Various proteins interact with these lipids via their basic domains to associate with organellar membranes (Lemmon, 2008). 3 Figure 1.1: Generic chemical structures of glycerophospholipid species in eukaryotic cells Indicated here are the chemical structures of various glycerophospholipids (PA, PI, PS, PE and PC) in eukaryotic cells. The length and saturation of the fatty acid chains can vary. The glycerophospholipid backbone is shown in black and the head groups attached to the glycerophospholipid background (hydrogen, inositol, serine, ethanolamine and choline respectively) are shown in red. Henry et al, Figure 1. From Henry, S. A., Kohlwein, S. D., & Carman, G. M. (2012). Metabolism and regulation of glycerolipids in the yeast 4 Saccharomyces cerevisiae. Genetics, 190(2), 317–349. https://doi.org/10.1534/genetics.111.130286. Used with permission from RightsLink / Elsevier. Sphingolipids contain fatty acids with either no, or few double bonds linked to a serine backbone (Figure 1.2). Fatty acids and the serine backbone they are linked to together make up the structure of ceramide, which is the basic structural unit of sphingolipids. In addition to the ceramide backbone, sphingolipids either contain a PC head group in the case of sphingomyelin or saccharides in the case of glycosphingolipids in mammals (Holthuis and Menon, 2014). Yeast contain inositol phosphosphingolipids instead of sphingomyelin, which are synthesized by the addition of inositol phosphates to ceramide (Fakas, 2017). These include inositol phosphorylceramide (IPC), mannosyl inositol phosphorylceramide (MIPC) and mannosyl di-inositol phosphorylceramide M(IP)2C (Megyeri et al., 2016). Sterols are another class of lipids in cellular membranes. They are apolar and contain a core formed by four fused rings (steroid backbone), with a hydroxyl group and a small branched hydrophobic tail (Figure 1.3). Cholesterol, ergosterol and phytosterol are respectively the major sterols in mammals, fungi, and plants (Hannich et al., 2011). 5 Figure 1.2: Chemical structures of glycosphingolipid species in fungal cells (A) Indicated here is the structure of a neutral glycosphingolipid, which is made up of a ceramide backbone linked to a sugar residue, highlighted in red. The ceramide backbone is made up of a sphingoid base, and a fatty acid chain, highlighted in blue and black respectively. (B) Indicated here is the structure of IPC, a simple acidic glycosphingolipid. Highlighted in orange are the structural features that differentiate a neutral and an acidic glycosphingolipid, i.e., an additional OH group, lack of a methyl group and unsaturation. Additionally, acidic glycosphingolipids are made up of a very long fatty acid (highlighted in purple) compared to shorter acyl chains found in neutral glycosphingolipids. Fernandes et al, Figure 1. From Fernandes Caroline Mota, Goldman Gustavo H., & Del Poeta Maurizio. (2018). Biological Roles Played by Sphingolipids in Dimorphic and Filamentous Fungi. MBio, 9(3), e00642-18. https://doi.org/10.1128/mBio.00642-18. https://creativecommons.org/licenses/by/4.0/. A B 6 Figure 1.3: Chemical structures of cholesterol and ergosterol Indicated here are the chemical structures of cholesterol and the fungal sterol, ergosterol. Both cholesterol and ergosterol contain four hydrocarbon rings fused to an acyl chain, and a 3’ hydroxyl group. The chemical structure of ergosterol differs from that of cholesterol by the presence of two additional double bonds and a methyl group in the acyl chain.Tierney et al, Figure 1. From Tierney, K. J., Block, D. E., & Longo, M. L. (2005). Elasticity and Phase Behavior of DPPC Membrane Modulated by Cholesterol, Ergosterol, and Ethanol. Biophysical Journal, 89(4), 2481–2493. https://doi.org/10.1529/biophysj.104.057943. Used with permission from RightsLink / Elsevier. Sterols are shielded from the aqueous cytosol and extracellular interface by interacting with other lipids such as sphingolipids and phospholipids such as PC and PS (Maekawa and Fairn, 2015; Ramstedt and Slotte, 1999). Moreover, it was shown using fluorescent sterols and an impermeable fluorescence quencher that the inner leaflet of the PM in S. cerevisiae is enriched in sterols compared to the outer leaflet (Solanko et al., 2018). In mammalian cells, using a bacterial pore-forming toxin that binds to sterols, it was found that there are three pools of sterols (Das et al., 2014). 7 These include an accessible pool and an inaccessible pool. The inaccessible pool can be further divided into sterols that interact with sphingolipids and sterols that interact with phospholipids (Das et al., 2014). Moreover, lipid asymmetry at the PM has been found to be essential for the retention of ergosterol in the yeast PM (Kishimoto et al., 2021). Further, in mammalian cells it was shown that PS is required for the retention of sterols in the inner leaflet using probes that interact with the sterols in the two leaflets of PM (Maekawa and Fairn, 2015). Thus, the interaction between different lipid species is required for cellular homeostasis. The PM in eukaryotic cells is enriched in sterols, sphingolipids, and saturated phospholipids (van Meer, 2008). The outer leaflet is enriched in sphingolipids and PC, while the inner leaflet is enriched in PS, PE, PA and PI species (van Meer, 2008). The ER membrane primarily contains unsaturated phospholipids and a small percentage of sterols and sphingolipids. It was shown through studies of vesicles with different lipid compositions, combined with molecular dynamics simulations, that the lipid composition of membranes is tightly linked to membrane thickness (Frallicciardi et al., 2022). Membranes containing a higher percentage of sterols, phospholipids with longer chain lengths and a higher degree of saturation were observed to be thicker than those with lower amounts of sterols, phospholipids with shorter chain lengths and lower degree of saturation (Frallicciardi et al., 2022). Hence, the PM is thicker than the ER membrane. The thickness of membranes might influence the lengths of the membrane spanning regions of proteins (transmembrane domains). In fact, it was found that lengthening the transmembrane domain (TMD) of proteins missorts mutant cytochrome b5 protein from the ER to the PM (Pedrazzini et al., 1996). More recently, Sharpe et al., (2010), found that the lengths of the transmembrane domains of proteins spanning the ER membrane are shorter compared to those spanning the PM. This was shown to be consistent between S. cerevisiae and H. sapiens proteins. In this study, a dataset of known transmembrane proteins containing single transmembrane domains (TMDs), and experimentally documented localization and topology was obtained from the literature. The sequences of the TMDs of the proteins was obtained using scanning algorithms and aligned to determine their lengths (Sharpe 8 et al., 2010). This shows how the membrane lipid composition influences protein localization in cells. Another property of membranes that the lipid composition of membranes influences is membrane curvature. It has been found that an addition of excess anionic lipids, such as PS, or the removal of cholesterol from membranes, increases the charge density of membranes and hence induces membrane curvature (Hirama et al., 2017). The negatively charged headgroups of anionic lipids repel one another due to close proximity (Hirama et al., 2017). This results in the curving of membranes and membrane interaction with specific proteins, which specifically interact with curved membranes. In cells supplemented with PS and in cells in which sterols were extracted using methyl-β-cyclodextrin, PS was redistributed from the PM to internal structures (Hirama et al., 2017). Furthermore, PS containing PM invaginations were generated in these cells. This indicates a role for membrane lipid composition in membrane curvature. 1.1.1.1 Techniques used to study lipid distribution in cells Some commonly used techniques used to study lipids in cells include lipidomics, using fluorescent lipids, lipid binding toxins, radioactive labelling and subcellular fractionation, antibodies, and fluorescent lipid-associated reporters (FLARES). Lipidomics employs mass spectrometry-based techniques to analyse the lipid content of cells (Han, 2016). This technique is useful to observe any alterations in the levels of cellular lipid species in mutant cells under different conditions. However, the various methods used to recover or extract lipids could influence the amount of lipids extracted. It is thus possible that some lipid species that are present in low abundance are barely detected in test samples (Bhaskar et al., 2022). Using appropriate controls or internal standards is very important to eliminate any variability that could occur due to the different methods used to recover lipids or the yield of lipids. However, internal standards might not be present for all the different lipid species found in the test samples. In the absence of appropriate internal standards, i.e., molecules with similar chemical, ionization, and fragmentation characteristics, differentiating between various lipid species is very difficult (Bhaskar et al., 2022). Furthermore, interpreting 9 data from complex samples could prove to be challenging. Fluorescent lipids are either lipids that can fluoresce naturally or lipids attached to fluorescent molecules. Even though these lipids help visualize the changes in lipid distribution, the fluorescent molecules have different properties compared to native lipids, which can affect the function of lipids in cells (Maekawa and Fairn, 2014). Fluorescently labelled or radiolabelled lipid binding toxins can be used to visualize lipids in the external leaflet of the PM. However, these toxins need to be modified so that they are not toxic. Additionally, fluorescently tagged versions of specific domains of pore forming toxins that bind to lipids can be either used with fixed cells or can be genetically encoded and used in live cell imaging (Maekawa and Fairn, 2014). Introducing radiolabelled lipid precursors to cells and combining it with sub-cellular fractionation can help determine how lipids traffic between membranes. However, sub-cellular fractionation can be cumbersome and obtaining pure fractions of membranes can be hard (Reinisch and Prinz, 2021). Antibodies are available for very few lipids and can be used only for fixed cells, preventing their use in understanding dynamic processes in cells (Maekawa and Fairn, 2014). Lipid FLARES are fluorescent lipid probes that localize to cellular membranes depending on their affinity for specific lipids. These probes include known lipid-binding protein domains fused to fluorescent molecules. Some notable examples of lipid FLARES used in yeast include GFP-P4C (PI4P-binding P4C domain of the Legionella virulence factor SidC; Luo et al., 2015), mCherry- or GFP-2xPH(PLCδ) (two tandem repeats of the PI(4,5)P2-binding PH domains of PLCδ), GFP-C2 (PS-binding C2 domain of Lactadherin; Yeung et al., 2008), GFP-evt-2PH (PS-binding PH domain of Evectin-2; Uchida et al., 2011), GFPenvy-D4H (derivative of the D4 sterol-binding domain of bacterial toxin perfringolysin O; Johnson et al., 2012; Kishimoto et al., 2021), GFP-C1 (DAG-binding C1 domain of protein kinase C δ; Ganesan et al., 2016), lysenin (a sphingomyelin binding pore forming toxin from earthworms; Nakai et al., 2000; Yamaji et al., 1998) and the PA-binding domains of Spo20 (Nakanishi et al., 2004) and Opi1 (Loewen et al., 2004). However, lipid FLARES could preferentially localize to membranes enriched in specific lipid species, other than the lipid species 10 that they detect, and can sequester lipids, affecting the functions of lipids in cells (Maekawa and Fairn, 2014). 1.1.2 Lipid biosynthesis is compartmentalized Most lipid biosynthetic and modification enzymes are present in the ER, while other lipid-synthesizing enzymes are present in non-ER organelles. Lipid biosynthetic pathways thus require the transport of lipid precursors between organelles. Most lipids are synthesized from acetyl-CoA. Cholesterol synthesis involves the conversion of acetyl-CoA to acetoacteyl-CoA, from which HMG-CoA is synthesized in the cytosolic leaflet of the ER (Holthuis and Menon, 2014). HMG-CoA is converted to mevalonate by HMG-CoA reductase (HMGCR) in a rate-limiting step, which regulates the abundance of sterols synthesized. The mevalonate synthesis pathway is conserved throughout eukaryotes (Jordá and Puig, 2020). A series of intermediates are produced resulting in the synthesis of cholesterol. In S. cerevisiae, ergosterol synthesis can be divided into three modules (Figure 1.4). The first module or the mevalonate pathway involves the synthesis of mevalonate from acetyl-CoA. This occurs in the vacuole and mitochondria. In the second module, mevalonate is converted to farnesyl pyrophosphate (farnesyl-PP), which is carried out in the vacuole. The last module is the late pathway, which is carried out mainly in the ER (Jordá and Puig, 2020). 11 Figure 1.4: Ergosterol biosynthesis pathway in S. cerevisiae The major steps in the ergosterol biosynthesis pathway in yeast are indicated in this figure. The different color boxes represent the three parts into which the pathway is divided. The mevalonate pathway is indicated in green and occurs in the vacuole and mitochondria; the farnesyl pyrophosphate (farnesyl-PP) biosynthesis pathway is indicated in blue and occurs in the vacuole and the late pathway is indicated in orange and occurs mainly in the ER. Farnesyl-PP is also used to synthesize ubiquinone, dolichol, heme and prenylated proteins, indicated in the grey box. The enzymes involved in the different steps, the intermediates generated, inhibitors and the requirements of oxygen, heme and iron molecules for different steps are indicated. Jordá and Puig, Figure 1. From Jordá, T., & Puig, S. (2020). Regulation of Ergosterol Biosynthesis in Saccharomyces 12 cerevisiae. Genes, 11(7), 795. https://doi.org/10.3390/genes11070795. http://creativecommons.org/licenses/by/4.0/. The first step in sphingolipid synthesis involves a condensation reaction between serine and palmitoyl-CoA, which is catalyzed by serine palmitoyltransferases Lcb1/2 and Tsc3 in yeast and serine palmitoyltransferase (SPT) in mammals, which results in 3-ketodihydrosphingosine in yeast and dihydrosphingosine in mammals (Blom et al., 2011; Holthuis and Menon, 2014). This step takes place at the cytosolic face of the ER. In yeast, 3-ketodihydrosphingosine is reduced to dihydrosphingosine (Toume and Tani, 2016). This step is followed by the synthesis of long chain bases (LCBs), which are converted to ceramides (Cers), a major intermediate in sphingolipid biosynthesis (Figure 1.5). Ceramide synthesis is catalyzed by ceramide synthase (CERS). Ceramides are then transported to the Golgi, where they are modified into sphingomyelin by sphingomyelin synthase (SMS) or glucosylceramide by glucosylceramide synthase (GCS) in mammals. In yeast, dihydrosphingosine and Cers containing dihydrosphingosine are hydroxylated. IPC, the simplest complex sphingolipid in yeast is generated by the transfer of the head group of phosphatidylinositol to Cers, MIPC is generated by an addition of mannose to IPC and M(IP)2C is generated by the addition of another phosphatidylinositol to MIPC (Toume and Tani, 2016). Sphingomyelin, glucosylceramide, and complex sphingolipids are transported from the Golgi to the PM, where they are enriched (Fagone and Jackowski, 2009). Thus, the synthesis of sphingolipids requires lipid transport between the ER and Golgi. 13 Figure 1.5: Sphingolipid biosynthesis pathway in S. cerevisiae The major steps in the sphingolipid biosynthesis pathway in yeast and the enzymes involved are indicated in this figure. Cers; Ceramides, IPCs; inositol phosphorylceramide, LCBs; Long chain bases, MIPC; mannosyl inositol phosphorylceramide, M(IP)2C; mannosyl di-inositol phosphorylceramide. Toume and Tani, Figure 1. From Toume, M., & Tani, M. (2016). Yeast lacking the amphiphysin family protein Rvs167 is sensitive to disruptions in sphingolipid levels. The FEBS Journal, 283(15), 2911–2928. https://doi.org/10.1111/febs.13783. Adapted with permission from RightsLink / Elsevier. PE, PS and PI are synthesized from PA by the cytidine diphosphate diacylglycerol (CDP-DAG) pathway (Fakas, 2017). PS synthesized in this way is transported to the mitochondria, where it is converted to PE by the PS decarboxylase enzyme and a part of the PE population is transported back to the ER (Holthuis and Menon, 2014). PC can be synthesized either by the methylation of PE (in yeast and mammalian 14 systems) or via the Kennedy pathway. Kennedy pathway is the only de novo pathway for PC synthesis in most eukaryotic cell types (McMaster, 2018). PI is transported to different organelles where it is converted into its derivatives via specific PI-kinases, which are localized to the specific membranes where the PI derivatives are enriched (Di Paolo and De Camilli, 2006; Fagone and Jackowski, 2009). These derivatives are transported to the ER, where they are dephosphorylated by phosphatases to maintain the ER-pool of PI (Di Paolo and De Camilli, 2006; Fagone and Jackowski, 2009). 1.2 Lipid transport in cells After lipids have been synthesized in either the ER or other organelle membranes, they need to be transported to their destination membranes. Moreover, lipids need to be transported between the leaflets of membranes to maintain the distinct composition of membrane leaflets. Lipids can be transported by a variety of mechanisms, such as vesicular transport, non-vesicular transport to specific leaflets by ATPases and non-vesicular transport at membrane contact sites (MCSs) (Figure 1.6). In the sections below, I highlight the various methods of lipid transport between organelle membranes and between membrane leaflets in cells. In section 1.2.1, I briefly describe vesicular transport and the factors involved in this mode of transport. In section 1.2.2, I describe the non-vesicular transport between membrane leaflets and the proteins that mediate this mode of transport. In section 1.2.3, I describe non-vesicular transport at MCSs and the key classical experiments that provided evidence for this mode of transport. 15 Figure 1.6: Mechanisms of lipid transport between membranes and leaflets. This figure indicates the different mechanisms of lipid transport between membranes or leaflets, which includes vesicular transport and non-vesicular transport. Further highlighted are the modes of non-vesicular transport such as, transport between leaflets by translocases and lipid transporter (LTP)-mediated transfer. Stefan et al., Figure 1. Modified from Stefan, C. J., Trimble, W. S., Grinstein, S., Drin, G., Reinisch, K., De Camilli, P., Cohen, S., Valm, A. M., Lippincott-Schwartz, J., Levine, T. P., Iaea, D. B., Maxfield, F. R., Futter, C. E., Eden, E. R., Judith, D., van Vliet, A. R., Agostinis, P., Tooze, S. A., Sugiura, A., & McBride, H. M. (2017). Membrane dynamics and organelle biogenesis-lipid pipelines and vesicular carriers. BMC biology, 15(1), 102. https://doi.org/10.1186/s12915-017-0432-0. https://creativecommons.org/licenses/by/4.0/legalcode. 1.2.1 Vesicular transport Vesicular transport of lipids or lipid intermediates synthesized in the ER to other organelles requires that these organelles be a part of the endomembrane system, which includes the Golgi apparatus, the PM, endosomes, and lysosomes. Additionally, 16 vesicular transport of lipids requires metabolic energy and an intact cytoskeleton in mammalian cells (Lev, 2012). Vesicular transport is mediated by fluid filled sacs called vesicles, which transport lipids and proteins from a donor compartment to an acceptor compartment. Lipids are either packaged into vesicles along with proteins or form a part of the lipid membrane that encloses the vesicle. After vesicles are packaged at donor sites, they bud off and are targeted to specific acceptor sites, where they fuse and deliver their contents. Lipids including sterols, ceramide and phospholipids are partially transported by vesicular transport (Funato et al., 2020). 1.2.2 Non-vesicular transport by translocases Non-vesicular transport of lipids between the two leaflets of a membrane bilayer is mediated by translocases such as flippases, floppases and scramblases. Flippases are proteins that catalyze the transfer of specific phospholipids from the extracellular/luminal leaflet to the cytosolic leaflet, while floppases catalyze the reaction in the opposite direction, and scramblases act in both directions (Hankins et al., 2015). Flippases and floppases use energy in the form of ATP to mediate lipid transfer, while scramblases act in an ATP independent manner. Distinct leaflet lipid compositions are required for specific proteins to associate with membranes and for signal transduction (Hankins et al., 2015). An example of a phospholipid that is transported between leaflets is PS. PS is synthesized in the cytosolic leaflet of the ER and is translocated rapidly by a scramblase to be enriched in the luminal leaflet (Holthuis and Menon, 2014). It is flipped to the cytosolic leaflet in the Golgi and maintains this orientation when transported to the PM (Hankins et al., 2015). Thus, flippases and other lipid translocases transport phospholipids like PS between leaflets, to maintain asymmetrical leaflet composition. 1.2.3 Non-vesicular transport at membrane contact sites Non-vesicular transport of lipids between organelle membranes occurs at membrane contact sites (MCSs), which are sites of close apposition (10-30nm) between organelle membranes (Lev, 2012). Non-vesicular transport at MCSs is especially important when organelles are not connected by vesicular transport. This form of transport is required to make rapid changes to the lipid composition of membranes, 17 without any changes in membrane protein composition (Wong et al., 2019). Initial indications for the non-vesicular transport of lipids came from studies in which cells were able to transport lipids faster than the transport of proteins and even on the blockage of the secretory pathway (Sleight and Pagano, 1983). In mammalian cells using radiolabelled PE precursors, it was shown that the transport of PE to the PM occurred faster than the transport of proteins, providing evidence for the transport of lipids independent of protein transport (Sleight and Pagano, 1983). This study further showed that this form of lipid transport did not require energy or the cytoskeletal network by using energy poisons and drugs to inhibit the cytoskeletal network respectively. Moreover, blocking the protein secretion through the Golgi by using BrefeldinA did not impact the transport of newly synthesized PE from either the ER or from the Golgi to the PM (Vance et al., 1991) and only slightly slowed down the transport of newly synthesized cholesterol to the PM (Kaplan and Simoni, 1985). In yeast cells, a deletion of Sec18, a protein required for most steps of the secretory pathway, did not alter the transport of sterols from the ER to the PM (Baumann et al., 2005). These studies provided evidence for the non-vesicular transport of lipids. 1.3 Lipid transport proteins are important for membrane contact site function Lipid transport proteins (LTPs) are found in both prokaryotes and eukaryotes. LTPs are required for the efficient non-vesicular transport of the three major classes of lipids between organelles at MCSs in eukaryotes. Eukaryotic LTPs were originally believed to be extracellular, until many intracellular LTPs were identified. Most of these intracellular LTPs localize to MCSs. A list of intracellular LTPs found in humans and yeast have been listed in Appendix A. There are 27 LTP families, with members of the same family possessing different lipid specificities (Wong et al., 2019). LTPs contain lipid transfer domains, which house a hydrophobic cavity to shield the lipid species from the cytosolic environment and ensure efficient transport. Many LTPs contain additional membrane localization domains or interact with the vesicular associated membrane protein (VAMP)-adaptor protein (VAP) proteins to tether apposing organelle membranes at MCSs (Kumar et al., 2018; Loewen et al., 2003; Wu et al., 2018). VAP proteins are highly conserved ER-resident proteins that interact 18 with proteins containing FFAT (two phenylalanines in an acidic tract) motifs (Loewen et al., 2003). These proteins have been further explained in section 1.7. LTPs can either move lipids one at a time between membranes, as shuttles or multiple lipids at a time, as lipid bridges (Wong et al., 2019). In section 1.3.1, I describe LTPs that function as shuttles and in section 1.3.2, I describe LTPs that function as bridges. Additionally, in both sections I describe the different lipid transfer domains and the lipids transferred by the shuttle and bridge-like LTPs with specific examples. 1.3.1 LTPs as shuttles LTPs that shuttle lipids between membranes can either adopt a box-like or rod-like conformation (Wong et al., 2019). LTPs that adopt a box-like conformation with a lid to shield their central hydrophobic cavity shuttle lipids to mediate lipid exchange at MCSs (Figure 1.6). However, some LTPs that fold into rod-like structures , which might not be long enough to span the distance between two membranes, could also function as shuttles. Shuttle-like LTPs in Gram negative bacteria are required for the transport of lipids across the periplasm. These include the Lol protein complex that shields the lipid chains in lipoproteins and transports them from the inner membrane to the outer membrane. This complex is composed of five units LolA-E. LolA shuttles lipoproteins (Matsuyama et al., 1995; Yakushi et al., 1998) and LolCDE use energy from ATP hydrolysis to release lipoproteins from the inner membrane and transfers them to LolA (Yakushi et al., 2000). Lipoproteins are transferred from LolA to LolB, which inserts lipoproteins into the outer membrane. In eukaryotes, the lipid transfer domains in the box-like LTPs first dock at donor membranes and the lid can either move from a closed conformation to an open conformation or from a partially open conformation to a fully open conformation to extract lipids from the membrane. The lipid transport domain then undocks from the donor membrane, diffuses cytosolically and then docks onto an acceptor membrane to deposit the lipid (Wong et al., 2019). If LTPs exchange two lipid species, the docking, extraction, undocking, diffusion, and deposition steps from the acceptor membrane follow. 19 Some LTP families where the LTPs fold into box-like structures and act as shuttles include the StARkin superfamily, OSBP-related protein (ORP) family and Sec14 protein (Appendix A; Wong et al., 2019). The StARkin superfamily includes the StAR-related transfer (StART) family that transports sterols and ceramides, phosphatidylinositol transfer proteins (PITPs) that transfer PI/PA or PI/PC and Protein of Relevant Evolutionary and Lymphoid Interest (PRELI) domain-containing proteins that bind PA, PS or both (Wong et al., 2019). The ORP family includes OSBP, its related proteins and homologs. These proteins are involved in the counter-transport of PI4P down its concentration gradient and another ligand, which could be PS or sterol or another lipid against its concentration gradient (Tong et al., 2013; Wong et al., 2019). The Sec14 proteins have been found to transport PI/PC by the shuttle mechanism in yeast (Sugiura et al., 2019). To ensure directional transport by shuttle LTPs, the transported lipids are either dephosphorylated by phosphatases, or converted into other intermediates, or associate with other lipids or proteins at acceptor membranes. This ensures that the LTP does not return to the donor membrane with the deposited lipid. 1.3.2 LTPs as lipid transfer bridges Some LTPs adopt a rod-like conformation and have an open ended elongated hydrophobic cavity that runs along the length of the protein (Figure 1.6). Many of these LTPs are multimers and the different subunits together form the hydrophobic lipid transport bridge. For example, the bacterial lipopolysaccharide transport protein (Lpt) assembles as two membrane sub-complexes to transport lipopolysaccharide from the inner to the outer membrane in Gram negative bacteria (Suits et al., 2008; Wong et al., 2019). The Lpt complex includes seven proteins, which are LptA-G. This complex uses energy in the form of ATP to transport lipopolysaccharide (Freinkman et al., 2012). LptBFGC is an inner membrane transporter that is physically connected to LptDE in the outer membrane by LptA, a periplasmic protein (Freinkman et al., 2012). LptC, A and D form the lipid bridge required to ferry proteins from the inner membrane to the outer membrane. It is thus possible that lipid bridges in eukaryotes could also use energy in the form of ATP to transport lipids at MCSs. 20 In eukaryotes, the TULIP and N-chorein domain-containing proteins form rod-like structures to transport lipid moieties between apposing membranes at MCSs (Lees and Reinisch, 2020; Reinisch and De Camilli, 2016; Wong and Levine, 2017). Intracellular TULIPs include the Synaptotagmin-like mitochondrial-lipid-binding domain (SMP)-containing proteins (Reinisch and De Camilli, 2016; Wong and Levine, 2017; Wong et al., 2019). SMP domains dimerize head-to-head and form lipid transport bridges. In the case of the mammalian E-Syts, which tether ER and PM, the bridges formed by SMP domain dimers are shorter than the distance between membranes at MCSs. Hence, it has been proposed that E-Syts shuttle lipids between membranes instead (Reinisch and De Camilli, 2016; Saheki and De Camilli, 2017). The ER-mitochondria encounter structure (ERMES) complex tethers the ER and mitochondria in yeast and consists of four members, of which three; Mmm1, Mdm12 and Mdm34 contain SMP domains. These domains not only form head-to-head dimers, but also heterotetramers that can transfer glycerophospholipids (AhYoung et al., 2015). It is also possible that the lipid bridge formed by SMP domains in the ERMES complex may not be static and could act as a shuttle to transport lipids at MCSs (AhYoung et al., 2015). Hence, both shuttle and lipid bridge mechanisms have been suggested for the SMP-domain containing proteins. N-chorein domains in the well-conserved S.cerevisiae Vps13 and Atg2 proteins have been found to transport lipids in bulk using lipid transfer assays. These assays used fluorescent lipid containing liposomes and FRET to determine whether Vps13 and Atg2 could transfer lipids between donor and acceptor liposomes (Kotani et al., 2018; Kumar et al., 2018; Lees and Reinisch, 2020; Li et al., 2020; Osawa et al., 2019). This domain forms a scoop-like structure with a hydrophobic cavity that can bind tens of glycerophospholipids. The full-length structure of the lipid bridge in these proteins is predicted to be composed of multiple such scoop-like regions. Other Vps13-like proteins have been identified since (Castro et al., 2022; John Peter et al., 2022; Neuman et al., 2022a; Toulmay et al., 2022). These have recently been grouped into a superfamily called the Repeating Beta Groove (RBG) Superfamily (Levine, 2022). These proteins and their functions will be discussed in sections 1.9, 1.10 and 1.11. 21 1.4 Tethers are required for MCS formation A prerequisite for lipids to be transported by LTPs at MCSs is that the apposing membranes be in close proximity. MCSs are bridged by protein tethers in cells. These proteins differ from LTPs in that they maintain MCSs and might not be required for lipid transport and could instead have other functions in the cell. However, any protein that localizes to MCSs or has domains that could localize to apposing membranes could function in tethering membranes. Many artificial tethers have been developed using protein domains that localize to different membranes from non-MCS proteins (Quon et al., 2018). These artificial tethers could rescue the growth defects of mutants lacking six ER-PM MCS tether proteins (Quon et al., 2018). This means that tethers can non-specifically function to tether membranes irrespective of other functions. Many tethers function as LTPs and many LTPs function as tethers. For example, an overexpression of VPS13A , a bulk lipid transporter, in HeLa cells has been found to increase ER-mitochondrial contacts (Kumar et al., 2018). This is characteristic of protein tethers, whose overexpression leads to an increase in contacts between organelles. Furthermore, in yeast, a deletion of both the ERMES complex (an ER-mitochondria tethering complex) and Vps13 renders cells inviable (Lang et al., 2015). This indicates that both the ERMES complex and Vps13 could function in tethering the ER and mitochondria, and a deletion of these proteins abrogates these contacts. The ERMES complex and Vps13 are implicated in lipid transport and could not only function as tethers at ER-mitochondria MCSs but also in lipid transport between these organelles. Tethers are modular in structure (Eisenberg-Bord et al., 2016). They are either a single protein with different domains that bind apposed membranes or proteins that interact with each other to bridge apposing membranes at MCSs. Deletion of tethers could lead to an abolishment of contacts and their overexpression to expansion (Eisenberg-Bord et al., 2016). For example, the deletion of Nvj1 and Vac8, which form the nuclear-vacuolar junction (NVJ), leads to an abolishment of the NVJ and overexpression of these proteins leads to its expansion (Pan et al., 2000). Some MCSs could require 22 multiple tethers for their formation, for example, at least six tether proteins are required to maintain ER-PM MCSs in yeast (Manford et al., 2012; Quon et al., 2018). Tether proteins may reside in a membrane and can be recruited via adaptors or interacting partners to the apposing membrane, allowing tethers to localize to multiple MCSs (Eisenberg-Bord et al., 2016). Deletion of the recruitment partner could prevent the protein from localizing to the MCSs. For example, Lam5/6 are ER-resident proteins, which are recruited to ER-mitochondrial MCSs by Tom70/71 and to ER-vacuole MCSs by Vac8 (Elbaz-Alon et al., 2014; Gatta et al., 2015; Murley et al., 2017). Deletion of Tom70/71 and Vac8 prevent Lam5/6 localization to ER-mitochondrial and ER-endosomal MCSs respectively. Tethers can tether membranes either constitutively or in some cases can be stimulated by cellular or external cues to tether membranees (Eisenberg-Bord et al., 2016). An example is the Extended synaptotagmin 1(E-Syt1) protein, which has been found to be recruited at ER-PM MCSs and reduce the distance between the two membranes in response to increased cytosolic calcium levels (Giordano et al., 2013). In this case, calcium levels act as the MCS recruitment signal. Tethers contain multiple domains that allow them to localize to or be recruited to organelle membranes and tether them. These domains include transmembrane domains (TMD) and hydrophobic hairpins that are integrated inside organelle membranes (Eisenberg-Bord et al., 2016). Another category of domains are those which mediate binding to membrane lipids. For example, C2 domains, pleckstrin homology (PH) domains or polybasic patches mediate binding to phospholipids in the organelle membranes. PX and FYVE domains mediate binding to PI(3)P in endosomal membranes. Tethers could additionally contain lipid transfer domains and many LTPs function in tethering membranes. In addition to having a structural role in bridging membranes and in lipid transport, tethers could play a role in signalling, maintaining membrane integrity, organelle biogenesis and dynamics (Collado et al., 2019; Eisenberg-Bord et al., 2016; Eisenberg-Bord and Schuldiner, 2017; English and Voeltz, 2013; Scorrano et al., 2019; Thomas et al., 2022; Wu et al., 2018). 23 1.5 ER structure, organization, and function in protein synthesis The ER is the major site of protein and lipid synthesis in eukaryotic cells. Proteins and some lipids synthesized in the ER are transported via vesicular transport, and other lipids are transported via the non-vesicular method from the ER to other organelles at MCSs. Many protein tethers and LTPs localize to the ER membrane and mediate MCS formation and lipid transport between the ER and other organelles. Some protein tethers preferentially localize to different structural domains of the ER, making it important to understand the structure and organization of the ER. In section 1.5.1, I describe the structure and organization of the ER. In section 1.5.2, I describe how the ER functions in protein synthesis. 1.5.1 ER structure and organization The ER is a large, continuous network of flattened cisternae/sheets and highly curved tubules enclosed by a single membrane bilayer (English and Voeltz, 2013). The ER is divided into the nuclear ER (nER), which is continuous with the outer nuclear membrane, and the peripheral ER, which includes a part of the ER that is in close contact with the PM, called the cortical ER (cER). Around 40% of the PM associates with the cER sheets and tubules in yeast (Manford et al., 2012), whereas a much less and variable proportion is found in mammalian cells, with differences between excitable and non-excitable cells (Chang et al., 2017). Many different proteins contribute to shaping the ER. The reticulon proteins (Rtns) and DP1 in mammals/Yop1 in S. cerevisiae contribute to shaping the cylindrical ER tubules and the curved edges of ER sheets (Zhang and Hu, 2016). These proteins contain a reticulon homology domain, which form hydrophobic hairpins that are embedded in membranes in the form of wedges. The wedges occupy more space in the outer leaflet of the ER membrane than the inner leaflet, thus curving the membranes. Climp-63 is an integral ER membrane protein whose luminal coiled-coil domains are proposed to dimerize and thus maintain the distance between ER sheets (Zhang and Hu, 2016). Integral ER membrane proteins kinectin and p180 are required to keep the ER sheets flattened. Atlastin in higher eukaryotes/Sey1 in S. cerevisiae are required for the fusion of ER tubules to form the tubular network of ER in cells. 24 Overexpression or deletion mutations that lead to changes in the levels of these proteins leads to either an increase or a decrease of different ER structures (Zhang and Hu, 2016). Changes in the ER structure might influence the localization of ribosomes and different proteins to the ER and thus the function of the ER. Some ER-PM MCS proteins localize to cER sheets and some to cER tubules. Elegant studies by Collado et al., (2019) and Hoffman et al., (2019) concluded that among the yeast ER-PM tethers, Scs2 and Ist2 primarily localize to cER sheets and Tcb1/2/3 localize to cER tubules, thus localizing to distinct sites at ER-PM MCSs. ER tubules are studded with a smaller proportion of ribosomes compared to ER sheets and mainly function in lipid synthesis and calcium signaling. ER sheets are studded with a larger proportion of ribosomes, owing to their flatter cisternae, and are involved in protein synthesis, protein modification and protein folding. Both ER sheets and ER tubules make contacts with other organelles (English and Voeltz, 2013; Wu et al., 2018; Zhang and Hu, 2016). 1.5.2 ER function in protein synthesis In addition to functioning in the synthesis of lipids, the ER is a major site for protein synthesis in cells. Proteins contain specific sequences that determine their localization to different organelles. Proteins can either be synthesized by the ribosomes attached to the ER or by ribosomes in the cytosol (Schwarz and Blower, 2015). Proteins can be imported either co-translationally or post-translationally into the ER . Proteins with longer transmembrane helices prefer a co-translational route. Proteins that are co-translationally imported into the ER contain an N-terminal signal peptide that is recognized by the signal recognition particle. The signal recognition particle binds to the signal recognition particle receptor and the growing peptide chain is imported into the ER by the Sec61 translocon complex (Schwarz and Blower, 2015). Some proteins (mostly ER lumen proteins or those meant to be trafficked to other organelles) contain a signal peptide that gets cleaved off, some proteins contain an additional hydrophobic sequence downstream of the signal peptide (which forms a transmembrane domain) for ER membrane insertion and other proteins contain a hydrophobic signal peptide (also called the signal sequence 25 and forming a transmembrane domain) that is not cleaved off, and instead inserts into the ER membrane, and some proteins contain multiple hydrophobic sequences (which form multiple transmembrane domains) (Schwarz and Blower, 2015). Some ER proteins are imported post-translationally, i.e., they are synthesized in the cytosol and are imported into the ER with the help of chaperones. The ER contains chaperone proteins that ensure the proper folding and other proteins that contribute to the post translational modification of newly synthesized peptide chains. The signal peptide has n (amino terminus), h (hydrophobic region) and c (carboxy terminus) regions and a cleavage site downstream of the c region. The n region is composed of upto five positive residues, while c region has upto seven polar residues and the central h region is hydrophobic and is composed of upto fifteen residues. (Owji et al., 2018). Transmembrane domains differ in that the positive residues can be either upstream or downstream of the hydrophobic sequence. Blocking the signal peptide or signal sequence leads to the mistranslocation of proteins. Proteins that localize to the Golgi, endosomes, lysosomes, the PM, and those which are secreted contain other specific sequences in addition to the ER signal sequence, which mediates their transport to the appropriate organelle membrane during vesicular transport (Schwarz and Blower, 2015). 1.6 The ER makes membrane contact sites with different organelles The ER makes contacts with almost all organelles in the eukaryotic cell and these contacts are bridged by various proteins (Figure 1.7). The ER contacts the Golgi, mitochondria, peroxisomes, endocytic organelles, lipid droplets (LDs), PM, and chloroplasts in plants. Many proteins that localize to ER contact sites are conserved throughout eukaryotes.In the following sections, I describe various proteins involved at the contacts between the ER and other organelles and the functions of these ER contact sites. In sections 1.6.1-1.6.6, I describe ER-Golgi MCSs, ER-mitochondria MCSs, ER-peroxisomes MCSs, ER-endosomes/lysosomes/vacuole MCSs, ER-LD MCSs and ER-PM MCSs respectively. 26 Figure 1.7: The ER makes contacts with different organelles. Shown here are the ER (in orange) MCSs with various organelle membranes including the plasma membrane, mitochondria (pink), Golgi (green), endosomes (purple) and lipid droplets (brown). The ER also makes contacts with lysosomes and peroxisomes (not shown here). Also indicated are some contact site proteins that bridge these MCSs. ER; endoplasmic reticulum; LD; lipid droplets; PM; plasma membrane; SR; sarcoplasmic reticulum. Zhang and Hu, Figure 2. From Zhang, H., & Hu, J. (2016). Shaping the Endoplasmic Reticulum into a Social Network. Trends in cell biology, 26(12), 934–943. https://doi.org/10.1016/j.tcb.2016.06.002. Used with permission from RightsLink / Elsevier. 27 1.6.1 ER-Golgi MCSs The primary function of these contacts is in the exchange of lipids between the ER and the Golgi membrane. CERT, Nir2, OSBP and FAPP2 are box-like LTPs that transport ceramides, PI/PA, PI4P/sterols, glucosylceramide respectively at ER-Golgi MCSs (D’Angelo et al., 2007; Wong et al., 2019). These proteins contain FFAT (two phenylalanines in an acidic tract) motifs to bind the ER-resident VAP proteins (Loewen et al., 2003) and a basic patch to interact with the phoshphoinositides on Golgi membranes. Nvj2 tethers ER-Golgi MCSs in yeast under stress conditions or when ceramides accumulate in the ER (Liu et al., 2017; Prinz et al., 2020). Nvj2 contains a PH domain, which binds phosphoinositides, and an SMP domain, which can transport lipids (Liu et al., 2017; Prinz et al., 2020). 1.6.2 ER-Mitochondria MCSs ER-mitochondria MCSs are required for lipid exchange, calcium exchange, mitochondrial fission, mitochondrial fusion, and mitochondrial inheritance. These MCSs are tethered by the ER-mitochondria encounter structure (ERMES) complex, Lam6-Tom70/71, EMC-Tom5 in S. cerevisiae and PDZD8 in mammals (Eisenberg-Bord and Schuldiner, 2017). The ERMES complex consists of members that are integral ER membrane and outer mitochondrial membrane proteins that are important for tethering. The proteins of the ERMES complex contain SMP domains, which participate in lipid transport (Reinisch and De Camilli, 2016). S. cerevisiae Lam6 contains transmembrane domains for ER localization, a StART-like domain for sterol transport between the membranes and GRAM domains that interact with Tom70/71 at the mitochondria (Eisenberg-Bord et al., 2016). Many other proteins are involved at these sites including VDAC-IP3R-GRP75, which are involved in calcium exchange at ER-mitochondria MCSs, Mfn2, a tether required for this calcium exchange, and VAPA/B-VPS13A (Kumar et al., 2018). Vps13 and VPS13A might also function as tethers at ER-mitochondria contact sites, as has been described in section 1.4. 28 ER-mitochondrial contacts are also involved in mitochondrial fission, with the ER tubules wrapping around the mitochondria and constricting it by recruiting dynamin-like factors (Wu et al., 2018). 1.6.3 ER-peroxisome MCSs ER-peroxisome contacts are required for lipid exchange between the ER and peroxisome membranes, peroxisome inheritance, fission and motility (Eisenberg-Bord et al., 2016; English and Voeltz, 2013). Proteins that tether these contacts include the ER-resident VAPA/B proteins which interact with the FFAT motif of peroxisome-resident ACBD5 in mammals (Hua et al., 2017). ACBD5 contains an acyl-CoA binding domain (ACBD) that could potentially transport fatty acids. In S. cerevisiae, ER-localized Pex3-Inp1-peroxisome localized-Pex3 tether ER-peroxisome MCSs and is required for peroxisome fission (Wu et al., 2018). 1.6.4 ER-endosomes/lysosomes/vacuole MCSs The MCSs between the ER and endosomes/lysosomes/ vacuoles are required for lipid exchange, endosomal fission, piecemeal microautophagy of nucleus (PMN), cargo sorting and endosome trafficking (Wu et al., 2018). Some proteins involved at these sites in mammals include VAPA/B-VPS13C, VAPA/B-STARD3, VAPA/B-ORP1L, VAPA/B-OSBP, GRAMD1b-NPC1(Kumar et al., 2018; Wu et al., 2018). FFAT motif containing VAPA/B interactors (VPS13C, STARD3, ORP1L, OSBP) are LTPs that transport glycerophospholipids, sterols, and PI4P/sterols respectively at these MCSs. NPC1-GRAMD1b interact with each other to tether ER-endocytic organelles and promote LDL-cholesterol egress to the ER (Höglinger et al., 2019). In S. cerevisiae, nuclear vacuolar junctions (NVJs) between the nER and vacuole are tethered by Nvj1-Vac8 and have a role in PMN (Pan et al., 2000). Lam5/6-Vac8 interact to tether the ER and vacuoles in yeast (Eisenberg-Bord et al., 2016; Gatta et al., 2015; Murley et al., 2017). A notable example of a disease involving proteins at ER-endosomes/lysosomes MCSs is the progressive childhood neurodegenerative disorder Niemann-Pick type C (NPC), which involves cholesterol and sphingolipid accumulation in lysosomes (Höglinger et al., 2019). Mutations in the proteins NPC1 and NPC2 have been 29 implicated in the disease (Höglinger et al., 2019). NPC1 has been found to localize at ER-lysosomal MCSs and interacts with GRAMD1b sterol transport protein, which could potentially directly transport excess cholesterol from lysosomes to the ER (Höglinger et al., 2019). More recently, using sphingosine probes, Altuzar et al., (2023), found that NPC1 binds to sphingosine and its absence leads to an accumulation of sphingosine in lysosomes (Altuzar et al., 2023). Mutations in NPC1 would thus prevent the transport of excess cholesterol and sphingosine from the lysosomes to the ER leading to an accumulation of cholesterol in lysosomes and hence disease phenotypes. Additionally, NPC1 has also been found to interact with ORP5 at ER-late endosomal/lysosomal MCSs for LDL-cholesterol egress from endocytic organelles to the ER (Du et al., 2011). These studies indicate a role for ER-late endosomal/lysosomal MCSs in the prevention of cholesterol and sphingolipid accumulation in lysosomes and thus the NPC disease. 1.6.5 ER-LD MCSs ER-LD contacts could have a role in LD biogenesis. Seipin proteins Sei1/Fld1 in yeast and BSCL2 in mammals are well-conserved ER transmembrane proteins that localize at ER-LD MCSs and may play a role in LD biogenesis (Wu et al., 2018). Mammalian proteins that localize to these contacts include VPS13A/C, which localize to the ER via an interaction with VAPA/B and to LDs via an amphipathic helix (AH) and could potentially transport lipids from the ER to LDs (Kumar et al., 2018). Another set of proteins include the ER acyl-CoA synthetase FATP1 and the LD localized diacylglycerol acyltransferase 2 (DGAT2). These two proteins could act as ER-LD tethers and are predicted to form a complex that could mediate LD expansion (Wu et al., 2018). 1.6.6 ER-PM MCSs ER-PM MCSs were one of the first MCSs to be discovered. These MCSs are required for lipid exchange, calcium homeostasis, ER inheritance in yeast, maintenance of membrane integrity under stress, store operated calcium entry (SOCE), calcium homeostasis, excitation-contraction coupling in muscles, and calcium modulation of excitability in neurons (Chang et al., 2017; Collado et al., 2019; Hoffmann et al., 2019; 30 John Peter et al., 2022; Loewen et al., 2007; Manford et al., 2012; Thomas et al., 2022; Wu et al., 2018). Various tethers and other proteins localize at ER-PM MCSs. 1.7 ER-PM MCS tether proteins ER-PM MCSs in yeast are tethered by at least six proteins, i.e., Scs2/22, Tcb1/2/3 and Ist2 (Manford et al., 2012; Quon et al., 2018), which are well conserved and have mammalian orthologs (Figure 1.8). However, the contact between the ER and the PM is not lost completely in a mutant that lacks these six proteins (Manford et al., 2012). Not all tethers are conserved from yeast to mammals and some ER-PM MCSs tethers, such as junctophilins are specifically expressed in excitable cells in mammals. The various ER-PM MCSs tether proteins are described in the following paragraphs. Figure 1.8: ER-PM MCS proteins Domain structures of various ER-PM MCS proteins in yeast and mammals, including their localization and lipid transport domains, are indicated in this figure. Indicated in blue and green are the PM and the ER membranes respectively. Junctophilins (mammals) are indicated in crimson, Tcb1/2/3 (S.cerevisiae) or E-Syt1/2/3 (mammals) in grey, Scs2/22 (S. cerevisiae) or VAPA/B (mammals) in pink. Ist2 (S. cerevisiae) or TMEM16 (mammals) contain a polytopic ER localization domain and a polybasic region for PM localization, as indicated by a violet oval with a ‘+’ to indicate polybasic region (Fischer et al., 2009; Manford et al., 2012). Osh3 (S. cerevisiae) is indicated in dark blue, Osh6/7 (S. cerevisiae) or ORP5/8 (mammals) in green, Lam2/4 (S. cerevisiae) or GRAMD1a-c (mammals) in brown. STIM1-Orai1(mammals) interact with each other at ER-PM MCSs. STIM1 protein contains multiple domains, EF hand calcium-binding domain in dark brown, sterile-α-motif (SAM) domains in dark purple, TMD in salmon, coiled 31 coil (CC) domains in pink, the STIM1 Orai activating region (SOAR) domains in gold (Stathopulos et al., 2008). The Orai1 channel is depicted in vermillion. Adapted from (Hewlett et al., 2021; Quon et al., 2018; Yuan et al., 2009). The Suppressor of Choline Sensitivity (Scs)2/22 in yeast or the vesicle-associated membrane protein-associated protein (VAP)A/B in mammals or VAP27 in plants are ER-PM tethers whose deletion reduces the contact between cER and PM in yeast and increases PI4P levels at the PM (Manford et al., 2012). Scs2/22 contains a C-terminal transmembrane domain that inserts into the ER and an N-terminal MSP domain that interacts with phenylalanines in an acidic tract (FFAT) motif-containing proteins (Loewen et al., 2003) (Figure 1.8). VAPA/B have additional coiled-coiled domains via which they can dimerize. Scs2 has additionally been implicated in cER inheritance in yeast, with its deletion significantly impairing the process (Loewen et al., 2007). Mutations in the VAP proteins, particularly P56S in VAPB, have been implicated in a form of familial amyotrophic lateral sclerosis (ALS), ALS8 (Teuling et al., 2007). Moreover, VAPs have many different FFAT motif-containing interaction partners, including LTPs and autophagy regulators, which might not be able to interact with VAPs in the P65S mutant (Loewen et al., 2003; Zhao et al., 2018). Thus, mutations in the FFAT-motif binding region of VAPs could disrupt cellular homeostasis and lead to diseases. Tricalbins (Tcb)1/2/3 in yeast or extended synaptotagmins (E-Syt)1/2/3 in mammals or SYT1 and SYT3 in plants have been found to be required for the maintenance of PM integrity on exposure to different stresses, such as heat stress (Collado et al., 2019; Thomas et al., 2022), osmotic stress (Lee et al., 2019; Ruiz-Lopez et al., 2021; Schapire et al., 2009), mechanical stress (Pérez-Sancho et al., 2015) and freezing (Ruiz-Lopez et al., 2021; Yamazaki et al., 2008). Tcb1/2/3 have been found to generate specialized cER peaks to maintain membrane integrity in response to heat shock (Collado et al., 2019), by regulating PS homeostasis at the PM (Thomas et al., 2022). SYT1 loss of function results in mechanical instability of cells in the plant model system Arabidopsis thaliana (Pérez-Sancho et al., 2015). These proteins localize to the ER via a hydrophobic hairpin and to the PM via C2 domains that bind to anionic 32 phospholipids in the PM (Creutz et al., 2004; Saheki and De Camilli, 2017; Schulz and Creutz, 2004). Additionally, they contain a SMP domain to mediate lipid transport (Saheki and De Camilli, 2017) (Figure 1.8). E-Syt1 responds to cytosolic calcium levels and increases the tethering between ER-PM and transports diacylglycerol (DAG) via the SMP domain (Giordano et al., 2013). Thus, tricalbins and their homologs have been found to act as ER-PM tethers, transport lipids, and have a role in maintaining membrane integrity under stress conditions. Increased sodium tolerance 2 (Ist2) is another protein that tethers cER-PM in yeast (Manford et al., 2012). It is well-conserved and the only yeast member of the TMEM16/ANO family, of which some members function as Ca2+-regulated ion channels of the PM and as scramblases at the PM (Fischer et al., 2009; Manford et al., 2012; Suzuki et al., 2010). Ist2 deletion increases the distance between the cER and the PM, thus reducing the extent of ER-PM contacts (Fischer et al., 2009; Manford et al., 2012). It has a modular structure characteristic of tethers and contains a polytopic N-terminus that inserts into the ER and a polybasic C-terminus that interacts with anionic phospholipids at the PM to tether the ER and the PM (Fischer et al., 2009; Manford et al., 2012) (Figure 1.8). Moreover, Ist2 has been found to recruit the PS/PI4P transporters Osh6/7 to cortical sites (D'Ambrosio et al., 2020; Wong et al., 2021). Junctophilins are a class of proteins that specifically tether ER-PM MCSs in excitable cells, which includes skeletal muscle cells, cardiac muscle cells and neurons (Chang et al., 2017). They are ER-transmembrane proteins that contain membrane occupation and recognition nexus (MORN) motifs, which bind to the PM, and are required for calcium signaling in excitable cells (Chang et al., 2017) (Figure 1.8). 1.8 Other ER-PM MCS proteins ER-PM MCSs house other proteins, which do not participate in tethering the membranes, but instead play roles in sterol and phospholipid metabolism and calcium homeostasis. Some ER-PM non-tether proteins such as Stromal interacting molecule 33 1 (STIM1)-Orai1, Lam1-4/GRAMD1a-c, GRAMD2a-b, Osh6/7, ORP5/8 and Osh3 (Figure 1.8) have been described below. STIM1 and Orai1 proteins are expressed in all kinds of mammalian cells and play a role in maintaining calcium levels. The ER-resident protein STIM1 senses a reduction in ER calcium levels via its calcium binding EF-hand and sterile α motif (SAM) domains (EF-SAM) in the ER lumen and localizes to ER-PM contact sites, where it oligomerizes and recruits the Orai1 calcium-channel via the STIM1 Orai activating region (SOAR) (Stathopulos et al., 2008; Yuan et al., 2009) (Figure 1.8). Calcium is an important second messenger required for apoptosis, signal transduction, muscle contraction, immune cell response and exocytosis of neurotransmitters (Chang et al., 2017). The ER is a major calcium store in mammalian cells and calcium needs to be efficiently transported the ER to other organelles, without any possible leakage into the cytosol. However, ER calcium levels are reduced in response to upstream signalling from growth factors, hormones and other signals. The STIM1-Orai1 proteins regulate the direct entry of calcium from the extracellular space into the ER, in a process termed Store Operated Calcium Entry (SOCE) (Stathopulos et al., 2008). This allows for the restoration of calcium levels in the ER. Lipid transfer protein Anchored at Membrane contact sites (Lam)1-4 in yeast or GRAMD1a-c, GRAMD2 and GRAMD3 in mammals are ER-PM MCS proteins that localize to the ER via their N-terminal TMD, to the PM via GRAM domains and contain sterol binding StART-like domains (Besprozvannaya et al., 2018; Gatta et al., 2015; Murley et al., 2017) (Figure 1.8). Lam 1-4 have been predicted to transport sterols via their StART-like domain from the PM to ER in yeast (Gatta et al., 2015). GRAMD1s have been observed to localize at ER-PM MCSs in response to increased levels of accessible cholesterol, and transport cholesterol from the PM to ER and thus contribute to cholesterol homeostasis in mammals (Naito et al., 2019; Sandhu et al., 2018). GRAMD1b mediated transport of HDL-cholesterol from the PM to the ER is important for cholesterol homeostasis in the adrenal cortex in mouse models (Sandhu et al., 2018). Gene knockouts in mice resulted in decreased storage of excess cholesterol in the cytosol of the adrenal cortex cells (Sandhu et al., 2018). In yeast, the Lam1-4 proteins form a specialized PM domain and regulate TORC2 signalling in 34 response to cellular stress (Murley et al., 2017). Lam 1-4 and its orthologs have thus emerged as retrograde sterol transporters that localize at ER-PM MCSs. OSBP-related proteins (ORP) family members that localize to ER-PM MCSs include Osh6/7 and Osh3 in yeast and ORP5/8 in mammals. They contain an OSBP-related protein domain (ORD) that binds to lipids. These proteins are involved in the transport of PI4P from the PM to the ER, where it is converted to PI by phosphatases and PS from the ER to the PM (Stefan et al., 2011). Osh6/7 have been found to localize to ER-PM MCSs by interacting with Ist2 (D'Ambrosio et al., 2020; Wong et al., 2021) and ORP5/8 localize to the ER via a TMD and to the PM via a PH domain (Stefan et al., 2011) (Figure 1.8). Another yeast ORP, Osh3, localizes to ER-PM MCSs by interacting with Scs2 to localize to the ER and via its PH domain to bind to PI4P in the PM (Stefan et al., 2011; Tong et al., 2013). Osh3-Scs2 interaction is required to recruit phosphatases that regulate PI4P levels at the PM (Stefan et al., 2011) (Figure 1.8). Different ER-PM MCS proteins localize to unique sites. For example, in mammalian cells it has been observed that GRAMD1a and GRAMD2a mark separate regions of the PM, with GRAMD2 colocalizing with E-Syt2/3 and marking a subset of ER-PM MCSs required for STIM1 recruitment, while GRAMD1a-c do not (Besprozvannaya et al., 2018). Similarly, yeast ER-PM MCSs proteins also localize to unique sites, where they localize to different ER structures. 1.9 Vps13 is a LTP that transports lipids at various MCSs Vps13 is a bridge-like LTP that transports lipids in bulk at MCSs. It has been found to function as a tether and localizes to various different MCSs. It is highly conserved and its mammalian orthologs are implicated in diseases, making it important to study these proteins. Many other bulk transporters of lipids have now been described at different MCSs. The study of Vps13 and other bulk lipid transporters forms the basis of this thesis, and in the following sections I have described important features of these 35 proteins. In section 1.9.1, I describe how Vps13 and its orthologs localize to various MCSs. In section 1.9.2, I describe the lipid transport activity of Vps13. 1.9.1 Vps13 localizes to different MCSs S. cerevisiae Vps13 has been found to localize to nuclear-vacuolar junctions (NVJ), vacuole-mitochondrial patches (vCLAMPS), ER-mitochondria contacts, endosomes, to the prospore membrane and to various other MCSs (Bean et al., 2018; Dziurdzik et al., 2021; Lang et al., 2015; Pan et al., 2000; Park et al., 2016) (Figure 1.9). It has 4 human orthologs, VPS13A-D (VPS13s). Mutations in VPS13A-D have been implicated in Chorea-acanthocytosis, Cohen syndrome, Parkinson’s disease, and spastic ataxia, respectively (Kolehmainen et al., 2003; Lesage et al., 2016; Rampoldi et al., 2001; Seong et al., 2018). Dziurdzik et al., (2020) showed that a spastic ataxia-causing mutation in VPS13D blocks adaptor binding to the Vps13 VAB domain and hence recruitment to MCSs when modelled in yeast. This indicates that mutations that prevent Vps13 recruitment to MCSs could lead to diseases. VPS13A has been found to localize at ER-mitochondria, ER-LD (Kumar et al., 2018) and mitochondria-endosome contacts (Muñoz-Braceras et al., 2019). VPS13B has been found to localize to the Golgi apparatus, endosomes and acrosomes (Da Costa et al., 2020; Koike and Jahn, 2019; Seifert et al., 2006). VPS13C has been found to localize at ER-late endosomes/lysosomes and ER-LD contacts (Kumar et al., 2018). VPS13D localizes to ER-mitochondria and ER-peroxisome contact sites and the Golgi apparatus (Guillén-Samander et al., 2021). 36 Figure 1.9: Vps13 and Atg2 are LTPs that localize to different MCSs Vps13 has been found to localize to various MCSs that are shown here. Vps13 and Atg2 proteins have been found to transport lipids at MCSs. In this figure, Vps13 is depicted in pink, Atg2 in light blue, ER in red, lysosomes/vacuole in grey, lipid droplets in yellow, endosomes in brown, isolation membrane (IM) in purple, the prospore membrane in dark blue, the Golgi apparatus in sky blue, peroxisomes in orange and the mitochondria are outlined in black. In some cases, Vps13 has been found to localize to single organelles, however it is inferred that this could be mediated by the ER (indicated by a transparent ER, light pink). Adapted from (Bean et al., 2018; Dziurdzik et al., 2021; Kumar et al., 2018; Li et al., 2020). S. cerevisiae Vps13 and VPS13s possess multiple conserved domains that mediate their interactions with other proteins and are important for their functions (Bean et al., 2018; Guillén-Samander et al., 2021; Kumar et al., 2018). These domains include the N-terminal chorein domain, Vps13 adaptor binding (VAB) domain, Aberrant Pollen Transfer 1 (APT1) domain, Autophagy-related protein 2 C-terminal (ATG2_C) domain and the PH domain (Bean et al., 2018; Dziurdik et al., 2021; Kumar et al., 2018). The N-terminal chorein domain has been found to be involved in lipid transport (described further in 1.9.2; Kumar et al., 2018) and the VAB domain is primarily a protein interaction domain (Bean et al., 2018). The APT1 domain was named after the Golgi-binding maize protein Aberrant Pollen Transfer 1 where it was first identified (Xu and Nucleus Vacuole Mitochondria Nucleus Endosomes Lysosomes LDs IM Mammals Yeast Mitochondria Endosomes ER ER ER? Peroxisomes ER? ER? IM Prospore membrane Peroxisomes ER? ER? 37 Dooner, 2006). This domain is C-terminal to the VAB domain (Kolakowski et al., 2020; Rzepnikowska et al., 2017), and is predicted to bind to PI3P in the case of Vps13 (Rzepnikowska et al., 2017) and to PI3P and PI5P in the case of VPS13A (Kolakowski et al., 2020). Vps13 is recruited to different MCSs and membranes via protein adaptors (Bean et al., 2018). These include Ypt35 (recruitment to endosomal and vacuolar membranes), Mcp1 (recruitment to mitochondrial membranes) and Spo71 (recruitment to prospore membrane) (Bean et al., 2018; Lang et al., 2015; Park et al., 2012). Ypt35 is an uncharacterized sorting nexin that was identified as an interactor of Vps13 in a high throughput proteomics study (Ho et al., 2002). A co-immunoprecipitation experiment confirmed an interaction between Ypt35 and Vps13 (Bean et al., 2018). Spo71 is a PH domain containing protein that is induced during sporulation and was found to have a role in prospore membrane growth during sporulation using mutageneis and microscopy-based experiments (Parodi et al., 2012). Vps13 was found to be recruited to the prospore membrane by Spo71 to promote prospore membrane closure (Park et al., 2013). These protein adaptors contain a Proline-X-Proline (PxP) motif that interacts with the VAB domain of Vps13 (Bean et al., 2018; Dziurdzik et al., 2021). The PxP motif was identified by a multiple sequence alignment of Saccharomycetales-level fungal homologs of Ypt35 (Bean et al., 2018). A Multiple Expectation Maximization Algorithms for Motif Elucidation (MEME) motif was then generated from the Saccharomycetales-level fungal homologs of Ypt35 and fed into a Find Individual Motif Occurrences (FIMO) search of the S. cerevisiae proteome (Bean et al., 2018). This search identified a PxP motif in Spo71, a known interactor of Vps13 (Park et al., 2013). A new MEME motif was generated from Saccharomycetales-level fungal homologs of Ypt35 and Spo71 and fed into a FIMO search (Bean et al., 2018). This identified Mcp1, an outer mitochondrial membrane protein as an interactor of Vps13, and its interaction with Vps13 was verified by co-immunoprecipitation experiments (Bean et al., 2018). The VAB domain contains six repeats of similar topology, consisting of β-strands, which arrange into a fishhook-like structure (Adlakha et al., 2022). Each of these modules has conserved asparagine residues, and the 38 asparagine residues of the first and sixth repeat are important for adaptor binding (Dziurdzik et al., 2020). The ER localization of Vps13 has not been investigated and no predicted FFAT motifs have been determined (Dziurdzik et al., 2021). The N-terminus of Vps13 and Atg2 have been found to have sequence and structural similarities (Osawa et al., 2019) and a helix in the N-terminus of Atg2 has been found to bind to the ER (Kotani et al., 2018). It is thus possible that Vps13 interacts with the ER via a helix in its N-terminus. VPS13A, C and D interact with the ER-resident VAP proteins via FFAT motifs to localize at ER MCSs (Guillén-Samander et al., 2021; Kumar et al., 2018). VPS13A and C contain C-terminal amphipathic helices via which they interact with mitochondria and LDs respectively (Kumar et al., 2018). The VAB domain in VPS13C interacts with late endosomes/lysosomes (Kumar et al., 2018). Miro, a GTPase, recruits VPS13D to the mitochondria and peroxisomes (Guillén-Samander et al., 2021). Vps13 and its orthologs have been found to be involved in sporulation (Park et al., 2012), autophagy (Chen et al., 2020; Lei et al., 2022), protein trafficking (Brickner and Fuller, 1997) and lipid transport (Kumar et al., 2018; Li et al., 2020). Mutating the contact site-binding regions of Vps13 and VPS13s disrupts the localization of these proteins and impairs their function in cells (Bean et al. 2018; Dziurdzik et al., 2020; Kumar et al., 2018). 1.9.2 Vps13 has glycerophospholipid transfer activity Vps13 was found to transport glycerophospholipids between donor and acceptor liposomes in vitro using FRET-based lipid transfer assays (Kumar et al., 2018). Fragments containing the residues 1-1350 of Vps13 were found to bind to tens of glycerophospholipids (Kumar et al., 2018). This region of Vps13 was found to bind to glycerophospholipids such as PS, PA, PE and PC, and transport PS in vitro (Kumar et al., 2018). Further, the residues 1-335 of Vps13 were found to fold into a utility scoop-like structure with a hydrophobic concave face that interacts with lipids via X-ray crystallography (Kumar et al., 2018). Li et al., (2020) used cryo-EM to show that residues 1-1390 of Vps13 forms a rod-like structure with a utility scoop at one end 39 (region 1-335), where lipid moieties dock to be transported between membranes. Moreover, they showed that mutating a band of hydrophobic residues in the lipid transport channel region blocks the lipid transport activity of Vps13. vps13Δ mutant cells are defective in sporulation (Park and Neiman, 2012) and the lipid transport activity of Vps13 could contribute to the formation of the prospore membrane in yeast. Li et al., (2020), found that the band mutant can no longer rescue the sporulation defective vps13Δ mutants. This indicates that lipids pass in bulk through the length of the Vps13 lipid bridge and this bulk transport of lipids is required for prospore membrane formation in yeast. Vps13 and VPS13s could transport multiple lipids from the ER, which could act as a lipid reservoir, to apposing membranes at MCSs. Vps13 and VPS13A have been found to interact with scramblases that could aid in the equilibration of lipids at both the donor and acceptor membranes (Adlakha et al., 2022; Ghanbarpour et al., 2021). This would ensure that lipids are transported to the appropriate leaflets of membranes at MCSs. Scramblase activity could accelerate lipid transport (Adlakha et al. 2022), which would be necessary for processes requiring rapid membrane expansion such as sporulation (Li et al., 2020). Mcp1 and XK (known interactors of Vps13 and VPS13A respectively) have been found to act as scramblases and scramble lipids such as PS, PC and PE non-specifically, using liposomes containing fluorescently tagged lipids and an impermeable fluorescence quencher (Adlakha et al., 2022). When the liposomes were incubated with the quencher, approximately 50% of the fluorescence of the liposomes was lost. However, when the scramblases i.e., either Mcp1 or XK were added to the liposomes (proteoliposomes), more than 50% of the fluorescence was lost. This reduction in fluorescence was a result of the scrambling activity of the scramblases as the fluorescent lipids from both leaflets were accessible to the quencher (Adlakha et al., 2022). Vps13 interacts with Mcp1 via its VAB domain and VPS13A interacts with XK via its PH domain, forming the VPS13A-XK complex (Park et al., 2022). Mutations in the interaction interface disrupts complex formation and the co-localization of both 40 proteins (Park et al., 2022). Moreover, the mutant alleles of VPS13A lacking the PH domain have been found in patients with chorea-acanthocytosis. Chorea-acanthocytosis and McLeod syndrome (caused by mutations in XK) have phenotypic similarities and might lack the VPS13A-XK complex, leading to disease phenotypes (Park et al., 2022). The role of Vps13 and VPS13s in lipid transport and their interaction with scramblases are thus important for the functioning of cells. Other Vps13-like proteins have been determined and have been recently classified as the Repeated Beta Groove (RBG) superfamily (Levine, 2022). These proteins are predicted to contain multiple repeating units with a five-stranded β-sheet meander and a sixth element, which is a helix followed by a loop over the meander (Neuman et al., 2022b). These proteins form rod-like structures predicted to transport lipids by bridging membranes at MCSs (Levine, 2022). Apart from Vps13, the RBG superfamily also includes Atg2, a protein involved in autophagy, which has been described below. 1.10 Atg2 transports the lipids required for autophagosome expansion Atg2 is a Vps13-like protein which shares significant sequence and structural homology with the Vps13 N-terminal regions (Kumar et al., 2018; Osawa et al., 2019; Valverde et al., 2019). It is a well-conserved protein with two mammalian orthologs ATG2A and ATG2B (ATG2s). Atg2 and its orthologs have been implicated in the bulk transport of lipids and the expansion of membranes similar to Vps13. This indicates a common function for bulk transporters in the expansion of membranes. Atg2 and ATG2s contain conserved domains such as an N-terminal chorein domain, APT1 domain and the ATG2_C domain (Dziurdzik et al., 2021; Kaminska et al., 2016). The N-terminal chorein region of Atg2 forms a structure similar to the Vps13 N-terminus and contains a hydrophobic cavity along which lipids travel between membranes (Osawa et al., 2019). Full length ATG2 folds into a rod-like structure of 20nm as seen by negative EM (Chowdhury et al., 2018) and can transport lipids in bulk by bridging the ER and isolation membrane (IM) at MCSs (Osawa et al., 2018). In vitro studies using FRET-based lipid transfer assays have shown that Atg2 can transport PE, PS and PC between donor and acceptor liposomes and thus exhibits little glycerophospholipid head group specificity (Osawa et al., 2019). 41 Atg2 has been implicated in autophagosome formation during autophagy. Autophagy is a well-conserved process in eukaryotes, which is involved in the degradation of bulk or selected components. The process involves the de novo biogenesis of a cup-shaped double membraned structure called the isolation membrane (IM) (Osawa and Noda, 2019). The core autophagy machinery consisting of around 20 proteins in yeast come together to form the phagophore assembly site (PAS) (Osawa and Noda, 2019). Atg9 is incorporated into a vesicle at the Golgi apparatus, which is recruited to the PAS on starvation and serves as the initial membrane source for the IM (Yamamoto et al., 2012). Atg9 then relocates to the edge of the IM along with Atg2 and Atg18, which form a contact site between the ER and the IM (Gómez-Sánchez et al., 2018). The IM then grows into an autophagosome by incorporating phospholipids, and completely encapsulates the components that are to be degraded (Osawa et al., 2019). The autophagosome fuses with the vacuole/ lysosome, where the degradation occurs. Defective autophagy has been implicated in neurodegenerative and neuronal disorders (Osawa and Noda, 2019). Bulk transport of lipids is required for the rapid expansion of the growing IM into autophagosomes. Atg2 tethers the ER to the growing IM and transports glycerophospholipids from the ER to its growing edge (Figure 1.9) (Osawa et al., 2019; Valverde et al., 2019). Atg2 forms a complex with Atg18, which interacts with PI3P and PI(3,5)P2 in autophagic membranes (Kotani et al., 2018). Atg2 localizes to the ER via basic residues in its N-terminus and contains a C-terminal amphipathic helix (AH) for IM localization (Kotani et al., 2018). The C-terminal AH of Atg2 is required for Atg18 binding to PI3P-rich membranes, thus recruiting the Atg2-Atg18 complex to the PAS. Atg2-Atg18 complex bind to Atg9 and are targeted to the Atg9 vesicles in the PAS. The interaction between Atg2 and Atg9 is important for Atg2 retention at the growing edge of the IM and ensures that lipid transport and hence membrane expansion occur at this site (Gómez-Sánchez et al., 2018). Atg9 interacts with Atg2 via its APT1 domain, which is predicted to bind to PI3P (Kaminska et al., 2016). ATG2A forms a complex with WIPI proteins (mammalian orthologs of Atg18) and interacts with the scramblases TMEM41B and VMP1 at the ER and ATG9 at the 42 growing autophagosome (Ghanbarpour et al., 2021; Maeda et al., 2019; Matoba et al., 2020; Morita et al., 2019). Not only do Atg2 and Vps13 share similarities in their N-terminus, but they also share similarities at their C-terminus. In the case of Atg2, similar to Vps13, the ER acts as a lipid reservoir and scramblases at the IM ensure that lipids are transported to the appropriate leaflet. Atg9 scrambles the phospholipids delivered by Atg2 to the luminal leaflet from the cytoplasmic leaflet of the IM (Matoba et al., 2020). Thus, Atg2 is a LTP that transports lipids in bulk from the ER to the growing edge of the IM, which is required for autophagosome formation. 1.11 Vps13-like proteins at ER-PM MCSs Proteins sharing structural similarities with Vps13 and Atg2 have been identified at ER-PM MCSs recently. These proteins are Csf1, Fmp27 and Hob2 (Ypr117w). When I started this project in 2018, not much had been studied about these proteins and my research question was to identify Vps13-like proteins at ER-PM MCSs. Owing to their structural similarites with Vps13 and Atg2, Csf1, Fmp27 and Ypr117w could function in the bulk transport of lipids at ER-PM MCSs. This could be required for rapid changes in lipid composition in response to stresses, as has been implicated for Csf1 or in membrane elongation during endocytosis. In the sections below, I describe the known roles of these proteins and their orthologs. In section 1.11.1, I describe the role of Csf1 in cold stress response and in section 1.11.2, I describe the putative lipid transport proteins Fmp27 and Hob2 (Ypr117w). 1.11.1 Csf1 is a Vps13-like protein with a conserved role in cold stress response Csf1 has recently been identified as a protein with secondary structure similarities to Vps13 and is a member of the RBG superfamily (Levine, 2022). It has been predicted to fold into a long rod-like structure that could potentially transport lipids (Toulmay et al., 2022). Moreover, it has been found to localize at ER-PM MCSs (Toulmay et al., 2022). Csf1 contains a transmembrane domain at its N-terminus that could mediate ER localization (John Peter et al., 2022), and it is possible that it is recruited to ER-PM MCSs via an adaptor, similar to Vps13 recruitment to MCSs. 43 The deletion of Csf1 confers a cold-sensitive phenotype to yeast, resulting in impaired growth at low temperatures (Tokai et al., 2000). John Peter et al., (2022) speculate that Csf1 could selectively transport saturated or unsaturated lipids to allow cellular adaptation to cold stress. CSF1 has well-conserved orthologs, TWEEK in D. melanogaster (Verstreken et al., 2009), KIAA1109 in H. sapiens (Kane et al., 2019) and LPD-3 in C. elegans (McKay et al., 2003). Tweek is required for normal synaptic vesicle recycling and affects PI levels (Verstreken et al., 2009). A mutation in the KIAA1109 gene has been implicated in Alkuraya-Kučinskas syndrome, a neurological malformation disorder and the protein has been found to have a role in endosomal trafficking (Kane et al., 2019). LPD-3 has recently been found to localize to ER-PM MCSs, with a role in normal phospholipid distribution, fatty acid desaturase expression, and cold stress resistance (Wang et al., 2022). Similar phenotypes were observed in the case of the Zebrafish LPD-3 ortholog and KIAA1109 (Wang et al., 2022), indicating a conserved role for Csf1 and its orthologs in bridging ER-PM MCSs and cold stress response. 1.11.2 Fmp27 and Hob2 (Ypr117w) are putative lipid transport proteins at ER-PM MCSs Fmp27 and Hob2 (Ypr117w) have been recently identified as a part of the RBG superfamily and are predicted to fold into rod-like structures (Castro et al., 2022; Neuman et al., 2022a; Toulmay et al., 2022). These proteins have been found to localize at ER-PM MCSs and could potentially transport lipids in bulk at these sites. However, not much is known about how these proteins are recruited to the ER-PM MCSs and their functions at these sites. Fmp27 was initially identitified in a screen to find the proteins in the mitochondrial proteome and hence named Found in Mitochondrial Proteome 27 (Reinders et al., 2006). FMP27 has well-conserved orthologs, some of which have been well studied. These include KIAA0100 in H. sapiens (Song et al., 2006), HOBBIT in D. melanogaster (Neuman and Bashirullah, 2018), SABRE (Aeschbacher et al., 1995; Cheng and Bezanilla, 2021; Pietra et al., 2015) and KIP (Procissi et al., 2003) in A. thaliana, and 44 APT1 in Z. mays (Xu and Dooner, 2006). FMP27 shares 32% sequence identity with KIAA0100 and a 25% sequence identity with HOBBIT. KIAA0100 has been found to be overexpressed in breast cancers and is associated with poor prognosis (Zhong et al., 2018). Hobbit was identified from a fly screen for animals with small body size (Neuman and Bashirullah, 2018). hobbit mutants are arrested at the pupal stage and have tiny pupae. These mutants fail to secrete insulin and mucin, indicating a role for Hobbit in the trafficking of secretory granule proteins (Neuman and Bashirullah, 2018). SABRE is required for the stabilization of root hair patterning (Pietra et al., 2015) and KIP (KINKY POLLEN) is involved in growth of pollen tubes and root tips (Procissi et al., 2003) in plants. APT1 (ABERRANT POLLEN TRANSMISSION 1) is involved in pollen tube growth in Z. mays (Xu and Dooner, 2006). This indicates a role in secretion, membrane trafficking and lipid transport for this family of proteins. 1.12 Summary and Overview Lipids are continually trafficked in cells to maintain lipid homeostasis. Lipids can be transported either via vesicular or non-vesciular routes and most lipids are transported non-vesicularly by LTPs. Non-vesicular transport in cells takes place at MCSs. These sites are enriched in lipid biosynthetic enzymes and LTPs, which are involved in the transport of sterols, sphingolipids, and phospholipids. Mutations in the proteins involved at MCSs cause cellular dysfunction and hence diseases. This makes it important to study lipid transport proteins at MCSs and uncover their mechanisms of action, to better understand diseases. Many MCS proteins are highly conserved across eukaryotes. Moreover, yeast share cellular pathways, organelles, and proteins with other higher eukaryotes like humans. This and the ease of manipulation, genetic tractability, ease of maintenance and availability of molecular tools to work with the budding yeast, Saccharomyces cerevisiae make it a great model system to study MCSs. Vps13 and its paralog Atg2 are bridge-like LTPs that transport lipids in bulk at different MCSs in eukaryotes (Kumar et al., 2018; Osawa et al., 2019). The bulk transfer of lipids can be required for rapid membrane elongation as in the case of prospore 45 membrane elongation during S. cerevisiae sporulation (Li et al., 2020; Park et al., 2016) or IM expansion into autophagosomes (Osawa et al., 2019; Valverde et al., 2019) mediated by Vps13 and Atg2 respectively or for rapid changes in lipid composition in response to stresses, as has been implicated for Csf1 (John Peter et al., 2022). A potential role in the bulk transfer of lipids makes the Vps13-family of LTPs unique and different from other LTPs known so far, which only transport one lipid at a time. When I first started working on my thesis, no Vps13 family member was known to localize to ER-PM MCSs. Bulk transport of lipids at ER-PM MCSs would be required for rapid changes in the composition of the PM in response to different stresses. The PM forms a protective barrier in cells and rapid changes in its lipid composition could aid the growth of cells in response to stresses. Additionally, bulk transport of lipids to the PM could have a role in endocytosis. Bulk transport of anionic lipids at the PM could increase the membrane charge density and create regions of membrane curvature and invaginations, which could have a role in endocytosis. This makes it important to identify Vps13-like proteins at ER-PM MCSs. Hence, my research question was to identify a Vps13-like protein that localizes to ER-PM MCSs, determine how it is recruited to these sites and elucidate its function. Through the course of working on this thesis, many studies have since found Vps13-like proteins i.e., Csf1, Fmp27 and Ypr117w that could possibly transport lipids in bulk at ER-PM MCSs (Castro et al., 2022; John Peter et al., 2022; Neuman et al., 2022a; Toulmay et al., 2022). However, these studies have not determined how these proteins are recruited to ER-PM MCSs and only the function of Csf1 has been determined. In this study we identified how Fmp27 is recruited to ER-PM MCSs and a possible function of Fmp27 at ER-PM MCSs. In this PhD thesis, I used the budding yeast, Saccharomyces cerevisiae, to investigate whether Vps13 and Atg2 have structural homologs that localize to ER-PM MCSs and could transport lipids. In chapter 2, I used HHpred, a bioinformatics tool, and identified Fmp27, Ypr117w and Csf1 as proteins with structural similarities to Vps13 and Atg2. I focused on Fmp27 as it was the highest probability hit in my search. I determined whether Fmp27 localizes to ER-PM MCSs using microscopy, and if it is recruited to 46 these sites via an interactor, similar to Vps13. I used information from chemogenomics databases to identify an interactor of Fmp27, Ybl086c, and determined its localization via microscopy. Moreover, I determined how Fmp27 and Ybl086c interact with each other, and the domains involved using co-immunoprecipitation experiments. I used a combination of bioinformatics, mutagenesis, and microscopy to identify the conserved residues in Ybl086c that are required for the recruitment of and interaction with Fmp27. In chapter 3, I investigated whether the Fmp27 and Ybl086c function in lipid homeostasis and stress response. I used spot assays to identify how the mutants of the Fmp27 and Ybl086c respond on exposure to different stress conditions and drugs that target lipid pathways. Further, I used plate reader-based growth curve analysis to determine the growth patterns of mutant strains to specific drugs. I used fluorescent lipid probes (FLARES) to determine changes in lipid distribution in the mutants of the Fmp27 and Ybl086c. I found that Fmp27 contains an N-terminal hydrophobic helix for ER localization and is recruited to ER-PM MCSs by Ybl086c. Ybl086c contains a conserved region in its C-terminus, which is required for an interaction with Fmp27. Furthermore, I found that Fmp27 and Ybl086c could have a role in the retention of sterols in a bound state at the PM. Fmp27 and Ybl086c are highly conserved proteins and investigating the interactions and functions of these proteins in yeast could shed light on the role of their mammalian orthologs. 47 Chapter 2 Characterization of the interaction of Fmp27, a Vps13-like protein with its binding partner 2.1 Synopsis Membrane contact sites (MCSs) are sites where organelle membranes are in close proximity to one another. These sites are maintained by protein tethers, which bridge MCSs and are involved in other functions at these sites. One such protein tether, Vps13, is highly conserved and is present at various contact sites where it is predicted to function as a bulk lipid transporter. Bulk transport of lipids might be important for rapid membrane elongation during the formation of the prospore membrane or autophagosomes or during endocytosis or for rapid changes in lipid composition at MCSs. This could be mediated by Vps13-like proteins, making it important to identify such proteins at other MCSs. I used HHpred, a bioinformatics tool, to search for proteins with secondary structure similarities to the Vps13 lipid transport channel region in the S. cerevisiae proteome and found three uncharacterized proteins, Fmp27, Hob2 (Ypr117w) and Csf1. Fmp27 and Ypr117w are highly conserved proteins and their plant and fly orthologs have been found to have a role in development and the human ortholog is implicated in breast cancer. I found that Fmp27 is anchored to the ER and localizes to ER-PM cortical sites. Furthermore, I identified its interacting partner, which recruits it to these cortical sites. A conserved region in the C-terminus of the Fmp27-interacting protein is necessary for binding to Fmp27 and recruiting it to cortical sites. Our findings suggest that Fmp27 could have a role in lipid transport at MCSs, owing to its secondary structural similarity with Vps13 and related proteins. 2.2 Introduction Membrane contact sites are sites of close contact between organelle membranes that occur in eukaryotes. The endoplasmic reticulum (ER) being the most extensive organelle in eukaryotic cells forms contacts with different organelles and membranes, such as the plasma membrane (PM), mitochondria, trans-Golgi network (TGN), lipid droplets (LDs), late endosomes, and peroxisomes (Wu et al., 2018). ER-PM MCS 48 proteins are well conserved throughout eukaryotes (Besprozvannaya et al., 2018; Fischer et al., 2009; Gatta et al., 2015; Loewen et al., 2003; Murley et al., 2017; Saheki and De Camilli, 2017; Scorrano et al., 2019). Membrane contact sites are maintained by protein tethers, which function in the maintenance of membrane integrity, membrane biogenesis, ion regulation and in lipid transport. S. cerevisiae Vps13 (hereafter Vps13) and its human orthologs, are protein tethers involved in the bulk transport of lipids, which might be important in processes such as sporulation, which require rapid expansion of membranes (Kumar et al., 2018; Li et al., 2020; Park et al., 2012). Additionally, Vps13 related proteins such as Atg2 have also been found to transport lipids in bulk (Osawa et al., 2019; Valverde et al., 2019). Vps13 has been found to localize to different membrane contact sites such as ER-endosome, ER-mitochondria, vacuole-mitochondria contacts (vCLAMPs) and the nuclear-vacuolar junction (NVJs) contact sites (Bean et al., 2018; Kumar et al., 2018; Lang et al., 2015; Pan et al., 2000; Park et al., 2016), but not to ER-PM MCSs. ER-PM contacts are implicated in the maintenance of membrane integrity (by an alteration of the PM lipid composition when the cell is exposed to stresses) and lipid homeostasis, processes which might require bulk transport of lipids, making it important to identify a Vps13-like protein at these sites. I use the budding yeast, Saccharomyces cerevisiae as a model system for this study. In this study I identified Fmp27 and Ypr117w as Vps13-like proteins using bioinformatics tools. I found that Fmp27 is anchored to the ER and is recruited to the PM by Ybl086c. Ybl086c interacts with Fmp27 and Ypr117w via a conserved region in its C-terminus. These proteins might be important for lipid homeostasis owing to their functional correlation with sterol transporter and sphingolipid biosynthesis genes, making it important to study them. 2.3 Results 2.3.1 Fmp27 is a protein with structural similarities to Vps13 and localizes to ER-PM MCSs To identify a Vps13-like protein that could localize at contact sites and transport lipids, I used HHpred (Söding et al., 2005; Zimmermann et al., 2018). HHpred is an online server that allows for the detection of distant homologs of proteins based on structural 49 similarities using hidden Markov models (HMMs). It allows one to search a wide variety of proteomes and structural databases (Söding et al., 2005). I used a part of the lipid transport channel region of Vps13 (residues 650-1390) as a query fragment to find the putative S. cerevisiae structural homologs of the Vps13 lipid transport module. This is a part of a larger region (residues 1-1390) of Vps13, whose cryo-EM structure was found to fold into a rod-like structure lined with hydrophobic residues required for the transport of glycerophospholipids (Adlakha et al., 2022; Li et al., 2020). I found three uncharacterized proteins and a known structural homolog of Vps13, Atg2, as hits from our query (Figure 2.1A). The three uncharacterized homologs are Fmp27, Ypr117w (Hob2) and Csf1. Scores and other details of the HHpred search can be found in appendix B1. Atg2 is a known LTP, with its N-terminus shown to have high sequence and structural similarity with the N-terminal region of Vps13 (Kumar et al., 2018; Li et al., 2020; Osawa et al., 2019; Valverde et al., 2019), while Fmp27, Ypr117w and Csf1 are large uncharacterized proteins. Csf1 has recently been found to have a role in lipid transport (John Peter et al., 2022; Toulmay et al., 2022; Wang et al., 2022). Like Vps13, all these proteins are highly conserved. On further analysis of the alignments obtained from our query, I found that the lipid transport module of Vps13 is structurally similar to parts of the predicted APT1 domain of Atg2, Fmp27 and Ypr117w. This indicates that the APT1 domain of these proteins could have a role in lipid transport. Vps13 also has an APT1 domain, but that did not come up in the list of hits. To determine whether the APT1 domain of Vps13 has any structural similarities with the Vps13 lipid transport region, I used it as a query in a new HHpred search. I found structurally similar regions in the lipid transport region of Vps13 as hits from this search (Figure 2.1B). Scores and other details of the HHpred search can be found in appendix B2. This indicates a structural similarity between the lipid transport module and the APT1 domain of Vps13. 50 Figure 2.1: HHpred identifies Fmp27 and Ypr117w as Vps13-like proteins in S.cerevisiae (A) A query for proteins with distant structural homology to a part of the Vps13 lipid transport channel (650-1390) identifies Atg2, a Vps13-like protein and three uncharacterized proteins Fmp27, Ypr117w and Csf1. The query region and exact regions of hits are marked in magenta. The percent homologies are also indicated. (B) A query for sequences with structural homology to Vps13 APT1 (pink) domain yields the lipid transport channel of Vps13, and the APT1 domains of Atg2, Fmp27 and Ypr117w. The query region and exact regions of hits are marked in magenta. The percent homologies are also indicated. All domains present in these proteins are indicated in the legend to the right. I used AlphaFold (Jumper et al., 2021; Varadi et al., 2022) to obtain the predicted structures of Fmp27 and the other hits (Figure 2.2, 2.3A) (Adlakha et al., 2022; Toulmay et al., 2022). When I started this study in 2018, it was only possible to make A B 51 predictions about parts of the Fmp27 structure using trRosetta, since AlphaFold structures were not available at that time. AlphaFold relies on multiple sequence alignments to evaluate the co-evolution of proteins and uses machine learning to predict the structures of proteins (Jumper et al., 2021). The confidence of the predicted models can be inferred based on the predicted local distance difference test (pLDDT) and predicted aligned error (PAE) scores (Jumper et al., 2021). The pLDDT score measures whether the predicted residue has similar distances with neighbouring Cα atoms, as would be in a true structure (Jumper et al., 2021). Regions with a pLDDT score of greater than 90 are predicted with high accuracy and are colored dark blue, regions with a score between 70-90 are modelled well and are colored in light blue, a score between 50-70 is low confidence and the region is colored yellow, while scores less than 50 are very low confidence and indicate disordered elements, and are colored orange (Jumper et al., 2021). The PAE score gives the AlphaFold’s expected position error at residue x, when the predicted and true structures are aligned on residue y. This helps determine the confidence of the relative position and orientation of the different domains of a predicted protein structure (Jumper et al., 2021). The AlphaFold predicted structures of Fmp27, Ypr117w and Atg2 indicate that these proteins fold into rod-like structures similar to Vps13 (Figure 2.2B, 2.3). The region of these proteins containing RBG multimers and forming the lipid transport region is predicted with high confidence, indicating an evolutionary conservation in this region of the three proteins. The extreme N-terminal and C-terminal helices are low confidence predictions, indicating that these regions might be disordered or highly flexible. The Alphafold structures of Fmp27, Ypr117w and Atg2 further indicates that there is no separate APT1 domain in these proteins and that the predicted APT1 domain forms a part of the rod-like structure (Figure 2.2A). The APT1 domain could thus be a part of the lipid transport channel, as predicted by the HHpred hits obtained with Vps13 APT1 domain as a query (Figure 2.1B). These results indicate that Fmp27 is a Vps13-related protein that could potentially have a role in lipid transport at MCSs. 52 Since Fmp27 was the highest probability hit in our HHpred searches and a highly conserved, yet uncharacterized protein, I proceeded to characterize its role in this study. Figure 2.2: Predicted structures of HHpred hits and Vps13-like proteins (A) AlphaFold2 powered ColabFold (ab initio protein modeling software; Mirdita et al., 2022) predicted structure of the Vps13 lipid transport module (650-1390), which was used as a query for HHpred search in 2.1A. AlphaFold predicted structures of hits, i.e., Fmp27 (2076-2469), Ypr117w (2118-2241), Atg2 (1119-1245) and Csf1 (531-1052). ColabFold prediction of Vps13 APT1 region (2575-2832) was used as 53 a query for Figure 2.1B. colabFold was used since Vps13 and Csf1 structures are not available in the AlphaFold database. The legend indicates the color scheme used for coloring the different secondary structure elements in the structural predictions. (B) Full-length AlphaFold predicted structures of Atg2, Fmp27 and Ypr117w indicate rod-like structures similar to Vps13, indicating that these proteins could transport lipids. The legend indicates the color scheme used for coloring the different secondary structure elements in the structural predictions. Figure 2.3: Predicted structures of Atg2, Fmp27 and Ypr117w and degree of confidence of the predictions (A) The AlphaFold predicted structures of Atg2, Fmp27 and Ypr117w has been colored according to pLDDT scores (Jumper et al., 2021) to indicate the degree of confidence of the predicted model. The legend to the right indicates the color scheme. (B) The PAE plot of the predicted structures of Atg2, Fmp27 and Ypr117w indicates AlphaFold’s expected position error at residue x, when the predicted and true structures are aligned on residue y.The legend indicates the expected position error in Angstroms represented by various shades of green. A B 54 2.3.2 Fmp27 is an ER protein that localizes to ER-PM contact sites To determine the localization of Fmp27, I C-terminally tagged Fmp27 with an ENVY tag. ENVY is a brighter version of GFP (Slubowski et al., 2015) and is helpful to visualize proteins expressed at lower levels. Fmp27 localized to cortical sites, which could be ER-PM MCSs (Figure 2.4A). Fmp27 is not highly expressed in cells and fluoresces very dimly. To address this, I overexpressed Fmp27 via the constitutive ADH1 promoter and found that overexpressed Fmp27-ENVY localizes to the ER, where it colocalizes with the Scarlet-tagged ER marker Sec63 (Figure 2.4A). Scarlet is a bright monomeric red fluorescent protein, used to tag proteins (Bindels et al., 2017). The ER localization of overexpressed Fmp27 indicates that Fmp27 contains an ER localization signal. This also suggests that Fmp27 is recruited to cortical sites by a binding partner, which is saturated on the overexpression of Fmp27. To determine whether Fmp27 is an ER-resident protein, I used Phobius, a transmembrane domain (TMD) and signal sequence prediction software (Käll et al., 2004), to find putative TMDs in Fmp27. I found that there is a putative signal sequence at the N-terminus of Fmp27, spanning residues 1-28, that forms a part of an N-terminal hydrophobic helix (residues 1-32) (Figure 2.4C, D). I overexpressed the 1-28 residues of Fmp27 via an ADH1 promoter-containing plasmid and found that the 1-28 residues of Fmp27 indeed localize to the ER, and colocalize with the ER marker Sec63 (Figure 2.4A, B). On comparing the localization of overexpressed full length Fmp27-ENVY and the 1-28 region, I found that overexpressed full length Fmp27-ENVY localizes uniformly throughout the ER (~65%; P<0.01; Figure 2.4B), while cells with overexpressed Fmp27 (1-28)-ENVY exhibit a brighter ENVY fluorescence intensity at the nER as compared to the cER (~84%; P<0.01; Figure 2.4B). This shows that the N-terminal 28 residues of Fmp27 are sufficient for ER localization. Some cells were classified as having an ambiguous localization of overexpressed Fmp27 or Fmp27 (1-28), which implies that either there was no ENVY signal in these cells, or the localization of overexpressed Fmp27 was unclear (Figure 2.4B). 55 Figure 2.4: Fmp27 localizes to cortical sites and contains a hydrophobic helix for ER localization (A) Fmp27-ENVY localizes to cortical sites, while overexpressed Fmp27 localizes to the ER, as do residues 1-28, which are sufficient for this localization. Genomically Scarlet-tagged Sec63 is used as an ER marker. Custom settings were used to show protein localization in cells. Insets are scaled identically to overexpressed Fmp27-ENVY (explained in Materials and Methods). White arrowheads indicate Fmp27-ENVY localization. Scale bar =2µm. (B) Unpaired two-tailed t-test with Welch’s correction comparing the percentage of cells with the specific localization shown by overexpressed Fmp27-ENVY and overexpressed Fmp27-ENVY (1-28). Number of cells>100, n=3, p**<0.01. The legend to the right indicates the protein localizations shown in cells either overexpressing Fmp27-ENVY or overexpressing Fmp27-ENVY (1-28). (C) Shown D C A B 56 here is a longitudinal cross-section and a ribbon model of Fmp27, derived using Pymol. These models have been derived from the AlphaFold predicted structure of Fmp27 and have been colored according to hydrophobicity (Pymol; color scheme has been indicated at the bottom right) and key residues have been indicated. The longitudinal cross-section shows that the interior of Fmp27 is hydrophobic, indicated by the residues colored in red. The ribbon model of the Fmp27 structure (to the right) shows that Fmp27 contains a hydrophobic helix at its N-terminus of which 1-28 residues were predicted to function as a signal sequence (Phobius). (D) Helical wheel from Heliquest for Fmp27 1-28 residues indicates a helix with a hydrophobic face. The legend indicates the color scheme used for different kinds of amino acids. FL; full length, OE; overexpressed. 2.3.3 Fmp27 is recruited to cortical sites by Ybl086c To identify putative Fmp27-interacting partners, I turned to large-scale chemogenomics studies (Hoepfner et al., 2014) to identify genes that are functionally correlated with FMP27. The chemogenomics screen used a growth-based assay to identify the growth sensitivities of S. cerevisiae homozygous and heterozygous gene deletion collections to 1800 drugs (Hoepfner et al., 2014). Genes were correlated on basis of their similar growth profiles over the range of drugs used. Similar growth profiles indicate that the correlated genes function in similar pathways in cells. On searching for functionally correlated genes with FMP27 using the homozygous deletion profiling (HOP) assays, I found an uncharacterized ORF, YBL086C, to be the most correlated with FMP27 (0.56 Pearson correlation; Figure 2.5A). I recorded the list of the ten most correlated ORFs with FMP27, which included ER-PM MCS protein genes such as the sterol transporters and related proteins YSP2/LAM2/LTC4, LAF1, LAM1, SIP3, oxysterol binding protein homolog OSH3, a fatty acid elongase ELO2 and S. cerevisiae VAP ortholog SCS2. The YBL086C ORF encodes a well-conserved 52kDa protein, with two human orthologs, FAM102A and FAM102B (15.5% and 17.5% sequence identity respectively and 23% and 26% secondary structure homology between the N-terminal regions respectively) (Figure 2.5B). Ybl086c has been predicted to contain an N-terminal C2 57 (NT-C2) domain (predicted by Pfam), and a helical and primarily unstructured C-terminus (AlphaFold [Jumper et al., 2021]; Figure 2.5C, 2.6A). The NT-C2 domain is predicted with high confidence (Figure 2.6A). However, only few helices in the C-terminus are predicted with high confidence, while the rest is unstructured and predicted with very low confidence (Figure 2.6A). C2 domains have been found to interact with anionic and zwitterionic lipids in membranes in a calcium-dependent or independent manner (Corbalan-Garcia and Gómez-Fernández, 2014). This suggests that the N-terminus of Ybl086c could interact with the PM. Figure 2.5: Ybl086c is a well-conserved protein with functional correlation to Fmp27 (A) Chemogenomics (Hoepfner et al., 2014) identifies YBL086C as the most highly correlated gene with FMP27 (B) Ybl086c is a highly conserved protein containing an NT-C2 domain, indicated in gold, with well-conserved orthologs. Shown here are the H. sapiens orthologs FAM102A and FAM102B and the percent identities of their NT-C2 domains compared to Ybl086c NT-C2, based on the 58 secondary structure, are indicated. (C) The AlphaFold predicted structure of Ybl086c contains an NT-C2 domain (predicted via Pfam) and a helical and primarily unstructured C-terminus. The structure has been colored with ConSurf (Ashkenazy et al., 2016) to indicate the regions of conservation with other fungal species. 59 A B 60 Figure 2.6: Ybl086c predicted structure and degree of confidence of the predicted structure (A) The AlphaFold predicted structure of Ybl086c has been colored according to pLDDT scores (Jumper et al., 2021) to indicate the degree of confidence of the predicted model. The legend below the predicted model indicates the color scheme. (B) The PAE plot of the predicted structure of Ybl086c indicates AlphaFold’s expected position error at residue x, when the predicted and true structures are aligned on residue y.The legend indicates the expected position error in Angstroms represented by various shades of green. I found that C-terminally ENVY-tagged Ybl086c localizes to cortical sites, which could be ER-PM MCSs, and shows a partial colocalization with Tcb3 tagged with tdimer2 (a variant of RFP), an ER-PM MCS protein (Manford et al., 2012) (Figure 2.7A, B). To test whether Ybl086c recruits Fmp27 to cortical sites, I overexpressed Ybl086c in a Fmp27 overexpression strain. Fmp27 tagged with ENVY and Ybl086c tagged with Scarlet were overexpressed via a constitutive ADH1 promoter. I found that overexpressed Fmp27-ENVY localized uniformly throughout the ER (~65% cells; Figure 2.7C, D). On overexpressing Ybl086c in an Fmp27 overexpression background I found that some cells exhibited uniform ER localization (~7%; P<0.001; Figure 2.7D), while some cells exhibited a cER only localization (~5%; P<0.05; Figure 2.7D) and in most cells, overexpressed Fmp27-ENVY localized to the cER strongly as compared to the nER (~38%; P<0.01; Figure 2.7D). This indicates that overexpressed Ybl086c can recruit overexpressed Fmp27 to cortical sites (Figure 2.7C, D). Some cells had either no ENVY signal or unclear signals and were classified as ambiguous. These results show that Ybl086c is necessary for Fmp27 recruitment to ER-PM MCSs. 61 Figure 2.7: Ybl086c localizes to cortical sites and recruits Fmp27 (A) Ybl086c-ENVY localizes to cortical sites and partially colocalizes with Tcb3, an ER-PM MCS marker. Scale bar =2µm. White arrow in the merge image indicates the starting point for the line scan. (B) Line scans indicate a partial colocalization between Ybl086c-ENVY and Tcb3-tdimer2. Arrows indicate regions of overlap (C) Fmp27-ENVY localizes to the ER on overexpression. Overexpression of Ybl086c recruits overexpressed Fmp27 from the ER to cortical sites. Scale bar =2µm. (D) Unpaired two-tailed t-test with Welch’s correction of the different localizations exhibited by A D C B 62 overexpressed Fmp27-ENVY compared to when both Fmp27-ENVY and Ybl086c-Scarlet are overexpressed. Only significant differences are indicated. Number of cells>100, n=3, p*<0.05, p**<0.01 and p***<0.001. The legend to the right indicates the localizations shown in cells overexpressing Fmp27-ENVY only and overexpressing both Fmp27-ENVY and Ybl086c-Scarlet. OE; overexpressed. 2.3.4 Ybl086c interacts with and recruits Fmp27 via its C-terminus To test whether Fmp27 and Ybl086c interact with each other, I performed co-immunoprecipitation on strains expressing these proteins. I used genomically tagged Fmp27-ENVY and Ybl086c-3HA (three repeats of the hemagglutinin, HA epitope). In some strains used for the co-immunoprecipitation experiment, proteins were expressed at endogenous levels, in others either Fmp27-ENVY or Ybl086c-3HA were overexpressed via an ADH1 promoter and lastly a strain in which both Fmp27-ENVY and Ybl086c-3HA were overexpressed via an ADH1 promoter was included in the experiment. Based on the results obtained in Figure 2.7C, D, I expected that when higher levels of the tagged proteins are present in cells, the interaction would be elevated compared to when the tagged proteins are expressed at endogenous levels. I found that Fmp27 and Ybl086c interact with each other (Figure 2.8A), and the extent of interaction significantly increases on their overexpression (~3 fold higher compared to when both proteins are expressed at endogenous levels; P<0.0001; Figure 2.8A, B). 63 Figure 2.8: Interaction between Fmp27 and Ybl086c increases upon overexpression (A) Anti-GFP immunoprecipitation of genomically ENVY-tagged Fmp27 or overexpressed Fmp27 from strains co-expressing genomically 3HA-tagged Ybl086c or overexpressed Ybl086c. (B) Quantification of the interaction between Fmp27-ENVY and Ybl086c-3HA in A by densitometry. One-way ANOVA with Dunnett’s correction comparing wild-type (WT) interaction between Fmp27-A B 64 ENVY and Ybl086c-3HA to other strains, n=3, p*<0.5, p****<0.0001. Only significant differences are indicated. OE; overexpressed. To further elucidate whether Ybl086c interacts with Fmp27/Ypr117w via the N-terminal C2 domain or the C-terminal region, I generated plasmids carrying ENVY-tagged Ybl086c N or C-terminal fragments. I found that the C-terminal fragment of Ybl086c-ENVY is necessary and sufficient for the interaction with Fmp27-3HA and Ypr117w-3HA (Figure 2.9A-D). Figure 2.9: Ybl086c interacts with Fmp27 and Ypr117w via its C-terminus (A) Anti-GFP immunoprecipitation of plasmid-expressed full-length Ybl086c-ENVY, Ybl086c N-terminus (1-216)-ENVY or C-terminus (216-466)-ENVY from strains co-expressing genomically 3HA-tagged Fmp27 (B) Quantification of Fmp27-3HA IP in A by densitometry. Indicated as band intensity (%) relative to the interaction between full length Ybl086c-ENVY and Fmp27-3HA, n=3 (C) Anti-GFP immunoprecipitation of plasmid-expressed full-length Ybl086c-ENVY or Ybl086c N-terminus (1-216)-ENVY and C-terminus (216-466)-ENVY from strains co-expressing genomically 3HA-tagged Ypr117w (D) Quantification of Ypr117w-3HA 65 IP in C by densitometry. Indicated as band intensity (%) relative to the interaction between full-length Ybl086c-ENVY and Ypr117w-3HA, n=3. FL; full-length. To test whether the C-terminus of Ybl086c is sufficient to recruit overexpressed Fmp27 to cortical sites, I used a PM recruitment assay. The C-terminus of Ybl086c does not localize to the PM and shows a diffuse cytosolic localization (unpublished observations), making it necessary to use a PM recruitment assay to recruit the C-terminus of Ybl086c to the PM. For this assay, I generated plasmids expressing different Ybl086c regions fused to a PM localization module (Figure 2.10A). This module included two tandem repeats of the PH domain of PLCδ (2xPH(PLCδ)), which is required for its recruitment to the PM (Lemmon et al., 2007). This would ensure that the different Ybl086c constructs are recruited to the PM, and if the C-terminus of Ybl086c is sufficient for the recruitment of Fmp27, it would recruit overexpressed Fmp27 from the ER to cortical sites. These 2xPH(PLCδ)-Ybl086c chimeras were N-terminally tagged with mCherry (mCh), a variant of RFP. On expressing the mCh-2xPH(PLCδ)-Ybl086c and mCh-2xPH(PLCδ)-Ybl086c C-terminal (216-466) plasmids in an overexpressed Fmp27 background, I found that overexpressed Fmp27-ENVY localizes to the cER primarily (complete recruitment; >60% cells; P<0.001; Figure 2.10A, B), while in some cells overexpressed Fmp27-ENVY localizes partially to the cER (partial recruitment; <10%; Figure 2.10A, B) compared to when the 2xPH(PLCδ) plasmid is expressed by itself in an overexpressed Fmp27 background (Figure 2.10A, B). In cells expressing the mCh-2xPH(PLCδ) plasmid in an overexpressed Fmp27-ENVY background, overexpressed Fmp27-ENVY localizes uniformly throughout the ER in a significantly higher percentage of cells (no recruitment; >60%; Figure 2.10A,B). Some cells had either no ENVY signal or unclear signals and were classified as ambiguous. These results show that the C-terminus of Ybl086c is sufficient for the recruitment of Fmp27 to ER-PM MCSs. 66 Figure 2.10: The C-terminus of Ybl086c is sufficient to recruit overexpressed Fmp27 to cortical sites (A) Overexpressed Fmp27-ENVY is recruited to cortical sites by full length Ybl086c and the C-terminal tail (216-466) of Ybl086c fused to mCh-2xPH(PLCδ). Scale bar =2µm. A schematic explaining the 2xPH assay has been shown to the right. (B) One-way ANOVA with Dunnett’s correction comparing the percentage of cells exhibiting the different localizations of overexpressed Fmp27-ENVY with either 2xPH(PLCδ)-Ybl086c full length or 2xPH(PLCδ)-Ybl086c C-terminus (216-466) relative to those expressed with 2xPH(PLCδ). Only significant differences are indicated. Number of cells>100, n=3, p***<0.001, A B 67 p****<0. 0001.The legend to the right indicates the different protein localizations of overexpressed Fmp27-ENVY in cells expressing different 2xPH(PLCδ) constructs. FL; full length, mCh; mCherry (a variant of RFP), OE; overexpressed. I found that the N-terminus (1-216) of Ybl086c fused to 2xPH(PLCδ) cannot recruit overexpressed Fmp27 to cortical sites (Figure 2.11B,C). I divided the C-terminus of Ybl086c into smaller sections to find the region that is sufficient to recruit Fmp27 to the PM (Figure 2.11A-C). I found that the 295-345 region is the smallest region that shows a partial recruitment of Fmp27 to cortical sites, indicating that the possible interaction interface lies in this region. These results indicate that the C-terminus of Ybl086c is necessary for its interaction with Fmp27 and Ypr117w and the recruitment of Fmp27 to cortical sites (ER-PM MCSs). 68 Figure 2.11: Mapping the region of Ybl086c C-terminus sufficient for recruiting Fmp27 to the PM (A) A schematic showing the different truncations of Ybl086c made to map the region required for recruiting Fmp27 to cortical sites. Conserved regions have been indicated. (B) One-way ANOVA with Dunnett’s correction comparing the percentage of cells exhibiting the different localizations of overexpressed Fmp27-ENVY with different 2xPH(PLCδ)-Ybl086c chimeras relative to those expressed with 2xPH(PLCδ). Only significant differences are indicated. Number of cells>100, n=3, p*<0.05, p**<0.01, p***<0.001, p****<0.0001. The legend on the top indicates the protein localizations shown in cells expressing different 2xPH(PLCδ)-Ybl086c chimeras in an overexpressed Fmp27 background. (C) The smallest region of Ybl086c that recruits overexpressed Fmp27-ENVY to cortical sites is 295-345 residues. Scale bar =2µm. FL; full-length, mCh; mCherry (a variant of RFP), OE; overexpressed. 2.3.5 Ybl086c interacts with and recruits Fmp27 via a conserved region in its C-terminus To identify a conserved region in the C-terminus of Ybl086c, I used AlphaFold and ConSurf (Ashkenazy et al., 2016) modeling to analyze the C-terminus of Ybl086c for conserved regions (Figure 2.12A). I found two conserved helices spanning residues 304-315 and 326-336. The latter helix is highly conserved throughout eukaryotes, while the former is partially conserved and present only in fungal species. I deleted these helices individually and used the 2xPH(PLCδ) recruitment assay to test whether these helix deletion mutants can recruit overexpressed Fmp27 to cortical sites. I found that these helices are not necessary individually to recruit Fmp27 to cortical sites, as overexpressed Fmp27 localized to cortical sites primarily even on the deletion of these helices (Figure 2.11). I examined these helices and their flanking regions for conserved residues, to identify the residues required for Fmp27 binding and recruitment. I found that residues 302, 305, 308, 309, 310, 313, 314, 316, 317, 327, 331, 332, 333, 334, 335, 337 and 338 are highly conserved in fungal species (Figure 2.12A). I mutated these residues to alanine, except for 317 and 327, which are prolines. Prolines act as helix breakers 69 and mutating these amino acids could destabilize the structure of the conserved helices, hence I did not mutate these residues. I used the 2xPH(PLCδ) recruitment assay and found that this mutant (hereafter 15A mutant) can no longer recruit Fmp27 to cortical sites (Figure 2.12B,C). Further, I found this mutant can no longer bind to Fmp27 by co-IP (Figure 2.13A,B). This indicates that the conserved residues in Ybl086c C-terminal region are required for interaction with and recruitment of Fmp27 to cortical sites. Figure 2.12: Ybl086c recruits overexpressed Fmp27 to cortical sites via a conserved region in its C-terminus (A) The AlphaFold predicted structure of Ybl086c showing the conserved helices Helix 1 (304-315 aa) and Helix 2 (326-336 aa) in the C-terminus of Ybl086c. The structure has been colored with ConSurf to 70 indicate regions of conservation with other fungal species. The panel also shows the sequence of the mutated regions. Conserved residues are marked in orange. (B) Overexpressed Fmp27-ENVY is recruited to cortical sites by either the full length or the C-terminal tail (216-466) of Ybl086c, but not by the N-terminus (1-216) fused to mCh-2xPH (PLCδ). A mutation of 15 conserved residues in the C-terminus of Ybl086c to alanine (15A) blocks the recruitment to cortical sites. Scale bar =2µm. (C) One-way ANOVA with Dunnett’s correction comparing the percentage of cells exhibiting the different localizations of overexpressed Fmp27-ENVY with 2xPH(PLCδ)-Ybl086c full length, 216-466,1-216 or 15A mutant relative to those expressed with 2xPH(PLCδ). Only significant differences are indicated. Number of cells>100, n=3, p***<0.001, p****<0.0001. The legend to the right indicates the protein localizations shown in cells expressing different 2xPH(PLCδ)-Ybl086c chimeras in an overexpressed Fmp27 background. FL; full-length, mCh; mCherry (a variant of RFP), OE; overexpressed. 71 Figure 2.13: Ybl086c interacts with Fmp27 via a conserved region its C-terminus (A) Anti-GFP immunoprecipitation of plasmid-expressed full-length Ybl086c-ENVY or Ybl086c N-terminus (1-216) or C-terminus (216-466) or the 15A mutant from strains co-expressing genomically 3HA-tagged Fmp27 (B) Quantification of Fmp27-3HA IP in A by densitometry.Indicated as band intensity (%) relative to the interaction between full length Ybl086c-ENVY and Fmp27-3HA, n=4. FL; full length. 72 2.4 Conclusion and Discussion Our aim was to identify Vps13-like proteins that could transport lipids in bulk at ER-PM MCSs. Through this study, I identified Fmp27 and its paralog Ypr117w as proteins with structural similarities to Vps13. I found that Fmp27 localizes to the ER and is recruited to ER-PM MCSs by Ybl086c. A conserved region in the C-terminus of Ybl086c is necessary for this recruitment. I also found that Fmp27 and Ybl086c are functionally correlated with proteins localizing to ER-PM MCSs, which are also involved in lipid homeostasis. Like Vps13, Fmp27 could have a role in lipid transport and mutating Ybl086c could impair this function, by blocking the recruitment of Fmp27 to cortical sites. Fmp27 and Ybl086c are well conserved proteins, and the interaction and function of these proteins could shed light on the function of these proteins in human cells. 2.4.1 Fmp27 and Ypr117w are Vps13-like proteins I identified Fmp27 as the top-most hit in a HHpred search using a part of the Vps13 lipid transport module as a query. Other hits included Atg2, Ypr117w (a paralog of Fmp27) and Csf1. This indicates a structural similarity between Vps13 and these proteins. Fmp27 and its paralog Ypr117w are now classified as lipid transfer bridges of the Repeating Beta Groove (RBG) superfamily, which also includes VPS13, ATG2, Tweek (Csf1) and SHIP164 (Levine, 2022). The lipid transfer module in all members of this superfamily consist of RBG multimers, and fold into rod-like structures with a hydrophobic interior and a hydrophilic exterior (Levine, 2022). Lipids are predicted to travel through the length of the rod-like transfer modules (Li et al., 2020). Fmp27 and Ypr117w are predicted to fold into rod-like structures made up of RBG multimers and could transport lipids through their hydrophobic interior similar to other RBG members. However, which lipids are transported by Fmp27 and Ypr117w would need to be identified. 2.4.2 Fmp27 localizes to the ER via its N-terminus and is recruited to ER-PM MCSs by Ybl086c I found that Fmp27 is an ER-resident protein with a hydrophobic helix at its N-terminus. RBG proteins such as Atg2 contain an N-terminal helix that interacts with the ER 73 (Osawa et al., 2019), while Csf1 contains a short N-terminal transmembrane domain predicted to localize to the ER (John Peter et al., 2022). Moreover, VPS13A, VPS13C and VPS13D localize to the ER by interacting with the ER-resident VAPA/B proteins via a FFAT motif (Guillén-Samander et al., 2021; Kumar et al., 2018). This indicates that most RBG proteins interact with the ER via their N-terminus. I found that Fmp27 interacts with Ybl086c and is recruited to ER-PM cortical sites via a conserved region in its C-terminus. This is reminiscent of Vps13 and Atg2, which possess distinct domains that mediate their interactions with specific regions in their binding partners (Bean et al., 2018; Kumar et al., 2018; Osawa et al., 2019). For example, Vps13 adaptor proteins contain a PxP (proline-x-proline) motif that interacts with the VAB domain of Vps13 and results in Vps13 recruitment to MCSs (Bean et al., 2018). Similarly, Atg2 interacts with the IM via a region in its C-terminus (Osawa et al., 2019). Fmp27 was initially identified in a screen for the mitochondrial proteome (Reinders et al., 2006). We and another group (Neuman et al., 2022a) did not find any colocalization between Fmp27 and mitochondrial markers. However, recent studies by Toulmay et al., (2022) found a colocalization between Fmp27 and a mitochondrial marker and also between Fmp27 and Mmm1 (a component of the ERMES complex). C-terminally tagged Fmp27 is hard to visualize using widefield microscopy. However, in this study, Toulmay et al., (2022) used seven copies of GFP11, a fragment of GFP that would self-assemble with GFP 1-10, which aided in the visualization of Fmp27 using a widefield microscope. It is possible that we were unable to detect the colocalization of Fmp27 with mitochondrial markers due to the lack of a stronger fluorophore. Further, Fujimoto et al., (2023) identified Fmp27 in a proximity labelling screen for ER-mitochondrial MCS proteins in S. cerevisisae. It is possible that Fmp27 could be recruited to ER-mitochondrial sites via other binding partners or under different growth conditions. Similar to Vps13, it might be possible that the different binding partners of Fmp27 could compete with one another to recruit Fmp27 to different MCSs. It is also possible that Ybl086c is more abundant than the protein that recruits Fmp27 to ER-mitochondrial MCSs. In the Fujimoto et al., (2023) study, only proteins in proximity to ER-mitochondrial contacts were being investigated. A 74 comparison between the amount of Fmp27 found in proximity to ER-PM MCSs vs ER-mitochondria MCSs would help determine the abundance of Fmp27 at different MCSs. I found that Fmp27 can no longer interact with or be recruited by the conserved residue mutant (15A mutant) of Ybl086c. Similarly, mutations in the PxP motifs of Vps13 adaptors abrogate the recruitment of Vps13 to contact sites (Bean et al., 2018; Kumar et al., 2018) and could affect lipid transport activity at MCSs. Whether the 15A mutant of Ybl086c impacts Fmp27 function in lipid transport remains to be evaluated. 2.4.3 How is Ybl086c recruited to the PM? An open question however is how Ybl086c is recruited to the PM. Unpublished observations indicate that the NT-C2 domain of Ybl086c itself is not sufficient for its localization to the PM. NT-C2 domains are related to C2 domains, which bind to anionic or zwitterionic phospholipids in membranes either in a calcium dependent or independent manner (Corbalan-Garcia and Gómez-Fernández, 2014). The NT-C2 domains are predicted to interact with membranes in a calcium independent manner, as they lack the calcium binding regions found in the calcium dependent C2 domains. C2 domains are found in ER-PM MCS proteins such as Tcb3 and its orthologs, which recruit proteins to the PM, and are sufficient for PM localization (Pérez-Sancho et al., 2015). C2 domains in tricalbin proteins and their orthologs have been found to interact with various anionic lipids in the PM (Giordano et al., 2013; Manford et al., 2012; Pérez-Sancho et al., 2015; Saheki and De Camilli, 2017). Moreover, the recruitment of Ybl086c to the PM could be regulated. Ybl086c recruitment to the PM might require interactions with other ER-PM MCS proteins or might be dependent on the levels of anionic lipids at the PM. 2.4.4 Do Fmp27 and Ybl086c form a complex? I found that Fmp27 and Ybl086c need to be expressed at similar levels in the cell for the complete recruitment of Fmp27 to ER-PM MCSs. Since Fmp27 and Ybl086c interact with each other to bridge ER-PM MCSs, both would need to be expressed at similar levels to see a cortical site localization. Overexpressing Fmp27 alone would saturate Ybl086c, preventing its recruitment to ER-PM MCSs. Similarly overexpressing Ybl086c (cytosolic; unpublished observations) alone would not allow 75 for a complete recruitment of Fmp27 to ER-PM MCSs. However, when both proteins are overexpressed, both localize to cortical sites. this implies that these proteins could form a complex and are interdependent on each other for cortical site recruitment. 2.5 Materials and Methods 2.5.1 Yeast media Yeast peptone dextrose (YPD) or synthetic dextrose (SD) media lacking certain amino acids for selection of plasmids was used to culture yeast strains. Yeast strains were cultured at 30°C. 2.5.2 Yeast strains and plasmids Yeast strains and plasmids used in this study are listed in Supplementary Tables 1 and 2 respectively. Polymerase chain reaction (PCR)-based homologous recombination was used to generate the strains used in this study as described in Janke et al., 2004; Longtine et al., 1998. Colony PCR was used to confirm gene deletions. Plasmids were made by homologous recombination in yeast by cotransforming linearized plasmids with PCR products generated using oligos listed in Supplementary Table 3. The plasmids were recovered in Escherichia coli and sequenced. 2.5.3 Bioinformatics AlphaFold (https://AlphaFold.ebi.ac.uk/) was used to obtain predicted structures and PAE plots of S.cerevisiae Atg2, Fmp27 and Ypr117w. Pymol (The PYMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC) was used to modify and color the AlphaFold generated structures. AlphaFold2 powered ColabFold (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb) was used for ab initio modeling of Vps13 and Csf1 fragments. Chemogenomics database (Hoepfner et al., 2014) was used to identify genes functionally correlated with FMP27. FMP27 was added as a query in the “Strain correlation across HOP assays tab”. The search gave a Correlation vs Rank graph, from which ten most highly correlated genes with FMP27 were selected and tabulated. 76 Consurf (https://consurf.tau.ac.il/) was used to generate protein structures with degrees of conservation according to all fungal species. Predicted AlphaFold model IDs and Clustal Omega generated alignments were used for the predictions. HHpred (https://toolkit.tuebingen.mpg.de/tools/hhpred) was used to identify the distant structural homologs of S. cerevisiae Vps13. Vps13 amino acid sequence was obtained from Uniprot (ID:Q07878) and added as a query sequence in HHpred. HHpred gives an option to search across the proteones of different organisms and structural databases. The query sequence was searched against the S. cerevisiae proteome and no specific structural database was selected for the search. The HHpred search identified Atg2, Csf1 , Fmp27 and Ypr117w as proteins with structural similarities to Vps13. Default HHpred parameters were used for the HHpred searches. HHpred search with Vps13 (650-1390) as a query was performed on 26th March 2021 and with Vps13 APT1 domain as query was performed on 10th June 2021. Initial searches with full length Vps13 sequence were performed in 2018, but after Li et al., (2020) published information about the Vps13 lipid transport regions, the searches were re-performed with a refined query. Further, the same tool was used to identify Fmp27 and Ybl086c human orthologs. OrthoDB v10.1 (https://www.orthodb.org/) was used to obtain the sequences of Fmp27 and Ybl086c orthologs. These sequences were added to the Clustal Omega Tool (https://www.ebi.ac.uk/Tools/msa/clustalo/) to generate a multiple sequence alignment. The alignment was exported to JalView and edited by hand to eliminate incomplete sequences. Phobius (https://www.ebi.ac.uk/Tools/pfa/phobius/) was used to predict transmembrane regions and a signal sequence in Fmp27 (Uniprot ID:Q06179). Uniprot IDs of Fmp27 (ID:Q06179), Ybl086c (ID:P38177), Ypr117w (ID:Q06116), KIAA0100 (ID:Q14667), FAM102A (ID:Q5T9C2) and FAM102B (ID:Q5T8I3) were searched against the Pfam database (https://pfam.xfam.org/, now https://www.ebi.ac.uk/interpro/) to determine domain architecture. Vps13 and Atg2 architecture were derived from (Bean et al., 2018; Dziurdzik et al., 2021; Kaminska et al., 2016). 77 2.5.4 Fluorescence microscopy For imaging, log phase yeast were grown in synthetic dextrose (SD)-based media lacking certain amino acids for selection of plasmids unless otherwise mentioned, transferred to concanavalin A–treated glass bottom MatriPlates (Corning) and imaged on a Dmi8 inverted microscope (Leica Microsystems) with a high-contrast Plan Apochromat 63×/1.30 NA Glyc CORR CS objective (Leica Microsystems), an ORCA-Flash4.0 digital camera (Hamamatsu Photonics), using MetaMorph 7.8 software (MDS Analytical Technologies). Images were acquired under the widefield mode using lasers, at room temperature. All counting was done on blinded raw images; for presentation, images were processed by using MetaMorph, with linear intensity scale changes uniformly applied to all images of a given fluorophore. In the case that some signals were either very bright or very dim, and could not be identically scaled, uniformly applied brightness settings are shown as insets and custom settings were used to show protein localization in the full-size image. Cropping and adjustment of brightness was performed in Adobe Photoshop 2020. For Figure 2.11, uniform linear intensity scale changes were applied to all images in both GFP and RFP channels using MetaMorph. Custom brightness adjustments were applied in Adobe Photoshop to show protein localization in both channels. 2.5.5 Image analysis For all experiments, three independent replicates were imaged. At least 100 cells in the GFP channel were counted using the Fiji (Schindelin et al., 2012) Cell Counter tool. For the analysis of the localization of overexpressed Fmp27-ENVY N-terminus, the percentage of cells exhibiting different localizations of either overexpressed Fmp27-ENVY or overexpressed Fmp27(1-28)-ENVY were calculated and an unpaired two-tailed t-test with Welch’s correction was performed using GraphPad Prism. For the analysis of the recruitment of overexpressed Fmp27-ENVY to cortical sites by overexpressed Ybl086c-Scarlet, the percentage of cells exhibiting different localizations of overexpressed Fmp27-ENVY were calculated and an unpaired two-tailed t-test with Welch’s correction was performed using GraphPad Prism. For the 2xPH(PLCδ) recruitment analysis, the percentage of cells with specific localizations 78 of overexpressed Fmp27-ENVY in the cell were calculated and a one-way ANOVA with Dunnett’s correction was performed using GraphPad Prism. 2.5.6 Co-immunoprecipitation and Western Blotting For Fmp27-ENVY co-IP, yeast cells were grown to log-phase in YPD media and 70 OD600 of cells were spun down for immediate processing. Cells were frozen at -80°C for 20 minutes, thawed and resuspended in 500μl lysis buffer (50 mM HEPES pH 7.4, 1% Tween-20, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 1X yeast/fungal Protease Arrest) and lysed by vortexing with 0.5 mm diameter glass beads. 50μl of lysate was set aside and resuspended in 4X Laemmli Sample Buffer (8% SDS, 40% glycerol, 240 mM Tris-Cl pH 6.8, 0.004% bromophenol blue) with 8M urea and 10% β-mercaptoethanol. The remainder of the lysates were incubated with polyclonal rabbit anti-GFP (EU2; Eusera) for 1 h followed by Protein A-Sepharose beads (GE Healthcare) for another hour at 4°C. Washed beads were resuspended in 50μl of Thorner buffer (8 M urea, 5% SDS, 40 mM Tris pH 6.8, 0.1 M EDTA, 0.4 mg/ml bromophenol blue) with 5% β-mercaptoethanol and proteins eluted by heating at 100°C for 5 min. Samples were run on SDS-PAGE gels, transferred to nitrocellulose membranes overnight, blocked for an hour with 5% skimmed milk in TPBS (0.1% Tween-20 in PBS), probed with monoclonal anti-mouse GFP or HA.11 primary antibodies and a secondary horseradish peroxidase-conjugated polyclonal goat anti-mouse (115–035-146; Jackson ImmunoResearch Laboratories) antibody. For co-IPs with Ybl086c-ENVY mutation and truncations, yeast cells were grown to log-phase in synthetic minimal media and 70 OD600 of yeast were lysed, and a procedure similar to that described above was followed. Antibodies used for co-Ips have been listed in Supplementary Table 4. All blots were developed with Amersham ECL (GE Healthcare) or Amersham ECL Prime (GE Healthcare) chemiluminescent reagents and exposed to Amersham Hyperfilm (GE Healthcare). Films were scanned, and densitometry was performed in Fiji. Even though both the IP HA and Pre-IP HA samples were run on the same gel and the same exposure was used to probe the blots, different exposures for the IP and Pre-IP GFP blots were used. Even in cases in which the same exposure conditions 79 were used, the Pre-IP bands were either hardly detectable, or the IP bands were overexposed. Hence % recovery values have not been indicated for the blots. 80 Chapter 3 Investigating the function of Fmp27/Ypr117w and their binding partner Ybl086c 3.1 Synopsis Fmp27 is a Vps13-like protein that localizes to ER-PM MCSs, which have been implicated in lipid homeostasis, calcium signaling and the maintenance of membrane integrity in response to stresses. Chemogenomics data indicates that FMP27 is correlated to genes involved in lipid transport. This indicates that Fmp27 could be involved in lipid transport in cells. Impaired lipid transport could impact the membrane properties of mutants, affecting cellular response to various stresses. Thus, Fmp27/Ypr117w and Ybl086c mutants could have defective lipid transport, which could disrupt cellular stress response and the cellular distribution of lipids. To test this, I exposed fmp27Δ, ybl086cΔ, ypr117wΔ, fmp27Δypr117wΔ, fmp27Δybl086cΔ and a strain in which both Fmp27 and Ybl086c were overexpressed to different stresses. I used lipid probes to test the role of Fmp27/Ypr117w and Ybl086c in the distribution of glycerophospholipids. Lastly, I exposed Fmp27/Ypr117w and Ybl086c mutants to drugs impacting the sterol and sphingolipid pathways. I found that Fmp27/Ypr11w and Ybl086c deletion and overexpression strains grow without any defects on exposure to various stresses, such as non-permissive temperatures, high salt, alkaline pH and detergent stress. Additionally, I found that Fmp27/Ypr117w and Ybl086c do not have a clear role in the distribution of PS, PI4P and PI(4,5)P2 in cells. I found that fmp27Δypr117wΔ, ybl086cΔ and fmp27Δybl086cΔ mutants exhibit sensitivity to amphotericin B, a drug that binds to excess accessible sterols at the PM. This might imply that these mutants have excess accessible sterols at their PM, and these sterols are no longer bound to partner phospholipids and sphingolipids, and hence are accessible. This indicates a role for Fmp27/Ypr117w and Ybl086c in retaining sterols at the PM in a bound state. 3.2 Introduction ER-PM MCS proteins have been found to have a role in sterol transport (Gatta et al., 2015; Murley et al., 2017; Naito et al., 2019), phospholipid transport regulation (Thomas et al., 2022; Wong et al., 2021), calcium signaling (Várnai et al., 2007) and 81 in response to abiotic stresses (Collado et al., 2019; John Peter et al., 2022; Ruiz-Lopez et al., 2021; Thomas et al., 2022). I identified that Fmp27 localizes to cortical sites and could be involved in lipid transport owing to secondary structure similarities to Vps13, a bulk lipid transporter (Kumar et al., 2018). Chemogenomics data (Hoepfner et al., 2014) correlates FMP27 with genes involved in sterol transport such as YSP2/LAM2/LTC4, LAF1, LAM1 and SIP3 (Murley et al., 2017; Topolska et al., 2020), in sphingolipid biosynthesis such as ELO2; in phosphatidylinositol-4-phosphate (PI4P) sensing such as OSH3 (Stefan et al., 2011) and the ER membrane protein SCS2. This indicates that Fmp27 could be involved in maintaining cellular lipid homeostasis by interacting with either the sterol transport, sphingolipid synthesis or PI4P sensing pathways. Sterols interact with sphingolipids and phospholipids, contributing towards maintaining the structure, fluidity and permeability of the plasma membrane (Guan et al., 2009; Kishimoto et al., 2021; Topolska et al., 2020). This helps protect cells against various stressors such as temperature, ions, detergents, and toxic compounds (Mioka et al., 2018). Yeast cells have various mechanisms via which they control the levels and properties of different lipids, allowing yeast to adapt to different stress conditions (John Peter et al., 2022; Klose et al., 2012; Swain et al., 2002). Increased fluidity of the PM increases its permeability resulting in reduced membrane integrity. This makes the cells susceptible to small molecules. It has been observed that perturbing the sphingolipid biosynthesis pathway using the drug myriocin leads to an inhibition of the retrograde sterol transporter Lam2 (Ysp2) and its interactors (including Laf1), causing cells to have more accessible sterols at the PM (Roelants et al., 2018; Topolska et al., 2020). This results in an increased susceptibility of lam2Δ and laf1Δ to amphotericin B (Roelants et al., 2018; Topolska et al., 2020), a polyene antifungal drug that binds to accessible sterols in the PM and causes cytotoxicity. These mutants are also resistant to myriocin, as the increased levels of accessible sterols possibly make up for the loss of sphingolipids. I found that Fmp27/Ypr117w and Ybl086c do not have a role in cellular stress response using spot assays and in the distribution of the glycerophospholipids tested 82 using lipid FLARES. However, I found that the fmp27Δypr117wΔ, ybl086cΔ and fmp27Δybl086cΔ mutants exhibit sensitivity to amphotericin B, which might indicate that these mutants are unable to retain sterols in a bound state at the PM and hence have an excess of accessible sterols. This indicates a role for Fmp27 and Ybl086c in maintaining lipid homeostasis at the PM. 3.3 Results 3.3.1 Fmp27/Ypr117w and Ybl086c do not have a role in the growth of cells in response to different stress conditions To test whether Fmp27/Ypr117w and Ybl086c show any growth defects when grown at low temperature i.e., 16°C and high temperature i.e., 37°C, I spotted serial dilutions of fmp27Δ, ybl086cΔ, ypr117wΔ, fmp27Δypr117wΔ, fmp27Δybl086cΔ, and Fmp27 and Ybl086c overexpressed via the ADH1 promoter (OE FMP27 OE YBL086C) onto YPD plates. I used the mss4 temperature sensitive strain (exhibits inhibited growth at high temperatures) and the cold sensitive csf1^NAT (Csf1 truncated after 1003 amino acid residue, replicates cold sensitive phenotype seen in Tokai et al., 2000) strains as controls, and grew the strains for 5 days. Mss4 is a PI4P 5-kinase that phosphorylates PI4P to PI(4,5)P2 and is essential for generating the PM pool of PI(4,5)P2 (Desrivières et al., 1998). It has been found to have a role in the heat shock response of yeast (Audhya and Emr, 2002). I found that none of the Fmp27/Ypr117w and Ybl086c deletion or overexpression strains showed any growth defects on exposure to high or low temperatures (Figure 3.1), indicating that they are viable at a range of temperatures. 83 Figure 3.1: fmp27Δ, ybl086cΔ and ypr117wΔ do not show any growth defects at different temperatures Growth profiles by spot growth assay.10-fold serial dilutions of yeast cell cultures were spotted onto YPD plates and grown for 5 days at 16°C or 30°C or 37°C. OE; overexpressed, WT; wild type (BY4741). To further learn about the phenotypes exhibited by fmp27Δ, ybl086cΔ, ypr117wΔ, fmp27Δypr117wΔ, fmp27Δybl086cΔ, OE FMP27 OE YBL086C, I spotted serial dilutions of these strains onto YPD plates containing NaCl, LiCl, Sorbitol and SDS at the indicated concentrations, and YPD adjusted to alkaline pH (Figure 3.2 and 3.3). I used rim101Δ and rim8Δ, which have been shown to exhibit reduced viability in the presence of salt stress and alkaline pH stress (Marqués et al., 2015), hog1Δ, which has been shown to exhibit reduced viability in high osmolarity conditions (Westfall and Thorner, 2006) and vps13Δ, which has been found to have growth sensitivity in the presence of SDS (Soczewska et al., 2020) as controls. I found that none of the Fmp27/Ypr117w and Ybl086c mutant strains showed any growth defects to the different stress conditions tested (Figure 3.2 and 3.3), indicating that they are viable even on exposure to various stresses. These results indicate that there are redundant factors present that aid the growth of cells on exposure to various stresses, even when Fmp27/Ypr117w or Ybl086c are either deleted or overexpressed. To understand the role of Fmp27 and Ybl086c in lipid 84 homeostasis, I used direct approaches such as determining the distribution of phospholipids in Fmp27/Ypr117w and Ybl086c mutants using fluorescent lipid probes. 85 Figure 3.2: fmp27Δ, ybl086cΔ and ypr117wΔ do not show any specific phenotypes in response to NaCl and LiCl (A) Growth profiles by spot growth assay.10-fold serial dilutions of yeast cell cultures were spotted onto YPD plates or YPD plates supplemented with either 0.4M NaCl or 0.2M LiCl and grown for 3 days at 30°C. (B) Growth profiles by spot growth assay.10-fold serial dilutions of yeast cell cultures were spotted onto YPD plates or YPD plates supplemented with 1M NaCl and grown for 3 days at 30°C. OE; overexpressed, WT; wild type (BY4741). 86 Figure 3.3: fmp27Δ, ybl086cΔ and ypr117wΔ do not show any specific phenotypes in response to alkaline pH, high osmolarity conditions and an ionic detergent SDS (A) Growth profile by spot growth assay.10-fold serial dilutions of yeast cell cultures were spotted onto YPD plates or YPD plates supplemented with 1.2M Sorbitol or adjusted to alkaline pH and grown for 2 days at 30°C. (B) Growth profiles by spot growth assay.10-fold serial dilutions of yeast 87 cell cultures were spotted onto YPD plates or YPD plates supplemented with 0.03% SDS grown for 3 days at 30°C. OE; overexpressed, WT; wild type (BY4741). 3.3.2 Fmp27/Ypr117w and Ybl086c do not have a clear role in phospholipid distribution To test whether Fmp27/Ypr117w and Ybl086c have a role in phospholipid distribution, I imaged the fmp27Δypr117wΔ, ybl086cΔ, and OE FMP27 OE YBL086C (OE; overexpressed via the ADH1 promoter) strains with phospholipid FLARES (fluorescent lipid-associated reporters). FLARES are fluorescent reporters that contain fluorescently tagged lipid-binding domains from various proteins. I used the fluorescently tagged P4C domain of SidC (GFP-P4C), Lactadherin (GFP-LactC2) and the PH domain of phospholipase C delta (mCherry-2xPH(PLCδ)), to detect the distribution of PI4P, PS and PI(4,5)P2 respectively, in cells. Vps13 has been shown to transport glycerophospholipids in vitro (Kumar et al., 2018). It is possible that due to its secondary structure similarities with Vps13, Fmp27 transports glycerophospholipids and regulates their distribution. Hobbit, the fly ortholog of Fmp27, has been found to have a role in the steady-state subcellular localization of PI(4,5)P2 (Neuman et al., 2022a), indicating that Fmp27 could have a role in the distribution of PI(4,5)P2. PI4P is phosphorylated to PI(4,5)P2 by PI4P 5-kinases. An altered distribution of PI(4,5)P2 in cells could be a result of altered PI4P levels. Moreover, ER-PM MCSs have recently been found to have a role in PS homeostasis in yeast cells (Thomas et al., 2022). Hence, I tested the distribution of these lipids in Fmp27/Ypr117w and Ybl086c mutants. Rapidly growing small daughter buds of yeast are enriched in PI4P compared to mother cells (Omnus et al., 2020). PI4P is rapidly dephosphorylated to PI at ER-PM MCSs to maintain the steady state levels of PI and PI4P. Small daughter buds lack the machinery to dephosphorylate PI4P to PI, thus exhibiting elevated levels of PI4P at their PM (Omnus et al., 2020). Osh3 has been proposed to regulate the PI4P phosphatase, Sac1, at ER-PM MCSs (Stefan et al., 2011). Hence, osh3Δ has been found to have an increase in PI4P levels in mother cells leading to a uniform 88 distribution of PI4P across mother and daughter cells, since the PI4P in the mother cells can no longer be dephosphorylated (Omnus et al., 2020; Stefan et al., 2011). Thus, I used osh3Δ as a control to observe the distribution of the GFP-P4C probe in Fmp27/Ypr117w and Ybl086c mutants. A deletion of the six ER-PM MCS tether proteins or Δ-tether (Manford et al., 2012) cells have been found to have an impaired transport of PS, which is exhibited by a reduced intensity of the PS FLARE at the PM (Thomas et al., 2022). Moreover, these cells have been found to have a significant decrease in the intensity of the PI(4,5)P2 FLARE at the PM. I used the Δ-tether as a control to test for altered PS and PI(4,5)P2 distribution in cells. I imaged fmp27Δypr117wΔ, ybl086cΔ, OE FMP27 OE YBL086C and appropriate controls with these FLARES at 30°C. I found that neither Fmp27/Ypr117w nor Ybl086c mutations affect the distribution of any of the indicated lipid FLARES (Figure 3.4-3.6). 89 Figure 3.4: fmp27Δ, ybl086cΔ and ypr117wΔ do not show differences in PI4P localization compared to WT, BY4741 (A) Localization of the PI4P probe (GFP-P4C) in mother and daughter cells in indicated strains. Scale bar =2µm. (B) One-way ANOVA with Dunnett’s correction of the ratio of GFP fluorescence exhibited by the daughter vs the mother cell of all strains compared to WT. Only significant differences are indicated. Total number of cells ~70-100, biological replicates n=3, **p<0.01. The method described in Omnus et al., (2020) was used for analysis. OE; overexpressed, WT; wild type. 90 Figure 3.5: fmp27Δ, ybl086cΔ and ypr117wΔ do not show differences in PS localization compared to WT, BY4741 (A) Localization of the PS probe (GFP-C2) in indicated strains. Scale bar =2µm. (B) One-way ANOVA with Dunnett’s correction of the ratio of GFP fluorescence exhibited by PM vs the cytoplasm in all strains compared to WT. Only significant differences are indicated. Number of cells per replicate =10, biological replicates n=3, ***p<0.001. The method described in Thomas et al., (2022) was used for analysis. OE; overexpressed, WT; wild type. 91 Figure 3.6: fmp27Δ, ybl086cΔ and ypr117wΔ do not show differences in PI(4,5)P2 localization compared to WT, BY4741 (A) Localization of the PI(4,5)P2 (mCherry-2xPH (PLCδ)) probe in indicated strains. Scale bar =2µm. (B) One-way ANOVA with Dunnett’s correction of the ratio of GFP fluorescence exhibited by the PM vs the cytoplasm in all strains compared to WT. Only significant differences are indicated. Number of cells per replicate =10, biological replicates n=3, ****p<0.0001. The method described in Thomas et al., (2022) was used for analysis. OE; overexpressed, WT; wild type. 92 3.3.3 Fmp27/Ypr117w and Ybl086c have a role in the retention of sterols in a bound state at the PM Chemogenomics data indicated that Fmp27 and Ybl086c might have a role in sterol and sphingolipid regulation (Hoepfner et al., 2014). Hence, I used drugs that directly impact sterol and sphingolipid pathways to test the phenotypes of Fmp27/Ypr117w and Ybl086c mutants on exposure to these drugs. I exposed the fmp27Δ, ybl086cΔ, ypr117wΔ, fmp27Δypr117wΔ, fmp27Δybl086cΔ, OE FMP27 OE YBL086C (OE; overexpressed via the ADH1 promoter) and appropriate control strains to fluconazole, valinomycin, myriocin and amphotericin B. I spotted serial dilutions of fmp27Δ, ybl086cΔ, ypr117wΔ, fmp27Δypr117wΔ, fmp27Δybl086cΔ, OE FMP27 OE YBL086C and appropriate controls onto YPD plates supplemented with drugs. It has been shown that cells with compromised integrity take up cytotoxic compounds much more easily than wild type cells (Mioka et al., 2018). Lem3, a non-catalytic subunit of a yeast flippase, has been found to be important in maintaining PM integrity (Mioka et al., 2018), and lem3Δ has been found to exhibit sensitivity on exposure to fluconazole (Mioka et al., 2018). Fluconazole is a drug that inhibits sterol synthesis by inhibiting Erg11 and perturbs membrane integrity (White et al., 1998). I exposed fmp27Δ, ybl086cΔ, ypr117wΔ, fmp27Δypr117wΔ, fmp27Δybl086cΔ, OE FMP27 OE YBL086C and lem3Δ to fluconazole and found that none of the Fmp27/Ypr117w and Ybl086c mutants exhibit sensitivity to fluconazole (Figure 3.7). This indicates that Fmp27/Ypr117w and Ybl086c might not have a role in the synthesis of sterols or redundant mechanisms might be at play. 93 Figure 3.7: fmp27Δ, ybl086cΔ and ypr117wΔ do not show sensitivity in response to fluconazole Growth profiles by spot growth assay.10-fold serial dilutions of yeast cell cultures were spotted onto YPD plates or YPD plates supplemented with the indicated concentrations of fluconazole (FLC) and grown for 2 days at 30°C. OE; overexpressed, WT; wild type (BY4741). Valinomycin was obtained as a potential hit in a chemogenomics database search for drugs conferring sensitivity to FMP27 and correlated genes (Hoepfner et al., 2014). Valinomycin is a potassium ion uniporter that directly impacts the mitochondria; however, the state of cellular sterols has been found to impact the phenotypes obtained on exposing gene deletions to this drug (Jakubkova et al., 2016). I found that fmp27Δypr117wΔ, ybl086cΔ, and fmp27Δybl086cΔ are sensitive in response to valinomycin (Figure 3.8), indicating that cellular sterols might be perturbed in these mutants. 94 Figure 3.8: fmp27Δypr117wΔ, ybl086cΔ and fmp27Δybl086cΔ show sensitivity in response to valinomycin Growth profiles by spot growth assay.10-fold serial dilutions of yeast cell cultures were spotted onto YPD plates or YPD plates supplemented with the indicated concentration of valinomycin and grown for 3 days at 30°C. OE; overexpressed, WT; wild type (BY4741). I used myriocin to inhibit sphingolipid synthesis and to test the response of fmp27Δ, ybl086cΔ, ypr117wΔ, fmp27Δypr117wΔ, fmp27Δybl086cΔ, OE FMP27 OE YBL086C and appropriate controls on exposure to this drug. Myriocin is a drug that inhibits serine palmitolytransferase (Figure 3.9A), which catalyzes the first step in sphingolipid synthesis (Miyake et al.,1995). This leads cells to be devoid of sphingolipids and inhibits their growth. It has been found that in cells exposed to myriocin, retrograde transport of sterols by Ysp2 (also called Lam2 or Ltc4) and related proteins (Laf1, Lam1, Sip3, Lam4) is inhibited, which would help cells retain excess accessible sterols at the PM and help maintain their integrity in the absence of sphingolipids (Murley et al., 2017; Roelants et al., 2018). Laf1Δ cells cannot transport sterols in a retrograde manner from the PM to ER and hence retain accessible sterols at their PM, contributing to myriocin resistance and amphotericin B sensitivity. I found that fmp27Δypr117wΔ, ybl086cΔ, and fmp27Δybl086cΔ are 95 slightly resistant to myriocin (Figure 3.9B), indicating a possible elevation of accessible sterol levels at the PM in these mutants. A B 96 Figure 3.9: fmp27Δ ypr117wΔ, ybl086cΔ and fmp27Δybl086cΔ show a slight resistance in response to myriocin (A) A schematic indicating the mode of action of myriocin on the sphingolipid pathway in yeast. Toume and Tani, Figure 1. From Toume, M., & Tani, M. (2016). Yeast lacking the amphiphysin family protein Rvs167 is sensitive to disruptions in sphingolipid levels. The FEBS Journal, 283(15), 2911–2928. https://doi.org/10.1111/febs.13783. Adapted with permission from RightsLink / Elsevier. (B) Growth profiles by spot growth assay.10-fold serial dilutions of yeast cell cultures were spotted onto YPD plates or YPD plates supplemented with the indicated concentration of myriocin and grown for 5 days at 30°C. OE; overexpressed, WT; wild type (BY4741). I exposed fmp27Δ, ybl086cΔ, ypr117wΔ, fmp27Δypr117wΔ, fmp27Δybl086cΔ, OE FMP27 OE YBL086C and appropriate controls to amphotericin B, to test whether these mutants have excess accessible sterols in their PM. Amphotericin B is a polyene that binds to accessible sterols (sterols not interacting with sphingolipids or phospholipids at the PM), sequesters them and forms pores to reduce membrane integrity of cells (Anderson et al., 2014). I found that fmp27Δypr117wΔ, ybl086cΔ, and fmp27Δybl086cΔ are sensitive to amphotericin B (Figure 3.10), indicating a possible increase in accessible sterols in these cells. An increase in accessible sterols would mean that these cells are unable to retain sterols in a bound state, indicating an alteration in either sphingolipid or phospholipid levels at the PM. 97 Figure 3.10: fmp27Δypr117wΔ, ybl086cΔ and fmp27Δybl086cΔ show sensitivity in response to amphotericin B Growth profiles by spot growth assay.10-fold serial dilutions of yeast cell cultures were spotted onto YPD plates or YPD plates supplemented with the indicated concentration of amphotericin B (AmB) and grown for 4 days at 30°C. OE; overexpressed, WT; wild type (BY4741). I further determined whether the growth rate of Fmp27/Ypr117w and Ybl086c deletion and overexpression strains is affected on exposure to amphotericin B. For this, I used a 96 well-based plate reader assay and assessed the growth patterns of wild type (WT; BY4741), laf1Δ, fmp27Δypr117wΔ, ybl086cΔ, fmp27Δybl086cΔ and OE FMP27 OE YBL086C (OE; overexpressed via the ADH1 promoter) strains. I found that fmp27Δybl086cΔ , ybl086cΔ, and fmp27Δypr117wΔ grow slower as compared to WT cells (Figure 3.11B) and have a significantly lower growth rate compared to WT (P<0.05; Figure 3.11C). I then tested whether the interaction between Fmp27 and Ybl086c, and hence the recruitment of Fmp27 to cortical sites is necessary for sterol retention in a bound state. For this I used plasmids expressing either full-length Ybl086c or Ybl086c 15A mutant (does not interact with Fmp27) and tested whether these could rescue the amphotericin B sensitive phenotype of ybl086cΔ. I found that full length Ybl086c could rescue the amphotericin B sensitive phenotype and restore growth, while the 15A mutant could 98 not (Figure 3.12). This indicates that the interaction between Fmp27 and Ybl086c is required for retaining sterols in a bound state at the PM. 99 Figure 3.11: laf1Δ, fmp27Δypr117wΔ, ybl086cΔ and fmp27Δybl086cΔ show sensitivity in response to amphotericin B (A) The indicated strains were grown to log phase and transferred to 96 well plates. Starting OD600=0.04. DMSO was used as the solvent control and the OD was monitored every 30 minutes for 19 hours. (B) The indicated strains were grown to log phase and transferred to 96 well plates. Starting OD600=0.04. The indicated concentration of amphotericin B (AmB) was added, and the OD was monitored every 30 minutes for 19 hours. (C) The growth rate was determined from log-phase (5.5-10.5 hours) OD600 values. One-way ANOVA with Dunnett’s correction was performed to compare the growth rate of all strains to the WT (BY4741). Only significant differences are indicated. N=3, p*<0.05, p***<0.001. OE; overexpressed. 100 Figure 3.12: Ybl086c 15A mutant cannot rescue the amphotericin B sensitive phenotype of ybl086cΔ (A) The indicated strains were grown to log phase and transferred to 96 well plates. Starting OD600=0.1. DMSO was added as the solvent control and the OD was monitored every 30 minutes for 22 hours. (B) The indicated strains were grown to log phase and transferred to 96 well plates. Starting OD600=0.1. The indicated concentration of amphotericin B (AmB) was added, and the OD was monitored every 30 minutes for 22 hours. (C) The growth rate was determined from log-phase (5.5-12.5 hours) OD600 values. One-way ANOVA with Dunnett’s correction was performed to compare the growth rate of all strains to the WT (BY4741). Only significant differences are indicated. N=4, p*<0.05, p**<0.01, p***<0.001. OE; overexpressed, WT; wild type (BY4741). 101 3.4 Conclusion and Discussion Fmp27 and Ypr117w are uncharacterized proteins despite some indications about the functions of their orthologs (Song et al., 2006; Neumann and Bashirullah, 2018). I found that Fmp27/Ypr117w and Ybl086c do not have a role in response to various stresses using spot assays and in the distribution of glycerophospholipids such as PS, PI4P and PI(4,5)P2 usigng lipid FLARES. I found that the fmp27Δypr117wΔ, ybl086cΔ and fmp27Δybl086cΔ mutants are sensitive on exposure to amphotericin B. This indicates that these mutants might have increased levels of accessible sterols at the PM. Fmp27/Ypr117w and Ybl086c could be involved in maintaining sterols in a bound state at the PM and could thus regulate lipid homeostasis. 3.4.1 Fmp27/Ypr117w and Ybl086c retain sterols in a bound state at the PM Chemogenomics data functionally correlates FMP27 with proteins involved in sterol transport (Hoepfner et al., 2014). Hence, I tested the phenotypes exhibited by Fmp27/Ypr117w and Ybl086c mutants in response to amphotericin B, a drug that sequesters excess accessible sterols at the PM (Anderson et al., 2014). Previous studies have shown that strains lacking sterol retrograde transporters that are functionally correlated to FMP27 and YBL086C are sensitive to this drug (Murley et al., 2017; Roelants et al., 2018; Topolska et al., 2020). This has been attributed to the presence of excess accessible sterols at the PM in these mutants. I found that fmp27Δypr117wΔ, ybl086cΔ, fmp27Δybl086cΔ exhibit a sensitive phenotype on exposure to amphotericin B both via spot assays and liquid-based growth assays. This implies that these mutants might have an excess of accessible sterols in their PM. Sterols are a major component of the eukaryotic PM and play a major role in the maintenance of membrane integrity. Sterols are retained in the PM in a bound state via their interactions with sphingolipids and phospholipids. Phospholipid asymmetry has been found to be important for sterol retention at the PM. Loss of phospholipid asymmetry leads to an internalization of accessible sterols and their subsequent esterification to form lipid droplets (Kishimoto et al., 2021). 102 Excess accessible sterols at the PM would indicate that fmp27Δypr117wΔ, ybl086cΔ, fmp27Δybl086cΔ mutants have impaired sphingolipid or phospholipid transport, thus preventing an efficient interaction of sterols with these lipids. It is also possible that fmp27Δypr117wΔ, ybl086cΔ, fmp27Δybl086cΔ mutants lack phospholipid asymmetry and hence contain excess accessible sterols at the PM. I also found that full length Ybl086c can rescue amphotericin B sensitivity, while the 15A mutant cannot. This indicates that Fmp27 and Ybl086c work together in sterol regulation, and Fmp27 needs to be recruited to ER-PM MCSs via Ybl086c to function in lipid homeostasis. Previous studies have found that VPS13A and Vps13 respectively interact with the scramblases XK (Guillén-Samandar et al., 2022; Park et al., 2020) and Mcp1 (Adlakha et al., 2022). It is speculated that Vps13 transports lipids from the ER, which acts as a lipid reservoir, to the acceptor membrane. ATG2 has been similarly found to transport lipids from the ER to the growing autophagosome and to associate with scramblases at both the ER (TMEM41B and VMP1) and autophagosome (ATG9) membranes (Ghanbarpour et al., 2021; Maeda et al., 2019; Matoba et al., 2020). Lipid scramblases ensure that lipid equilibrium is maintained at both donor and acceptor membranes. Fmp27 could similarly transport lipids that interact with sterols from the ER to the PM, where it could interact with a scramblase to ensure the transfer of lipids to the appropriate PM leaflet. When either Fmp27/Ypr117w or Ybl086c are absent, or can no longer interact with each other, the transport of the lipids that interact with sterols to the PM will be impaired. This could lead to an accumulation of excess accessible sterols at the PM, as sterols would be present in an unbound state due to the lack of shielding lipids. An accumulation of accessible sterols at the PM would lead to their subsequent internalization mediated by retrograde sterol transporters. Amphotericin B sensitivity in fmp27Δypr117wΔ, ybl086cΔ, fmp27Δybl086cΔ mutants indicates that these mutants might have elevated levels of accessible sterols at the PM and are thus unable to retain sterols in a bound state at the PM. This indicates a role for Fmp27/Ypr117w and Ybl086c in the transport of lipids that interact with sterols and the retention of sterols at the PM. Further, studies in the lab have shown that in fmp27Δypr117wΔ and ybl086cΔ cells, mCherry-D4H, a probe that binds to 103 accessible sterols in the cytosolic leaflet of membranes, accumulates in internal punctate structures primarily, which could be endocytic compartments (S. Dziurdzik, unpublished results). Moreover, in a mutant with a deletion of LAF1 in an ybl086cΔ background mCherry-D4H localizes to the PM and not to punctate structures (S. Dziurdzik, unpublished results). This might indicate that Ybl086c could act downstream of Laf1 (S. Dziurdzik, unpublished results). The retrograde sterol transporters could also interact with other proteins like Fmp27/Ypr117w and Ybl086c to mediate the transport of phospholipids that interact with the excess sterols at the PM. Similarly, in Schizosaccharomyces pombe, Ltc1 (an ortholog of S. cerevisiae Lam proteins, which localizes to ER-PM MCSs) has been found to have a role in the internalization of excess accessible sterols into sterol rich compartments called STRICs (Marek et al., 2020). Ltc1Δ mutants are unable to accumulate the D4H probe in STRICs and instead accumulate the probe in the PM. Moreover, this mutant is sensitive to amphotericin B, similar to its S. cerevisiae counterparts (Marek et al., 2020). This could mean that when there is an excess of accessible sterols in the PM in cells, retrograde transporters and their interacting partners could mediate an internalization of these sterols into internal punctate structures via the ER. 3.4.2 Which glycerophospholipids are transported by Fmp27/Ypr117w? I found using lipid FLARES that Fmp27/Ypr117w and Ybl086c do not have a role in the distribution of the glycerophospholipids PS, PI4P and PI(4,5)P2. I focused on these phospholipid species specifically as the depletion of different ER-PM MCS proteins has been found to impact the distribution of these lipids in cells (Omnus et al., 2020; Thomas et al., 2022). However, the use of lipid FLARES has some limitations (mentioned in section 4.7). It is possible that lipid FLARES might not be capturing the changes in the levels of the glycerophospholipids tested and we might need to use techniques such as lipidomics to determine the changes in glycerophospholipid levels in Fmp27/Ypr117w and Ybl086c mutants. 104 Moreover, it is possible that Fmp27 may not be involved in the transport of the lipids tested and could instead transport or have a role in the distribution of either PE, PC, PA similar to Vps13, Atg2 and Csf1 or sphingolipids.Vps13, Atg2 and Csf1 have been found to transport glycerophospholipids, with evidence for the transport of PS in the case of Vps13 (Kumar et al., 2018), PE, PC and PS in case of Atg2 (Osawa et al., 2019) and PE in the case of Csf1 (Toulmay et al., 2022). 3.5 Materials and Methods 3.5.1 Yeast strains and plasmids Yeast strains and plasmids used in this study are listed in Supplementary Tables 1 and 2 respectively. Polymerase chain reaction (PCR)-based homologous recombination was used to generate the strains used in this study as described in Janke et al., 2004; Longtine et al., 1998. Colony PCR was used to confirm gene deletions. Plasmids were made by homologous recombination in yeast by cotransforming linearized plasmids with PCR products generated using oligos listed in Supplementary Table 3. The plasmids were recovered in Escherichia coli and sequenced. 3.5.2 Chemicals Drugs were purchased in powdered form and solubilized in specific solvents to make the stock solutions. Fluconazole, valinomycin and amphotericin B were purchased from Sigma, myriocin was purchased from Cayman Chemicals. Sodium chloride, sodium hydroxide and DMSO were purchased from Fisher Scientific, sorbitol and lithium chloride were purchased from Sigma and SDS from BioRad Laboratories. 3.5.3 Growth Assays To examine cell growth phenotypes, cells were grown in the appropriate media overnight and sub-cultured for 4 hours to achieve OD600 between 1-2 units and then 10-fold serial dilutions were spotted onto YPD plates or YPD supplemented with salts or drugs and grown at the indicated conditions. All experiments were performed at least three times to confirm the results. 105 For liquid culture growth assays, the method from Hung et al., 2018 was adapted. Briefly, cells were grown overnight in either YPD or synthetic dextrose (SD) media at 30°C. Overnight cultures were reseeded to an OD600=0.1 in 5ml YPD or SD and were grown to log phase. Cultures were diluted to OD600=0.2 in the appropriate media and 100µl of diluted cultures were transferred to a 96 well plate. Drugs were diluted in the assay media to twice the desired final concentration. 100µl of diluted chemicals were transferred to corresponding wells of the 96 well plate. Similarly, an equivalent amount of solvent was added as the untreated control. Some wells were also reserved for blank readings and 200µl of media was added to these. All wells were pipette mixed three times. OD600 readings were taken every 30 minutes, for at least 19 hours, with intermittent orbital shaking at 360 rpm for 2 minutes. Three technical replicates were included. The plate was covered with a lid, to prevent evaporation, which was removed by the instrument before taking every reading. The lid was treated with 0.05% Triton-X and 20% ethanol and air dried to prevent condensation (Jung et al., 2015). After the experiment was completed, the blank OD600 readings were subtracted from the sample OD600 readings, the three technical replicates were averaged and OD600 vs time (in hours) was plotted. OD600 values corresponding to the log phase of growth were used to calculate growth rate, which was averaged and plotted. A one-way ANOVA with Dunnett’s correction was performed using GraphPad Prism. 3.5.4 Fluorescence microscopy For imaging, log phase yeast were grown in SD-based media lacking certain amino acids for the selection of plasmids unless otherwise mentioned, transferred to concanavalin A–treated glass bottom MatriPlates (Corning) and imaged on a Dmi8 microscope (Leica Microsystems) with a high-contrast Plan Apochromat 63X/1.30 NA Glyc CORR CS objective (Leica Microsystems), an ORCA-Flash4.0 digital camera (Hamamatsu Photonics), under the widefield mode using lasers, at room temperature. MetaMorph 7.8 software (MDS Analytical Technologies) was used to acquire the images. For presentation, images were processed by using MetaMorph, and cropping and adjustment of brightness was performed in Adobe Photoshop 2020. 106 3.5.5 Image analysis For lipid FLARE localization assay, three independent replicates were imaged for each sample. The method described in Omnus et al., (2020) was used to quantify the distribution of GFP-P4C. Briefly, cells with a daughter bud were selected and a line was drawn through mother and daughter cells using Fiji. Fluorescence intensities were plotted and the values corresponding to the PM localization of GFP-P4C were averaged to obtain the fluorescence intensity of the mother cell (Fm) and the fluorescence intensity of the daughter cell (Fd). Ratio of Fd to Fm was calculated, plotted and analyzed using a one-way ANOVA with Dunnett’s correction, which was performed using GraphPad Prism. The method described in Thomas et al., (2022) was used to quantify the distribution of GFP-C2 and mCh-2xPH(PLCδ). Briefly, a line was drawn through cells using Fiji. Fluorescence intensities were plotted and the values corresponding to the PM localization of the lipid FLARES were averaged to obtain the fluorescence intensity of the PM. Similar lines were drawn through the cytoplasm of the cell to obtain the fluorescence intensity of the cytoplasm and in the background to obtain the fluorescence intensity of the background. The background fluorescence intensities were subtracted from the PM and cytoplasm intensities. Ratio of FPM-FBackground to FCytoplasm-FBackground was calculated, plotted and analyzed using a one-way ANOVA with Dunnett’s correction, which was performed using GraphPad Prism. 107 Chapter 4: Discussion and Future Directions 4.1 Summary of key findings In this study, I found a putative Vps13-related lipid transport protein Fmp27, and its paralog Ypr117w. I identified Ybl086c, a previously uncharacterized protein, as a binding partner for both these proteins. I characterized the interaction between Fmp27 and Ybl086c and investigated the function of these proteins. I found that Fmp27 localizes to ER-PM cortical sites. It contains an N-terminal hydrophobic helix to insert into the ER and is recruited to the PM by Ybl086c. These proteins might function in the retention of sterols at the PM in a bound state, as their deletion mutants exhibit sensitivity on exposure to amphotericin B. Sensitivity to amphotericin B might imply an elevation in the levels of accessible sterols at the PM. This indicates that Fmp27/Ypr117w and Ybl086c have a role in lipid homeostasis in cells. 4.2 Characterizing the recruitment of Ybl086c and Fmp27 to ER-PM MCSs I identified that Ybl086c interacts with and recruits Fmp27 to the PM. However, it remains to be investigated how Ybl086c is recruited to the PM. Ybl086c contains an N-terminal C2 domain and is highly conserved across eukaryotes. C2 domains in proteins have been found to bind to anionic and zwitterionic lipids in membranes in a calcium dependent or independent manner (Corbalan-Garcia and Gómez-Fernandez, 2014). C2 domains that are recruited to membranes in a calcium dependent manner contain calcium binding regions, which also contain basic residues to interact with phospholipids (Lemmon, 2008). C2 domains that interact with membranes in a calcium independent manner contain basic patches to interact with phospholipids in membranes (Lemmon, 2008). C2 domain sequences of different proteins are highly variable. When we aligned the sequences of Ybl086c from different organisms, we did not find any calcium binding regions, but we found conserved basic patches (E. Conibear, S. Dziurdzik unpublished observations). It is possible that the NT-C2 domain of Ybl086c binds to the PM by interacting with anionic lipids via these patches, and mutating these conserved regions would help determine whether these patches are required for the interaction of the NT-C2 domain to the PM. 108 When I express the C2 domain of Ybl086c by itself, I find that it does not localize to the PM (unpublished observations). This indicates that the recruitment of Ybl086c to the PM could be regulated and that the C2 domain is not sufficient for this recruitment. The recruitment of Ybl086c could be mediated by an interaction with other proteins. To find other proteins that interact with Ybl086c, apart from Fmp27 and Ypr117w, in a future experiment we would perform the immunoprecipitation of epitope tagged Ybl086c and appropriate controls from yeast lysates and use mass spectrometry to identify the interactome of Ybl086c. This would be followed by microscopy-based experiments to test whether Ybl086c is recruited to cortical sites in the absence of potential interactors (top hits from mass spectrometry analysis after filtering out contaminants using data from the CRAPome database). Further, we would test whether Ybl086c interacts with the top hits via co-immunoprecipitation experiments. Levels of certain lipids could potentially influence the localization of Ybl086c. For example, E-Syt2/3 (C2 domain containing proteins; human orthologs of yeast tricalbins) are unable to localize to the PM when PI(4,5)P2 is depleted in COS7 cells (Giordano et al., 2013). Similarly, Ybl086c recruitment to ER-PM MCSs could be influenced by the levels of specific anionic lipids. Hence, the Fmp27-Ybl086c complex would be recruited to ER-PM MCSs only when specific lipids are present at certain levels at the PM. To test this, we would use yeast mutants which have an altered expression of lipids, such as those lacking the enzymes required to generate anionic phospholipids. For example, we would observe the localization of fluorescently tagged Fmp27 and Ybl086c in mss4 and stt4 temperature sensitive mutant strains, which produce lower levels of PI(4,5)P2 and PI4P respectively at high temperatures (Audhya and Emr, 2002), and in cho1Δ, dgk1Δ and psd1/2Δ mutants, which have lower levels of PS, PA, and PE respectively. This would allow us to identify which lipids affect the recruitment of Ybl086c to the PM. Under the conditions required for Ybl086c recruitment to the PM, the conserved basic patches could mediate the interaction with the PM. I found that a conserved region in the C-terminus of Ybl086c is necessary to interact with and recruit Fmp27 to ER-PM MCSs. Some outstanding questions remain, including how Fmp27 interacts with Ybl086c and which regions of Fmp27 are required 109 for this interaction. To test this, we would use the AlphaFold predicted structure of Fmp27 and Consurf to determine the conserved regions in Fmp27 and design strains with Fmp27 truncations. We would then use the 2xPH recruitment assay to test which truncations are recruited to the PM by Ybl086c. This would determine how Fmp27 is recruited by Ybl086c and which regions of Fmp27 are necessary for this interaction. 4.3 Investigating the role of Fmp27/Ypr117w and Ybl086c in stress response PM integrity and resistance to certain stresses depends on the composition of lipids in the PM. Cells have various mechanisms to alter membrane composition which helps in their survival on exposure to stresses. Cells with compromised membrane integrity due to altered PM lipid composition exhibit phenotypes on exposure to different stresses such as temperature, salt, osmolarity, pH and detergents (Mioka et al., 2018, Soczewka et al., 2019). This is found in the case of cells with mutations in genes with roles in lipid transport, maintenance of lipid asymmetry and lipid homeostasis. These include bulk lipid transporters like Vps13 and related proteins. For example, Csf1 and its orthologs have been found to have a role in cold stress response, which has been attributed to the transport of unsaturated lipids by these proteins (John Peter et al., 2022; Wang et al., 2022). Vps13Δ has been found to exhibit hypersensitivity to the ionic detergent SDS (Soczewka et al., 2019). Since Fmp27 is structurally related to Vps13 and Csf1, it could have a role in the response of cells to various stresses. I found using spot assays that Fmp27/Ypr117w and Ybl086c do not have a role in cellular response to the different abiotic stresses tested. It is also possible that a combination of stresses could pose a bigger growth challenge to Fmp27 and Ybl086c mutants. Fmp27/Ypr117w and Ybl086c could have a role in the maintenance of PM lipid composition, similar to other Vps13-like proteins. We could use lipidomics to determine changes in the lipid composition of cells with Fmp27/Ypr117w and Ybl086c mutations on exposure to stressors. This would indicate which lipid species are undergoing changes on either the deletion or overexpression of Fmp27/Ypr117w and Ybl086c. Fmp27/Ypr117w and Ybl086c might not directly impact membrane integrity. One way to test membrane integrity would be to assess the uptake of cell impermeable 110 dyes such as propidium iodide (Collado et al., 2019) in Fmp27/Ypr117w and Ybl086c deletion mutants. This would give a direct indication of the role of these proteins in the maintenance of membrane integrity in response to abiotic stress. 4.4 Investigating the role of Fmp27/Ypr117w and Ybl086c in lipid transport RBG proteins have been found to transport glycerophospholipids in bulk, which is required for membrane expansion or rapid changes in lipid composition in response to stress. Vps13 and Atg2 have been found to transport glycerophospholipids which might be required for prospore membrane formation (Li et al., 2020), and autophagosome biogenesis (Osawa et al., 2019) respectively. Csf1 has been found to transport PE required for GPI anchor synthesis at the ER (Toulmay et al., 2022) and its orthologs in C.elegans, Zebrafish, mouse and human cells are speculated to have a role in the transport of phospholipids (Wang et al., 2022). This indicates that Fmp27 and Ypr117w could have a role in glycerophospholipid transport at ER-PM MCSs. Future directions would involve determining which lipid species are transported by Fmp27/Ypr117w. A direct way to test which lipid species are regulated by Fmp27/Ypr117w and Ybl086c at ER-PM MCSs would be to use lipidomics. Lipidomics would indicate which lipid species’ levels are significantly altered in Fmp27/Ypr117w and Ybl086c mutant cells compared to wild type. Liposome-based, gel shift-based and chromatography assays have been used to determine the species of lipids bound by and transported by Vps13 (Kumar et al., 2018), Atg2 (Osawa et al., 2019) and Csf1 (Toulmay et al., 2022). Liposome-based studies either used purified full-length proteins or specific regions of proteins. Vps13 Chorein domain and full length Atg2 (1592 aa) were purified and used for liposome-based assays (Kumar et al., 2018; Osawa et al., 2019). To determine the species of lipids being transported by Fmp27, purified Fmp27 protein would be required. Since Fmp27 is a very large (2628 aa) membrane protein, its purification may pose challenges. Fmp27 is predicted to form a continuous rod-like structure, without an obvious Chorein domain, hence purifying a small region of Fmp27 might impact its stability, 111 causing it to aggregate. We could start by purifying small fragments of Fmp27, incubating them with fluorescently tagged lipid species and using antibodies to probe for a fluorescence signal in western blots. This would indicate whether Fmp27 binds lipids and which lipid species it can bind to. The fragments of Fmp27 that bind to lipids can then be used in FRET based lipid tranfer assays to test whether these fragments can transfer lipids between donor and acceptor liposomes. Like Vps13 and Atg2, we might need to tether Fmp27 fragments to donor and acceptor liposomes to observe any lipid transfer activity (Kumar et al., 2018; Osawa et al., 2019). Similar to the experimental setup used by Kumar et al., (2018), we could tag Fmp27 fragments with a hexahistidine tag that would bind to the NTA-tagged lipids on the donor liposome and an upstream PH domain to interact with PI(4,5)P2 on the acceptor liposomes. 4.5 Investigating the role of Fmp27/Ypr117w and Ybl086c in retaining sterols at the PM Around 70% of total cellular ergosterol is present at the PM in S.cerevisiae cells (Solanko et al., 2018). Sterols can be either bound to sphingolipids or phospholipids (inaccessible) or unbound (accessible) (Naito et al., 2019). Excess accessible sterols could be toxic to the cells, resulting in their internalization by retrograde sterol transporters and subsequent storage as lipid droplets (Jordá and Puig, 2020; Kishimoto et al., 2021). Additionally, excess accessible sterols could trigger the production of phospholipids or sphingolipids (Swain et al., 2002), or trigger feedback mechanisms to prevent the production of additional sterols (Jordá and Puig, 2020; Marie et al., 2008; Yang et al., 2015). PM asymmetry has been found to be important in retaining sterols at the PM (Kishimoto et al., 2021). Thus, the presence of excess accessible sterols at the PM could indicate defects in retrograde sterol transport (Gatta et al., 2015; Naito et al., 2019) or altered distribution of sphingolipids or phospholipids that interact with sterols (Maekawa and Fairn, 2015) or a lack of phospholipid asymmetry (Kishimoto et al., 2021) or defects in feedback mechanisms. Yeast cells with excess accessible sterols are sensitive to amphotericin B (Gatta et al., 2015; Murley et al., 2017; Roelants et al., 2018; Topolska et al., 2020). I found 112 that fmp27Δypr117wΔ, ybl086cΔ and fmp27Δybl086cΔ are sensitive to amphotericin B via spot assays and growth curve assays. I also found that full length Ybl086c was able to rescue the ybl086cΔ sensitive growth phenotype on exposure to amphotericin B. However, a mutant defective in binding to Fmp27 (15A mutant) was not able to rescue this phenotype. This indicates that the interaction between Fmp27 and Ybl086c and the recruitment of Fmp27 to the PM is necessary for retaining sterols in a bound state at the PM. Further, fmp27Δ and ypr117wΔ single deletions share a similar phenotype to wild type cells via spot assays, while fmp27Δypr117wΔ is sensitive, indicating redundant functions for Fmp27 and Ypr117w. These proteins may not be involved directly in the transport of sterols and could instead function in the transport of glycerophospholipids that bind to sterols at the PM, with Ybl086c recruiting Fmp27/Ypr117w to ER-PM MCSs. Thus, either the deletion of Fmp27/Ypr117w or Ybl086c results in excess accessible sterols at the PM. However, a sensitivity to amphotericin B does not necessarily indicate an elevation of accessible sterols at the PM. It could also indicate defects in membrane thickness, membrane fluidity and membrane packing. To test whether the levels of accessible sterols are elevated at the PM or whether the distribution of accessible sterols has changed in cells, we could use probes that bind to accessible sterols in membranes such as the genetically encoded fluorescently tagged-D4H (Kishimoto et al., 2021), which contains a modified version of the sterol binding D4 domain of the bacterial pore forming toxin Perfringolysin O (Maekawa and Fairn, 2015). This probe interacts with membranes containing more than 20% accessible sterols (Maekawa and Fairn, 2015). Experiments in the lab using mCherry-D4H have shown that this probe is localized to internal punctae primarily in fmp27Δypr117wΔ and ybl086cΔ mutants, as opposed to the PM in wild type cells (S. Dziurdzik, unpublished results). This probe binds to accessible sterols in the cytosolic leaflets of membranes. Another probe that can be used to visualize accessible sterols in cells in filipin. Filipin is a naturally fluorescing antibiotic that binds to accessible sterols in cells (Börnig and Geyer, 1974). It is membrane permeable and can be used to visualize accessible sterols in internal compartments (Miller, 1984; Ohno-Iwashita et al., 2010). It stains both leaflets of membranes making it hard to distinguish changes in 113 accessible sterols in individual leaflets of membranes. It photobleaches quickly and is not suitable to study dynamic changes in sterol distribution, however it can be used to observe changes in the distribution of accessible sterols in fixed cells (Maekawa and Fairn, 2014). One outstanding question is whether the known retrograde sterol transporters and sterol esterification proteins, i.e., the acyl-CoA:cholesterol acyltransferases Are1 and Are2, that catalyse the synthesis of steryl esters, function efficiently in the Fmp27/Ypr117w and Ybl086c mutant cells, as a defect in these pathways could also lead to elevated levels of accessible sterols in cells. It has been previously shown that PM phospholipid asymmetry is required for sterol retention at the PM (Kishimoto et al., 2021). This is primarily attributed to the distribution of PE and PS to the inner leaflet of the PM, mediated by PE/PS flippasesI used lipid FLARES to observe changes in phospholipid distribution in Fmp27/Ypr117w and Ybl086c mutants. However, I did not use probes to test PE distribution in these mutants. Drugs such as papuamideB and duramycin could be used to determine whether PM asymmetry is impaired in Fmp27/Ypr117w and Ybl086c mutants (Kishimoto et al., 2021; Mioka et al., 2018). A sensitivity to these drugs would indicate impaired PM asymmetry (Kishimoto et al., 2021; Mioka et al., 2018). This would give an indication of how Fmp27/Ypr117w and Ybl086c are involved in retaining sterols in a bound state in yeast. 4.6 Characterization of the interaction and the function of Fmp27/Ypr117w and Ybl086c in other model organisms Fmp27/Ypr117w and Ybl086c are highly conserved throughout eukaryotes. It would be important to expand this study to other model organisms to understand whether their interaction and functions are conserved. For example, some key experiments would be to test whether the human ortholog of Fmp27/Ypr117w, KIAA0100 and the human ortholog of Ybl086c, FAM102A/B interact with each other, which regions are necessary for the interaction, where these proteins localize and whether FAM102A/B recruits KIAA0100 to cortical sites. KIAA0100 has been found to be overexpressed in breast cancer cells (Song et al., 2006) and is associated with acute monocytic leukemia (Chen et al., 2005). 114 FAM102A, also named as early estrogen-induced gene 1 (EEIG1) has been found to have a role in RANK signaling, and in RANK-mediated osteoclast formation (Choi et al., 2013). The RANK signaling pathway includes the receptor activator of the nuclear factor-κB (RANK) and its ligand RANKL. This pathway is the master regulator of osteoclastogenesis and is involved in bone homeostasis (Dougall et al., 1999; Kong et al., 1999; Yasuda et al., 1998). RANKL binds to RANK expressed on hematopoietic osteoclast precursors and triggers a cascade of signals, which includes the recruitment of FAM102A, subsequently leading to the expression of genes characteristic of active osteoclasts (bone resorbing cells) (Boyle et al., 2003; Choi et al., 2013). During bone metastases, the metastatic tumor cells in the bone express osteoclastogenic factors that promote bone resorption, and have an upregulation of RANKL (Casimiro et al., 2021). Elevated bone resorption fuels tumor cells by feeding factors from the bone matrix contributing to bone metastases. Inhibiting the RANK/RANKL pathway abrogates cancer-induced osteoclastogenesis and is effective in treating bone metastases (de Groot et al., 2018; Lacey et al., 2012). The RANK pathway is also implicated in breast cancer (Gonzalez-Suarez et al., 2010; Schramek et al., 2010; Tanos et al., 2013). Since FAM102A is stimulated by RANKL, its overexpression could have a role in bone metastases and breast cancer. This indicates a possible role for KIAA0100 and FAM102A in human diseases such as cancer and in bone diseases. Similar to the functional correlations obtained in yeast via chemogenomics, DepMap (https://depmap.org/portal/depmap/) data correlates KIAA0100 with WDR44 (human ortholog of LAF1) and various genes involved in lipid homeostasis indicating a shared function in lipid homeostasis. A future goal for this study would be to investigate the function of KIAA0100 in sterol retention and lipid homeostasis. Cancer cells have altered lipid profiles and exhibit a loss of lipid asymmetry, which reduces their permeability, predicted via molecular simulations to increase their resistance to drugs such as cisplatin (Rivel et al., 2019). If KIAA0100 transports phospholipids that are required for the retention of sterols in a bound state at the PM, it is possible that its overexpression might lead to increased levels of inaccessible sterols at the PM. This would decrease PM permeability making 115 it resistant to anti-cancer drugs. Overexpression of KIAA0100 has been found to increase the tolerance of HEK293 cells to microtubule targeting drugs (Zhong et al., 2018). However, an opposite effect might be true when KIAA0100 is down regulated, making cancer cells susceptible to drugs, and thus reducing the aggressive behavior (cell aggregation, reattachment, metastasis, and invasion) of cancer cells. To test the distribution and abundance of accessible sterols in cells either overexpressing or lacking KIAA0100, we would use probes such as the fluorescently tagged GRAM domain of GRAMD1b (human ortholog of the yeast Lam retrograde sterol transporters) (Naito et al., 2019) or GFP-D4H (Maekawa and Fairn, 2015). These probes bind to accessible sterols in the PM and show a strong PM localization in cells with elevated levels of accessible sterols (Maekawa and Fairn, 2015; Naito et al., 2019). We would express these probes in wild-type HeLa cells, cells lacking GRAMD proteins (shows stronger fluorescence of GRAMD probe at the PM; control [Naito et al., 2019]), cells overexpressing KIA00100 by itself and those overexpressing both KIAA0100 and FAM102A/B. Experiments in yeast indicate that cells overexpressing both Fmp27 and Ybl086c exhibit phenotypes similar to wild type cells implying that they might not have an alteration in the levels of accessible sterols. Hence, it is possible that we do not find any differences in the levels of accessible sterols in cells overexpressing KIAA0100 and FAM102A/B compared to wild type cells. We would also include cells in which KIAA0100, and FAM102A/B have been knocked out to test whether these cells show elevated levels of accessible sterols similar to the experiments in yeast. Some mammalian counterparts of yeast RBG proteins i.e., ATG2 and VPS13 have been found to transport lipids from the ER to growing membranes in cells (Kumar et al., 2018, Li et al., 2020; Maeda et al., 2019). They have been found to interact with scramblases at the acceptor membrane to ensure that lipids are distributed properly among the leaflets of the donor and acceptor membranes (Adlakha et al., 2022; Ghanbarpour et al., 2021; Guillén-Samandar et al., 2022; Matoba et al., 2020; Park et al., 2020). KIAA0100 and FAM102A/B could exhibit similar interactions with scramblases to maintain lipid homeostasis. Maintaining lipid homeostasis is key to the proper functioning of cells and hence organisms. Extending our studies to 116 human cells could thus shed light on the role of these proteins in diseases such as cancer. 4.7 Limitations of the study The various methods used in this study are not devoid of limitations. This section includes some of the limitations of the techniques used in this study. Firstly, I used co-immunoprecipitation experiments to determine that Fmp27 and Ybl086c interact with each other. However, in these experiments, we were probing for epitope tagged Fmp27 or Ybl086c and could have missed out on identifying other interaction partners of these proteins. Further, co-immunopreciptation experiments do not probe the interaction between proteins in a living yeast cell. Other methods that can be used to probe for the interactome of Fmp27 and Ybl086c and to probe the interaction between Fmp27 and Ybl086c in a living cell have been described below. The interactome of Fmp27 and Ybl086c could include many other interactors, with a difference in the interactome of both proteins. To identify the interactome of Fmp27 and Ybl086c, we would perform immunoprecipitation of epitope tagged Fmp27 and Ybl086c and appropriate controls from yeast lysates and use mass spectrometry to identify the interactome of these proteins. This would be followed by microscopy-based experiments to test whether the top hits from the mass spectrometry analysis colocalize with either Fmp27 or Ybl086c. A validation of direct interaction between Fmp27 and Ybl086c and their interactors (top hits from mass spectrometry experiments) can be done using the split-ubiquitin yeast two hybrid assay. In a traditional yeast two hybrid, the two proteins whose interaction needs to be tested are fused to the activation and DNA binding domains of a transcription factor. An interaction between the two proteins results in a functional transcription factor that binds to upstream activator sequences, leading to the subsequent expression of a reporter gene. A traditional yeast two hybrid would not work in case of Fmp27, as it is a membrane protein that is anchored to the ER and hence will not be imported into the nucleus. In a split-ubiquitin yeast two hybrid, the two proteins whose interaction needs to be tested are fused to the N terminal fragment of ubiquitin and the C terminal fragment of ubiquitin fused to a transcription factor (Fetchko and 117 Stagljar, 2004). An interaction between the two proteins results in the formation of functional ubiquitin molecules that cleave off the transcription factor, which binds to upstream activator sequences, resulting in the subsequent expression of a reporter gene (Fetchko and Stagljar, 2004). This interaction takes place in the cytosol of cells and hence does not require the import of the proteins into the nucleus. This technique would not only help determine whether Fmp27 and Ybl086c directly interact with one another but also help determine other interactors of these proteins. Moreover, this technique helps determine protein interactions in a living yeast cell, as opposed to co-immunoprecipitation, and would give an indication about the interaction between Fmp27 and YBl086c in living yeast cells. Fmp27 and Ybl086c are hard to visualize via widefield microscopy. This makes it hard to use automated quantification to ascertain colocalization with other proteins. In the case of Ybl086c, I used line scans to depict a partial colocalization between Ybl086c and Tcb3. The line scans have been done for more than one cell, but only one line scan is included in the figure. Another limitation is with regards to using hand counting based approaches to quantify the localization of Fmp27-ENVY via the PM recruitment assay. We had initially used Metamorph based journals (quantification programs) to determine the number of cells with different localizations of Fmp27-ENVY. Briefly, this was based on using either a PM or a nuclear marker to mask the respective organelles and to determine the fluorescence signal in cells. However, this approach failed to capture the various localizations exhibited by Fmp27-ENVY. Another approach could be to use line scans and obtain the ratio of the fluorescence intensity between the signal at the PM (for cER readings) and the nER. This would involve drawing a line through the PM and nER in yeast cell images, obtaining an average of the highest intensity readings (to obtain fluorescence readings corresponding to the PM and nER) and taking a ratio between the PM and nER fluorescence. This would give an estimate of the number of cells with a cER only or nER=cER or nER<cER or ambiguous localization of Fmp27-ENVY. This approach is limited by the number of cells for which these ratios can be calculated and a human 118 bias regarding which cells to pick to obtain the ratios between the fluorescence intensity of the PM and nER. Thirdly, I used spot assays to determine how cells respond to various stresses. We could additionally used plate reader-based growth assays to test the differences in growth of various mutant strains to difrerent stresses. Further, we could use lipidomics to determine a change in the lipid composition of cells in response to different stresses in Fmp27/Ypr117w and Ybl086c mutants. This would give an indication of whether Fmp27/Ypr117w and Ybl086c have a role in maintaining the lipid composition of cells during stress conditions. A no stress control would help determine whether the mutation of Fmp27/Ypr117w and Ybl086c by itself affects the proportion of lipid species in cells. Lastly, I used lipid FLARES to determine the distribution of various phospholipid species in Fmp27/Ypr117w and Ybl086c mutants. Lipid FLARES bind to accessible lipids and sequester them. Moreover, some lipid FLARES preferentially interact with membranes containing lipids other than the ones they bind to. These probes tend to have a high affinity for the lipids that they sense and hence to observe a change in distribution of lipids, a large change in the levels of lipids must occur. Combining lipidomics based approaches with sub cellular fractionation would allow us to obtain information of the changes in the lipid composition of different organelles and about the lipid distribution. However, obtaining pure membrane fractions could prove cumbersome. 4.8 Conclusion In this study, I identified that Ybl086c recruits Fmp27 to ER-PM MCSs, where they function in a coordinated manner to retain sterols in a bound state. This could be achieved by Fmp27/Ypr117w, by transporting lipids that interact with sterols, from the ER, which could act as a lipid reservoir. The transport of lipids would be important for the maintenance of membrane integrity and maintaining lipid homeostasis in cells. In an event that Fmp27 can no longer be recruited to the PM by Ybl086c, Fmp27 can no longer transport lipids that interact with sterols, causing an increase in the levels of accessible sterols at the PM. Similar studies could be done in human cells to 119 understand the role of the human orthologs of Fmp27/Ypr117w and Ybl086c in disease. 120 Appendices Appendix A: Supplementary material for Chapter 1 Appendix A: List of intracellular LTP families A list of intracellular LTPs found in yeast and humans, their domains involved in lipid transport, the lipids transported by them and some examples in humans and yeast [denoted by (s)] have been indicated. BLTP; Bridge-like lipid transfer protein, PRELI; Protein of Relevant Evolutionary and LTP family Domain involved in lipid transport Lipids transported Examples ORP/Osh(s) ORD Sterol, PI4P, PS OSBP, ORP1-11, Osh1-7(s) StART StART Sterol, glycerophospholipids, ceramide STARD1-15 PITP PITP PI, PC, PA Nir2, Nir3, Sec14(s) GRAMD/Lam(s) StART-like Sterol GRAMD1a-c, GRAMD2, GRAMD3, Lam1-6(s) PRELI (+TRIAP) PRELI PA and PS PRELID1, PRELID3a, PRELID3b, Ups1(s), Ups2(s), Ups3(s), TRIAP1/Mdm35 (s) SMP SMP Glycerphospholipids E-Syt1-3, Tcb1-3(s), Nvj1(s), Mmm1(s), Mdm12(s) and Mdm34(s) NPC1 N-terminal domain NPC1 N-terminal domain Sterols NPC1, Ncr1(s) NPC2 NPC2 Sterols NPC2, Npc2(s) BLTP N-Chorein/ RBG Glycerophospholipids BLTP1, BLTP2, BLTP3B, BLTP4A, BLTP5C, Csf1(s), Fmp27(s), Hob2(s), Atg2(s), Vps13(s) 121 Lymphoid Interest, TRIAP; TP53-regulated inhibitor of apoptosis. Adapted from (Wong et al., 2019). 122 Appendix B: Supplementary material for Chapter 2 Hit Query region matched Hit region matched to Prob (H%) Score E-value Aligned cols Identities (%) Similarity Fmp27 693-1034 2076-2469 93.96 46.36 4.3 296 13 0.16 Ypr117w 1065-1184 2118-2241 88.81 50.78 1.2 112 10 0.164 Csf1 894-1116 839-1052 86.82 41.44 19 196 11 0.139 Csf1 933-1363 531-997 83.99 40.1 26 386 9 0.036 Atg2 1075-1184 1119-1244 83.96 47.14 1.9 105 10 0.178 Appendix B1: HHpred results for the query Vps13 650-1390. The various hits from HHpred search using Vps13 650-1390 residues as a query. The regions mapped, the probability of homology and other parameters have been included. Smaller regions included within the hit regions and a hit corresponding to the exact query, i.e., Vps13 650-1390 (100% probability) have not been included. This search was performed on 26th March 2021. 123 Appendix B2: HHpred results for the query Vps13 APT1 domain. The various hits from HHpred search using Vps13 APT1 domain as a query. The regions mapped, the probability of homology and other parameters have been included. Smaller regions included within the hit regions and a hit corresponding to the exact query, i.e., Vps13 APT1 (100% probability) have not been included. This search was performed on 10th June 2021. Hit Query region matched Hit region matched to Prob (H%) Score E-value Aligned cols Identities (%) Similarity Atg2 2610-2832 1126-1340 99.84 170.23 1.10e-19 200 9 0.095 Fmp27 2582-2832 2338-2589 99.79 163.04 6.8e-18 228 14 0.166 Ypr117w 2576-2828 2111-2400 99.35 111.38 1.1e-10 227 11 0.068 Vps13 2575-2832 505-647 96.72 56.84 0.013 132 11 0.14 Vps13 2575-2832 1056-1183 88.91 40.98 1.9 123 9 0.158 Vps13 2579-2717 893-1033 85.69 39.89 2.6 130 6 0.148 124 Tables Supplementary Table 1: List of yeast strains used in Chapter 2 and 3 Strain ID Genotype Source LC 3589 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Giaever et al., 2002 LC 4065 MATa vps13Δ::kanMX4 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Lab collection LC 5428 MATa osh3Δ::kanMX4 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Lab collection LC 5432 MATa Ybl086c-ENVY::HIS3 Tcb3-tdimer2::URA3 leu2Δ0 met15Δ0 This study LC 5437 MATa YPR117W-3HA::hph ybl086cΔ::kanMX4 leu2Δ0 met15Δ0 ura3Δ0 This study LC 5486 MATa FMP27-ENVY::HIS3 kanMX4::ADH1 YBL086C-3HA::hph leu2Δ0 met15Δ0 ura3Δ0 This study LC 5488 MATa kanMX4::ADH1 YBL086C-3HA::hph his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 This study LC 5490 MATa Fmp27-3HA::hph ybl086cΔ::kanMX4 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 This study LC 5491 MATa fmp27Δ::hph ybl086cΔ::kanMX4 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 This study LC 5497 MATa fmp27Δ::kanMX4 ypr117wΔ::hph his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 This study LC 5501 MATa YBL086c-3HA::hph his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 This study 125 LC 5502 MATa Ypr117w-3HA::hph his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 This study LC 5507 MATa FMP27-ENVY::HIS3 leu2Δ0 met15Δ0 ura3Δ0 This study LC 5508 MATa YBL086c-ENVY::HIS3 leu2Δ0 met15Δ0 ura3Δ0 This study LC 5510 MATa FMP27-ENVY::HIS3 YBL086C-3HA::hph leu2Δ0 met15Δ0 ura3Δ0 This study LC 5511 MATa Fmp27-3HA::hph his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 This study LC 5514 MATa ymr102cΔ::kanMX4 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 This study LC 5532 MATa ist2∆::HisMX6 scs2∆::TRP1 scs22∆::HisMX6 tcb1∆:KanMX6 tcb2∆::KanMX6 tcb3∆::HisMX6 leu2∆3 ura3∆52 his3∆200 trp1∆901 lys2∆801 suc2∆9 (Δ-tether) Dr. Christopher Beh, SFU, Canada* LC 5534 MATa natNT2::ADH1 FMP27-ENVY::HIS3 leu2Δ0 met15Δ0 ura3Δ0 This study LC 5536 MATa Sec63-Scarlet::URA3 his3Δ1 leu2Δ0 met15Δ0 Lab collection LC 5537 MATa FMP27-ENVY::HIS3 Sec63-Scarlet::URA3 leu2Δ0 met15Δ0 Lab collection LC 5562 MATa kanMX4::ADH1 FMP27-ENVY::HIS3 natNT2::ADH1 YBL086c-Scarlet::URA3 met15Δ0, leu2Δ0 Lab collection 126 LC 5566 MATα mss4∆::HIS3 suc2D9 ura3-52 trp1-D901 lys2-801 YCplac111mss4ts-102-LEU Dr. Christopher Stefan, UCL, UK** LC 5623 MATa natNT2::ADH1 FMP27-ENVY::HIS3 Sec63-Scarlet::URA3 leu2Δ0 met15Δ0 This study LC 5624 MATa natNT2::ADH1 FMP27-ENVY::HIS3 YBL086C-3HA::hph leu2Δ0 met15Δ0 ura3Δ0 This study LC 5626 MATa fmp27Δ::kanMX4 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Lab collection LC 5627 MATa ypr117wΔ::kanMX4 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Lab collection LC 5628 MATa ybl086cΔ::kanMX4 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Lab collection LC 5632 MATa natNT2::ADH1 FMP27 kanMX4::ADH1 YBL086C his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 This study LC 5657 MATa natNT2::ADH1 FMP27-ENVY::HIS3 kanMX4::ADH1 YBL086C-3HA::hph leu2Δ0 met15Δ0 ura3Δ0 This study LC 5704 MATa csf1^NAT csf1(1003::NAT::2108) his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Lab collection LC 5851 MATa rim8Δ::kanMX4 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Lab collection LC 5852 MATa rim101Δ::kanMX4 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Lab collection LC 5909 MATa hog1Δ::kanMX4 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Lab collection 127 Giaever, G., Chu, A. M., Ni, L., Connelly, C., Riles, L., Véronneau, S., Dow, S., Lucau-Danila, A., Anderson, K., André, B., Arkin, A. P., Astromoff, A., El-Bakkoury, M., Bangham, R., Benito, R., Brachat, S., Campanaro, S., Curtiss, M., Davis, K., Deutschbauer, A., … Johnston, M. (2002). Functional profiling of the Saccharomyces cerevisiae genome. Nature, 418(6896), 387–391. Https://doi.org/10.1038/nature00935. (n.d.). * Manford, A. G., Stefan, C. J., Yuan, H. L., Macgurn, J. A., & Emr, S. D. (2012). ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology. Developmental cell, 23(6), 1129–1140. Https://doi.org/10.1016/j.devcel.2012.11.004. (n.d.). ** Stefan, C. J., Manford, A. G., Baird, D., Yamada-Hanff, J., Mao, Y., & Emr, S. D. (2011). Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites. Cell, 144(3), 389–401. Https://doi.org/10.1016/j.cell.2010.12.034. (n.d.). 128 Supplementary Table 2: List of plasmids used in Chapter 2 and 3 Plasmid ID Description Source pLC 695 pRS413-HIS3 Sikorski and Hieter, 1989 pLC 1407 pRS415-LEU2 Sikorski and Hieter, 1989 pLC 2259 pRS416-GFP-C2-URA3 Sergio Grinstein, UoT, Canada** pLC 3450 pRS413-UTR-YBL086C-ENVY (HIS3, CEN) This study pLC 3477 pRS413-UTR-YBL086C (216-466)-ENVY (HIS3, CEN) This study pLC 3524 pRS413-UTR-YBL086C (1-216)-ENVY (HIS3, CEN) This study pLC 3577 pRS416-pGPD-GFP-P4CSidC-URA3 Chris Stefan, UCL, UK* pLC 3578 pRS415-GPD-mCh-2xPH(PLCδ) (LEU2, CEN) Chris Stefan, UCL, UK* pLC 3581 pRS416-ADH1 FMP27(1-28)-ENVY (LEU2, CEN) This study pLC 3606 pRS415-GPD-mCherry-2xPH(PLCδ)-YBL086C (LEU2, CEN) This study 129 pLC 3607 pRS415-GPD-mCherry-2xPH(PLCδ)-YBL086C (216-466) (LEU2, CEN) This study pLC 3609 pRS415-GPD-mCherry-2xPH(PLCδ)-YBL086C (1-216) (LEU2, CEN) This study pLC 3610 pRS415-GPD-mCherry-2xPH(PLCδ)-YBL086C (245-466) (LEU2, CEN) This study pLC 3636 pRS415-GPD-mCherry-2xPH(PLCδ)-YBL086C (345-466) (LEU2, CEN) This study pLC 3665 pRS415-GPD-mCherry-2xPH(PLCδ)-YBL086C (216-245) (LEU2, CEN) This study pLC 3672 pRS415-GPD-mCherry-2xPH(PLCδ)-YBL086C (245-345) (LEU2, CEN) This study pLC 3673 pRS415-GPD-mCherry-2xPH(PLCδ)-YBL086C (216-345) (LEU2, CEN) This study pLC 3674 pRS415-GPD-mCherry-2xPH(PLCδ)-YBL086C (216-455) (LEU2, CEN) This study pLC 3675 pRS415-GPD-mCherry-2xPH(PLCδ)-YBL086C (245-455) (LEU2, CEN) This study pLC 3676 pRS415-GPD-mCherry-2xPH(PLCδ)-YBL086C (345-455) (LEU2, CEN) This study pLC 3687 pRS415-GPD-mCh-2xPH(PLCδ)-YBL086C delta 304-315 (LEU2, CEN) This study pLC 3688 pRS415-GPD-mCh-2xPH(PLCδ)-YBL086C delta 326-336 (LEU2, CEN) This study 130 pLC 3701 pRS415-GPD-mCh-2xPH(PLCδ)-YBL086C (245-295) (LEU2, CEN) This study pLC 3782 pRS415-GPD-mCh-2xPH(PLCδ)-YBL086C (15A) (LEU2, CEN) This study pLC 3702 pRS415-GPD-mCh-2xPH(PLCδ)-YBL086C (295-320) (LEU2, CEN) This study pLC 3703 pRS415-GPD-mCh-2xPH(PLCδ)-YBL086C (320-345) (LEU2, CEN) This study pLC 3704 pRS415-GPD-mCh-2xPH(PLCδ)-YBL086C (295-345) (LEU2, CEN) This study pLC 3779 pRS413-UTR-YBL086C (15A)-ENVY (HIS3, CEN) This study * Omnus, D. J., Cadou, A., Thomas, F. B., Bader, J. M., Soh, N., Chung, G. H. C., Vaughan, A. N., & Stefan, C. J. (2020). A heat-sensitive Osh protein controls PI4P polarity. BMC biology, 18(1), 28. Https://doi.org/10.1186/s12915-020-0758-x. (n.d.). ** Yeung, T., Gilbert, G. E., Shi, J., Silvius, J., Kapus, A., & Grinstein, S. (2008). Membrane phosphatidylserine regulates surface charge and protein localization. Science (New York, N.Y.), 319(5860), 210–213. Https://doi.org/10.1126/science.1152066. (n.d.). 15A mutant, 15 residues 302,305,308,309,310,313,314,316,331,332,333,334,335,337,338 mutated to alanine in full length YBL086C. Sikorski, R. S., & Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics, 122(1), 19–27. Https://doi.org/10.1093/genetics/122.1. (n.d). 131 Supplementary Table 3: List of oligos used in Chapter 2 and 3 Primer ID Primer Sequence LC 2376 pRS swap F ACCTCTGACACATGCAGCT LC 2377 pRS swap R ACTCAACCCTATCTCGGTCT LC 4510 YPR117W S1 CCATAATATCTAGTAGCTGGAGTATCCTGTTTATTGCAAGAAAAAAAATCTACACGTACGCTGCAGGTCGAC LC 4675 YLR454W (FMP27) F5 GAAGCAACTCACAACTTATAATTCCTACACGGAGCCTGAAGAACTTCGT GGTGACGGTGCTGGTTTA LC 4676 YLR454W (FMP27) R3 GATGCTACATTAAGAGATATGAAAAACTATAAATTTTTTTTGAATGTTT TCGATGAATTCGAGCTCG LC 4921 YBL086C F5 CAGATAGAATAAGAGAAAACAAAAACTGGTCTATAATAACCCCATCGGGCGGTGACGGTGCTGGTTTA LC 4922 YBL086C R3 CAGATAGAATAAGAGAAAACAAAAACTGGTCTATAATAACCCCATCGGGCATCGATGAATTCGAGCTCG LC 4935 fmp27 KANMXF AATTAGATCAGAACATAAGAATCCTTAGAAAAGCCCTTTACCTCGGTATATGTAG ACATGGAGGCCCAGAATACCCT LC 4936 fmp27 KANMXR TGTATGATGCTACATTAAGAGATATGAAAAACTATAAATTTTTTTTGAATGTTTCAGTATAGCGACCAGCATTCAC LC 4937 ybl086c KANMXF GCTTAGAAGTATTAGGTACCCAAGAAAGGTATAATACCGCTCACAAAATATTAAG ACATGGAGGCCCAGAATACCCT LC 4938 ybl086c KANMXR TAGTGATTGGTATTGCAAGCTTAAGATTATAATTACATAAAACAATACAAATATT CAGTATAGCGACCAGCATTCAC 132 LC 4939 ypr117w KANMX F TCCATAATATCTAGTAGCTGGAGTATCCTGTTTATTGCAAGAAAAAAAATCTACA ACATGGAGGCCCAGAATACCCT LC 4940 ypr117w KANMX R ATACAATTACTATATAACTGAAGAATATTTACGCTAGACACATGTGTGTGTTTCC CAGTATAGCGACCAGCATTCAC LC 4952 ymr102c KANMX F CTCTTGGCATATTGTATACGGTAGTGGCGTATAATATAGTACGAGAATTAGAAAT ACATGGAGGCCCAGAATACCCT LC 4953 ymr102c KANMX R AAGATTAATATGCATTTAAAGGTATCATCCACGGAAAAAAAATACTCCATCAGCG CAGTATAGCGACCAGCATTCAC LC 5010 FMP27 S1 CATAAGAATCCTTAGAAAAGCCCTTTACCTCGGTATATGTAG CGTACGCTGCAGGTCGAC LC 5011 FMP27 S4 CTGCAAATATAAGCCACTTGTACAGTAGAACATTAATCGGAAACATCATCGATGAATTCTCTGTCG LC 5018 YBL086C S3 TTTAACAGATAGAATAAGAGAAAACAAAAACTGGTCTATAATAACCCCATCGGGC CGTACGCTGCAGGTCGAC LC 5019 YBL086C S2 GCAAGCTTAAGATTATAATTACATAAAACAATACAAATATT ATCGATGAATTCGAGCTCG LC 5070 FMP27 S3 ACCACTGAAGCAACTCACAACTTATAATTCCTACACGGAGCCTGAAGAACTTCGT CGTACGCTGCAGGTCGAC LC 5071 FMP27 S2 AAGAGATATGAAAAACTATAAATTTTTTTTGAATGTTTTTA ATCGATGAATTCGAGCTCG LC 5072 YPR117W S3 TGGAAATGGAGTAAAGCCATTTTACCCTGTTACGAGTGAGTTTTCAAAAAATAAA CGTACGCTGCAGGTCGAC LC 5073 YPR117W S2 CTGAAGAATATTTACGCTAGACACATGTGTGTGTTTCCTCA ATCGATGAATTCGAGCTCG 133 LC 5285 pRS413-YBL086C F GCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGAATGTCGTTTCCGACTACG LC 5286 YBL086C NotI R CTCGCCTTTAGACATGTTAATTAAACCAGCACCGTCACCGCGGCCGCCGCCCGATGGGGTTATTATAGACC LC 5309 TCB3 F5 GACAAGAATGGTCAGGTACCTCCCGTGCCAGAAGTTCCTCAAGAATACACGCAG GGTGACGGTGCTGGTTTA LC 5310 TCB3 R3 GTTAACACACCAAATGTGCCCTTATTGAGCGTATAAAAGAATAGTTTTCACTGTTTA TCGATGAATTCGAGCTCG LC 5311 YBL086C Internal R GTTCAATCTCAATGCCTGTAAATCG LC 5312 YBL086C 216 F GGTACCCAAGAAAGGTATAATACCGCTCACAAAATATTAAGATGGGCCAGCTCGCTAATTACAG LC 5358 primextF YBL086C (1-216) GCTAGTTAGAGGCTCTTACGAAGATTTCAACATTTCCAAATCGTTTACTAATGGCGGCGGCCGCGGTGAC LC 5359 primextR YBL086C (1-216) CCTGTAAACAATTCCTCGCCTTTAGACATGTTAATTAAACCAGCACCGTCACCGCGGCCGCCGCCATTAG LC 5489 ADHpr_FMP27_F CTAGAACTAGTGGATCCCCCCCGGACGACAGAGAATTCATCATGATGTTTCCGATTAATGTTCTACTGTAC LC 5548 FMP27 28-ENVY R CCTCGCCTTTAGACATGTTAATTAAACCAGCACCGTCACCCCTTAGTAAAATTTTACAGCTCCATAAGAAAGTCACTGC 134 LC 5580 Sec63 F5 CTGATATCGATACGGATACAGAAGCTGAAGATGATGAATCACCAGAA GGTGACGGTGCTGGTTTA LC 5581 Sec63 R3 GATGAAATATATACGTCTAAGAGCTAAAATGAAAAACTATACTAATCACTTATAT TCGATGAATTCGAGCTCG LC 5606 2XPHPLCD_LINKER_YBL_F CTGCGCAAGATCATCCACCACTCCGGCTCCATGGACCAGCGGCAGAAGGGTTCTGGTGGTTCTGGCATGCCATTTAATCATAATAGCAAGGCAAAAAG LC 5606 2XPHPLCD_LINKER_YBL_F CTGCGCAAGATCATCCACCACTCCGGCTCCATGGACCAGCGGCAGAAGGGTTCTGGTGGTTCTGGCATGCCATTTAATCATAATAGCAAGGCAAAAAG LC 5607 pRS backbone YBL_R GGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACCTAGCCCGATGGGGTTATTATAGACC LC 5607 pRS backbone YBL_R GGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACCTAGCCCGATGGGGTTATTATAGACC LC 5607 pRS backbone YBL_R GGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACCTAGCCCGATGGGGTTATTATAGACC LC 5607 pRS backbone YBL_R GGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACCTAGCCCGATGGGGTTATTATAGACC LC 5608 2XPH_LINKER_YBL 216-466 F TCATCCACCACTCCGGCTCCATGGACCAGCGGCAGAAGGGTTCTGGTGGTTCTATGGGCCAGCTCGCTAATTAC LC 5608 2XPH_LINKER_YBL 216-466 F TCATCCACCACTCCGGCTCCATGGACCAGCGGCAGAAGGGTTCTGGTGGTTCTATGGGCCAGCTCGCTAATTAC 135 LC 5634 pRS backbone Ybl 1-216 R GGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACCTAGCCATTAGTAAACGATTTGGAAATGTTG LC 5644 2XPH linker YBL086C 245-466 F TCATCCACCACTCCGGCTCCATGGACCAGCGGCAGAAGGGTTCTGGTGGTTCTATGTCACCCAAGAATACATTCTCC LC 5644 2XPH linker YBL086C 245-466 F TCATCCACCACTCCGGCTCCATGGACCAGCGGCAGAAGGGTTCTGGTGGTTCTATGTCACCCAAGAATACATTCTCC LC 5644 2XPH linker YBL086C 245-466 F TCATCCACCACTCCGGCTCCATGGACCAGCGGCAGAAGGGTTCTGGTGGTTCTATGTCACCCAAGAATACATTCTCC LC 5683 pRS backbone Ybl 216-245 R GGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACCTACATCTGGTTTGTTGTTGATGTGGGG LC 5684 pRS backbone Ybl 245-345 R GGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACCTACTCGTTCTTAGCCCAACCGTTTC LC 5685 2XPH_LINKER_YBL 345-466 F TCATCCACCACTCCGGCTCCATGGACCAGCGGCAGAAGGGTTCTGGTGGTTCTATGGAGAAGGGCATAAATTTAATCGATTTACAGG LC 5685 2XPH_LINKER_YBL 345-466 F TCATCCACCACTCCGGCTCCATGGACCAGCGGCAGAAGGGTTCTGGTGGTTCTATGGAGAAGGGCATAAATTTAATCGATTTACAGG 136 LC 5723 pRS backbone YBL086C 455 R GGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACCTATTCTCTTATTCTATCTGTTAAAAAGTCCGTGGG LC 5759 YBL086C delta 304-315 primextF CAACAAAGCCAGGGCTGTCAACAACAATATCTTCCTCTATGAGCTTACCGTGGGATCCTCGACCAGGT LC 5760 YBL086C delta 304-315 primextR GTAAAATATCTTCCACGCATTCTCTTGGCGTAAATTCACCTGGTCGAGGATCCC LC 5761 YBL086C delta 326-336 primextF CCACTGATTGAGAGCTTGTACCAGAAAACGTTTAAATTACCGTGGGATCCTCGACCAGGTGAATTTGG LC 5762 YBL086C delta 326-336 primextR CGATTAAATTTATGCCCTTCTCGTTCTTAGCCCAACCGTTTCCGCCAAATTCACCTGGTCGAGG LC 5793 2XPH YBL086C 295 F TCATCCACCACTCCGGCTCCATGGACCAGCGGCAGAAGGGTTCTGGTGGTTCTATGTCAACAACAATATCTTCCTCTATGAGC LC 5794 pRS backbone YBL086C 295 R GGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACCTATGACAGCCCTGGCTTTGTTG LC 5795 2XPH YBL086C 320 F TCATCCACCACTCCGGCTCCATGGACCAGCGGCAGAAGGGTTCTGGTGGTTCTATGCCTCGACCAGGTGAATTTAC 137 LC 5796 pRS backbone YBL086C 320 R GGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACCTAAGGATCCCACGGTAATTTAAACG LC 5979 YBL086C mut fusion R CCTGGTCGAGGATCCCACGGAGCTTTAGCAGCTTTCTGAGCAGCAGCCTCAATAGCTGGGCTAGCAGAGGAAGATATTGTTGTTGACAGC LC 5980 YBL086C mut fusion F CCGTGGGATCCTCGACCAGGTGAATTTACGCCAAGAGAATGCGCTGCTGCTGCTGCTCAAGCTGCTAACGGTTGGGCTAAGAACG LC 5980 YBL086C mut fusion F CCGTGGGATCCTCGACCAGGTGAATTTACGCCAAGAGAATGCGCTGCTGCTGCTGCTCAAGCTGCTAACGGTTGGGCTAAGAACG LC 5995 YBL086C internal mut fusion F CCCAAGAATACATTCTCCAATTTTAACG LC 5996 YBL086C internal mut fusion R GCCGTATTTTCGTTATCCTCG 138 Supplementary Table 4: List of antibodies used in chapter 2 Host Target Clonality Source Mouse GFP Monoclonal Roche Mouse HA.11 Monoclonal Covance Rabbit GFP Polyclonal L. 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Thesis/Dissertation
eng
Cell and Developmental Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Conibear, Elizabeth
Investigating the role of the putative lipid transport protein Fmp27 at ER-PM membrane contact sites
Text