Molecular analysis of oxysterol binding proteins in yeast

MOLECULAR ANALYSIS OF OXYSTEROL BINDING PROTEINS IN YEAST WANG PENGHUA (Bachelor of Science (Hons), Zhongshan University, People’s Republic of China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2005 I ACKNOWLEDGEMENTS I am deeply indebted to my wife, Zhou Yuping, who has been always encouraging and supporting me since I met her. It is she who pushed me to sit for GRE and TOEFL, and applied to NUS. I thank her for sharing my ups and downs, happiness and unhappiness. I thank her for accompanying me for the past four difficult years. I also thank my dear son, who brings me laughs everyday. I wish to express sincere gratitude to my supervisor Dr Robert Yang Hongyuan for his serious, responsible supervision on my project all the way during the course of my candidature. His scientific attitude towards research and professionalism has impressed me and will benefit me for life. I thank all the former and current members in our laboratory including: Li Hongzhe, Chieu Hai Kee, Zhang Yong, Woo Wee Hong, Zhang Qian, Zhang Shao Chong, Xiao Han, Liew Li Phing, Fei Weihua, Zheng Li, Jaspal Kaur Kumar, Ho Zi Zong and Low Choon Pei for their help with my experiments. I appreciate Dr Munn L Alan’s invaluable suggestions and reagents for my project. Lastly, I thank NUS very much for giving me the opportunity to pursue a higher degree and offering me research scholarship. II PUBLICATIONS The major part of this work has been published in: 1. AAA ATPases Regulate Membrane Association of Yeast Oxysterol Binding Proteins and Sterol Metabolism. Penghua Wang, Yong Zhang, Hongzhe Li, Hai Kee Chieu, Alan L. Munn and Hongyuan Yang. The EMBO Journal (2005) 24, 2989–2999. 2. Molecular Characterization of Osh6p, an Oxysterol Binding Protein Homolog in the Yeast Saccharomyces cerevisiae. Penghua Wang, Wei Duan, Alan L. Munn and Hongyuan Yang. FEBS Journal (2005) 272(18), 4703-15. Other publications as co-author: 1. Simvastatin reverses the downregulation of dopamine D1 and D2 receptor expression in the prefrontal cortex of 6-hydroxydopamine-induced Parkinsonian rats. Wang Q, Wang PH, McLachlan C, Wong PT. Brain Res (2005) 1045(1-2),229-33. Manuscripts in preparation: 1. A role for the yeast oxysterol binding protein Osh1p and its interactors in maintaining sterol homeostasis. Penghua Wang, Wilson Cong Jin Low and Hongyuan Yang. 2005. 2. Acute glucose starvation reveals a critical role of Ncr1p in subcellular sterol transport in the yeast Saccharomyces cerevisiae. Shaochong Zhang, Li Zheng, Qian Zhang, Penghua Wang and Hongyuan Yang. 2005. III TABLE OF CONTENTS Chapter I: Introduction Part Intracellular Cholesterol Homeostasis 1.1 Overview…………………………………………………………………………… 1.2 Regulation of cholesterol biosynthesis ……………………………………… … 1.3 Intracellular cholesterol transport…………………………………………… 7 1.3.1 Regulated cholesterol transport…………………………………………………… 1.3.2 Mechanisms of intracellular transport…………………………………………… 11 1.3.3 General intracellular transport pathways………………………………………… .12 1.3.4 Sterol transport in yeast……………………………………………………………….15 14 16 1.4 ACAT and cholesterol homeostasis……………………………………………….16 1.5 Oxysterols and cholesterol homeostasis………………………………………… 18 17 1.6 LXRs……………………………………………………………………………… .19 18 Part 2The OxySterol Binding Protein (OSBP) family 2.1 Overview ………………………………………………………………………… 21 20 2.2 The molecular function of OSBP…………………………………………… .2122 2.3 The molecular function of ORPs……………………………………………….….24 23 2.4 The molecular function of OSHs 2.4.1 Secondary structures of Osh proteins…………………………………….…… .26 25 2.4.2 The role of Osh proteins in maintaining sterol homeostasis………………… .29 26 2.4.3 The role of Osh proteins in membrane trafficking…………………………………30 27 2.4.4 Roles of Osh proteins in other cellular activities ……………………………… … .34 29 2.5 Membrane targeting of OSBP proteins 2.5.1 Multiple membrane-targeting of OSBP proteins…………………………… .… .35 30 2.5.2 Requirement of phospholipids for membrane targeting ……………………… …37 32 2.5.3 Regulation of membrane association of Osh proteins by AAA ATPases…… … 39 33 Part Significance and Objectives of This Study 3.1 Intracellular cholesterol homeostasis and diseases ………………………… 41 36 3.2 Yeast as a good research model for the study of lipid metabolisms…………… 42 37 3.3 Why study intracellular cholesterol homeostasis and OSBP family…………….43 38 3.4 The objectives of this study 3.4.1 Characterize the cellular localization of Osh6p………………………………… 44 39 3.4.2 Determine the lipid ligand(s) of Osh6p ………………………………………… .44 39 3.4.3 Examine the role of Osh6p in maintaining sterol homeostasis ………………… 45 39 3.4.4 Investigate the role of Osh6p in membrane trafficking……………………………45 40 3.4.5 Determine the physical interaction between Osh proteins and AAA ATPases… 45 40 3.4.6 Examine the functional interactions between Osh proteins and AAA ATPase.… 45 40 3.4.7 Examine the roles of Vps4p and Afg2p in maintaining sterol homeostasis…… .45 40 IV Chapter II Materials and Methods Part Materials 1.1 Preparation of cell culture media………………………………………………….47 41 1.2 Drugs and special chemicals……………………………………………………….47 41 1.3 Antibodies………………………………………………………………………… 48 43 1.4 Radio chemicals…………………………………………………………………….48 43 Part Methods 2.1 DNA manipulation 44 2.1.1 PCR (polymerase chain reaction)………………………………………………….49 47 2.1.2 Subcloning…………………………………………………………………………52 47 2.1.2.0 Strategy………………………………………………………………………… 52 47 2.1.2.1 Purification of PCR products…………………………………………………….52 48 2.1.2.2 Digestion of DNA……………………………………………………………… 53 48 2.1.2.3 Gel purification of DNA…………………………………………………………53 48 2.1.2.4 Ligation………………………………………………………………………… 53 48 2.1.2.5 Preparation of DH5α chemical competent cells…………………………………53 49 2.1.2.6 E.coli transformation…………………………………………………………….54 49 2.1.2.7 Mini-preparation of plasmid DNA………………………………………………54 50 2.1.2.8 Diagnostic digestion ……………………….……………………………………55 50 2.1.2.9 DNA sequencing……………………………………………………………… .55 51 2.1.2.10 Midi-preparation of plasmid DNA…………………………………………… 56 2.1.2.11 Filling 5’-protruding ends (Blunting)………………………………………….52 57 52 2.1.3 Construction of yeast strains………………………………………………………57 52 2.1.3.1 Preparation of chemical competent yeast cells………………………………….57 52 2.1.3.2 Yeast transformation…………………………………………………………….57 53 2.1.3.3 PCR-based gene disruption…………………………………………………… .58 54 2.1.3.4 Rapid isolation of chromosomal DNA………………………………………….59 2.2 Protein expression, purification and production of antibody 2.2.1 Protein expression in E.coli and purification by affinity chromatography……… 59 54 55 2.2.2 Production of antibody…………………………………………………………….60 2.3 Fluorescence microscopy 2.3.1 FM4-64 staining………………………………………………………………… .61 56 2.3.2 Lucifer yellow staining……………………………………………………………61 56 2.3.3 GFP imaging………………………………………………………………………62 57 2.3.4 Filipin staining…………………………………………………………………….62 57 2.3.5 DAPI staining…………………………………………………………………… 57 62 2.3.6 Nile Red staining………………………………………………………………….58 63 2.4 Lipid assay 2.4.1 Analysis of ergosterol by GC-MS……………………………………………… .63 58 2.4.2 Sterol esterification……………………………………………………………… 64 59 2.4.3 Measurement of the rate of sterol biosynthesis………………………………… .64 59 2.4.4 TLC chromatography……………………………………………………………. 60 65 V 2.5 Biochemical assay 61 2.5.1 Extraction of yeast total proteins………………………………………………….66 2.5.2 Differential centrifugation……………………………………………………… 62 67 63 2.5.3 Sucrose density gradient centrifugation…………………………………………. .68 2.5.4 Membrane extraction…………………………………………………………… 64 69 65 2.5.5 Membrane floatation………………………………………………………………70 65 2.5.6 Pulse-chase radiolabeling and immunoprecipitation……………………………. .70 2.5.7 GST pull-down……………………………………………………………………66 72 2.5.8 SDS-PAGE electrophoresis……………………………………………………….73 67 2.5.9 Western blotting………………………………………………………………… .74 69 2.6 Protein-lipid overlay assay……………………………………………………… .75 70 2.7 Two-hybrid analysis 2.7.1 Principles of yeast two-hybrid…………………………………………………… 76 71 2.7.2 X-gal plate assay………………………………………………………………… .76 71 2.7.3 Liquid assay with ONPG……………………………………………………… 77 72 Chapter III Results and Discussion Part1 Characterization of Osh6p 1.1 Expression and purification of GST fusion proteins…………………………….85 80 1.2 Production of anti-Osh6p antiserum………………………………………………86 81 1.3 Osh6p is localized to cytosol and membranes 1.3.1 Construction of Osh6p-GFP…………………….…………………………… 88 82 1.3.2 Osh6p localizes to both cytosol and membrane structures……………………… .89 82 1.4 Osh6p is a peripheral membrane protein……………………………………… 94 87 1.5 Conclusions ……………………………………………………………………… 96 89 Part Functional Studies on Osh6p 2.1 Osh6p is required for sterol homeostasis……………………………………… 97 90 2.2 Osh6p is not essential for fluid-phase endocytosis or endocytic trafficking of membrane markers from the cell surface to the vacuole…….10698 2.3 Osh6p is not essential for CPY maturation………………………………… .110 102 2.4 Characterization of the functional domains of Osh6p…………………………111 103 2.5 Characterization of the lipid ligands and lipid-binding domain of Osh6p……112 104 2.6 Characterization of the membrane targeting domains of Osh6p…………… 116 108 2.7 Conclusions……………………………………………………………………….119 109 Part Interaction between Osh6p and Vps4p 3.1 Osh6p physically interacts with Vps4p………………………………………….120 110 3.2 Vps4p regulates the membrane disassociation of Osh6p………………………121 111 3.3 Conclusions……………………………………………………………………… 131 118 Part Interaction between Osh1p and Afg2p 4.1 Afg2p interacts with Osh1p …… ………………………………………………132 119 VI 4.2 Afg2p regulates Osh1p/2p disassociation from the ER membrane 121 4.2.1 Ag2p regulates Osh1p disassociation from the ER membrane…………………134 125 4.2.2 Scs2p is required for Osh1p targeting to the ER in afg2-ts……………………139 126 4.2.3 Afg2p regulates Osh2p disassociation from the ER membrane.………………140 127 4.3 Deletion of OSH1, 2, affects sterol esterification……………………….… 142 4.4 Conclusions…………………………………………………………………… 144 129 Part AAA ATPases and lipid metabolism 5.1 Afg2p and Vps4p regulate cellular sterol metabolism………………………145 130 5.2 Functional interaction of Osh6/7p with Vps4p……………………………….152 135 5.3 Conclusions…………………………………………………………………… 157 139 Part Discussion 6.1 Localization of Osh6p………………………………………………………… 158 140 6.2 Lipid ligands and functional domains of Osh6p…………………………… 160 142 6.3 Molecular function of Osh proteins 6.3.1 Osh6p and sterol homeostasis…………………………………………………162 144 6.3.2 Osh6p and membrane trafficking………………………………………………164 147 6.3.3 Osh1p and sterol homeostasis………………………………………………….165 147 6.4 Osh proteins, AAA ATPases and lipid transport…………………………….166 148 6. 4.1 AAA ATPases and lipid metabolism………………………………………… 167 149 6. 4.2 Regulation of Osh6p membrane association by Vps4p………………………169 151 6. 4.3 Regulation of Osh1p membrane association by Afg2p……………………… 171 153 6. 4.4 Functional interaction between Osh6/7p and Vps4p…………………………173 155 6.5 A proposed working model for concerted action of Osh and AAA proteins in regulating lipid transport………………… …………………… 174 156 REFERENCES………………………………………………………………………177 159 VII SUMMARY Oxysterol binding protein (OSBP) and its homologues constitute a large family of conserved proteins present in many eukaryotes, and they have been shown to regulate lipid metabolism and vesicular transport. The seven yeast homologues (OSH) collectively are essential for sterol homeostasis and cell viability, but each OSH exhibits distinct function. Here, we examined the: 1. Localization of Osh6p. Osh6p was largely soluble with a small pool peripherally associated with membranes, possibly the endosomal membranes. 2. Roles of Osh6p in sterol metabolism and vesicular transport. Osh6p played a role primarily in maintaining sterol homeostasis, not in endocytic trafficking or protein transport to the vacuole. Deletion of OSH6 caused a significant increase in total ergosterol; whereas overexpression of OSH6 reduced total ergosterol and lipid bodies, and caused nystatin resistance. The rate of sterol esterification was mildly decreased in both ∆osh6 and overexpression cells; while sterol biosynthesis was enhanced in ∆osh6. Free sterols were found to accumulate in the intracellular compartments in ∆osh6. However, deletion or overexpression of OSH6 showed no effect on Lucifer yellow internalization, and FM4-64 transport. Furthermore, ∆osh6 exhibited no defect in carboxypeptidase Y (CPY) transport and maturation. Osh6p was shown to bind phosphoinositides and phosphatidic acid through its conserved OSBP related domain (ORD). The ORD of Osh6p was shown to associate with endosomes, whereas the Cterminal putative coiled coil (CC) localized to the nucleoplasm. However, both the ORD and CC domains were indispensable for the in vivo function of Osh6p. VIII 3. Interaction between AAA ATPases and Osh proteins. We showed that a subset of AAA ATPases regulated the membrane association of Osh proteins. First, Osh6p interacted with Vps4p, a member of the AAA protein family, both in vitro and in vivo. Deletion of VPS4 induced a dramatic increase in the membrane associated pool of Osh6p. In addition, the ATPase activity of Vps4p was essential for this regulatory process. Second, the ORD domain-containing fragment of Osh1p interacted with the ATPase domain-containing fragment of Afg2p, another member of the AAA family, in vivo. Interestingly, when Afg2p was inactivated, a significant pool of Osh1p was redistributed from the cytosol to the ER membrane. 4. Roles of AAA ATPases in lipid metabolism. Deletion of VPS4 resulted in a significant decrease in sterol esterification. Inactivation of Afg2p led to a substantial reduction of oleate incorporation into both sterol esters (SE) and triacylglycerol (TAG), and accumulation of free sterols in the intracellular compartments. 5. Functional interaction between Osh6/7p and Vps4p. Overexpression of Osh6/7p suppressed the defect in sterol esterification in vps4∆. Overexpression of the coiled coil motif of Osh7p resulted in a multi-vesicular body sorting (MVB) defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. 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Eur J Biochem. 267(4):1075-82. 196 [...]... results in the soluble NH2-terminal domain of SREBP, which enters the nucleus as an active positive transcription factor Normal or high levels of cholesterol block the incorporation of SREBP and SREBP-cleavage activation protein (SCAP) into COPII vesicles by retaining their interaction with an ER membrane protein-INSIG (insulin-induced protein); however, low cholesterol level promotes the release of SREBP... termed OSHs (oxysterol binding protein homologue), which are encoded by open reading frames: YHR001w, YKR003w, YHR073w, YDL019c, YAR042w, YPL145c and YOR237w respectively (Beh et al., 2001) All ORPs and Osh proteins share a highly conserved “fingerprint” sequence EQVSHHPP, which is within the ligand binding domain (ORD domain) at the carboxyterminus Most ORPs except ORP2 and three long Osh proteins (Osh1p,... Osh3p) also contain a pleckstrin homology domain (PH domain, β-sandwich module) at their amino-termini, which targets these proteins to membranes by interaction with 20 phosphoinositides in the membranes (Lemmon and Ferguson, 2000) Some proteins also have ankyrin repeats, which are believed to mediate protein-protein interactions (Sedgwick and Smerdon, 1999; Letho and Olkkonen, 2003) Osh proteins also have... their C-termini, which are believed to mediate protein-protein interactions (Beh et al., 2001; Kranz et al., 2001) Although it has been more than one decade since the cloning of OSBP, the molecular function of OSBP proteins remains to be defined Because of its high affinity for oxysterols and the potency of oxysterols as feedback regulators, OSBP is believed to mediate the feedback control of the mevalonate... Protein in humans; OSBP: Oxysterol Binding Protein; OSH: OSBP Homologues in Saccharomyces cerevisiae; PA/PtdOH: Phosphatidic Acid; PAGE: PolyAcrylmide Gel Electrophoresis; PBS: Phosphate Buffered Saline; PC/PtdCho: PhosphatidylCholine, PCR: Polymerase Chain Reaction; PE/PtdEth: Phosphatidyl Ethanolamine; PH: Pleckstrin Homology; PIK1: yeast PhosphatidylInositol 4-Kinase; PI/PtdIns: PhosphatidylInositides;... early recycling compartment and for delivery of cholesterol to the inner mitochondrial membrane in steriodogenic cells (Strauss et al., 1999) A family of carrier proteins with high-affinity for lipids has been identified, one of which is the steroidogenic acute regulatory protein (StAR/StarD1) containing a lipid- 9 binding domain (START) (Stocco, 2001) StAR has been implicated in the delivery of cholesterol... bulk of oxysterols to high density lipoprotein particles in the plasma Since oxysterols play an important role in sterol biosynthesis and homeostasis, they must be subject to regulation; then what are their regulators and mediators? Two protein families including some of the steroid hormone nuclear receptors FXR / LXRs (Russell, 1999) and oxysterol binding proteins (OSBPs) appear to mediate many of the... The molecular function of ORPs Like OSBP, ORPs have been shown to play a role in cholesterol homeostasis and intracellular membrane trafficking ORP1 transcription levels were strikingly high in cortical areas of human brain and were regulated by sterols in a cultured human neuroblastoma cell line (Laitinen et al., 1999), which suggests ORP1 plays an important role in maintaining sterol homeostasis in. .. high-density plasma lipoproteins (Fielding and Fielding, 1995) However, hepatocytes with active LDL internalization have few caveolae (Fielding and Fielding, 1997)，suggesting caveolae are largely involved in cholesterol trafficking to the plasma membrane 1.3.3 General intracellular transport pathways Nascent cholesterol transport out of ER: The ER is the site of cholesterol synthesis but it maintains a low steady-state... macromolecular complex containing SREBP, SCAP and Insig-1 in the ER in the presence of normal or higher cellular cholesterol levels (b), (c) Disassociation of Insig-1 from the complex stimulated by a low level of cholesterol, allowing SCAP and SREBP to be packaged into COP II-coated vesicles and then delivered to the Golgi apparatus (d) Processing of SREBP in the Golgi apparatus by S1P and S2P 6 1.3 Intracellular . Factor; OB: Oxysterol Binding domain; O.D: Optical Density; OPI1: OverProduction of Inositol; ORD: OSBP Related Domain; ORP: OSBP Related Protein in humans; OSBP: Oxysterol Binding Protein; OSH:. molecular function of OSHs 2.4.1 Secondary structures of Osh proteins ………………………………….…… 26 2.4.2 The role of Osh proteins in maintaining sterol homeostasis………………… 29 2.4.3 The role of Osh proteins. I MOLECULAR ANALYSIS OF OXYSTEROL BINDING PROTEINS IN YEAST WANG PENGHUA (Bachelor of Science (Hons), Zhongshan University, People’s Republic of China)