CHARACTERIZATION AND FUNCTION STUDIES OF Ncr1p: A YEAST ORTHOLOG OF MAMMALIAN NIEMANN PICK C1 PROTEIN (NPC1) ZHANG SHAOCHONG (B.S WUHAN UNIV.) (M.S HUAZHONG AGRICULTURAL UNIV.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHYLOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2006 -1- Acknowledgement This thesis is the main part of research work done for four and half years whereby many people have helped and supported me during this period It’s my pleasure to formally express my gratitude to all of them Firstly, I would like to thank my mentor, Dr Hongyuan Yang for his dedicated supervision since January, 2001 His overly enthusiasm and active thinking on science has made a deep impression on me I sincerely thank him for showing me the way of research It is difficult to estimate how much I have learned from him Therefore, I am really glad to know Dr Yang in my life My colleagues in Dr Yang’s lab gave me the harmonious feeling of being at home at work They are Woo Wee Hong, Jaspal Kaur Kumar, Zhang Qian, Ren Jihui, Li Hongzhe, Lai Liyun, Low Soo Mei, Liew Li Phing, Fei Weihua, Zheng Li, Ho Zi Zong, Chieu Hai Kee, Low Choon Pei, and Wang Peng Hua At the same time, I am extremely happy to be part of you all I greatly appreciate Dr Zhang Qian for taking care of my studies and living in Singapore with tremendous energy I also especially thank Dr Jaspal Kaur Kumar for revising this thesis with terrible patience I am grateful to Dr Alan Munn for his help to study sucrose density gradient centrifugation techniques I also thank Dr Liang Fubo from Professor Chang’s Lab who helped me to learn SDS-PAGE electrophoresis of membrane proteins It was my pleasure to work with them and the students and employees from their labs Deeply from my heart, I would like to thank my wife for her love and support, -2- even at the most difficult time, I was happy and peaceful It is a pleasure to share my happiness and sorrows with her With the support of all of them part of our results have been published in Traffic 2004; 5: 1017-1030 (see bibliography), which is also part of my thesis -3- Table of Contents Acknowledgement Table of Contents Summary 10 List of Tables 12 List of Figures 13 Abbreviations and Symbols Used 15 Chapter Introduction 20 1.1 Biochemistry of Cholesterol .20 1.1.1 Chemical Structure of Cholesterol .20 1.1.2 The Features and Functions of Cholesterol 23 1.1.3 Biosynthesis of Cholesterol 26 1.1.4 Cholesterol Metabolism .26 1.2 Intracellular Cholesterol Homeostasis 29 1.2.1 Introduction to Cholesterol Homeostasis 29 1.2.2 A Key Domain and Protein Involved in Intracellular Cholesterol Homeostasis 32 1.2.2.1 Sterol-sensing Domain (SSD) Proteins 33 1.2.2.2 Oxysterol and OSBP .34 1.2.3 The Role of Cholesterol Metabolism, Esterification and Cholesterol Transport in Cholesterol Homeostasis .36 1.2.3.1 Regulation of HMG-CoA Reductase 37 -4- 1.2.3.2 The Activation of SREBPs 39 1.2.3.3 The Cholesterol Esterification Enzyme: ACAT 42 1.2.3.4 Cholesterol Transport through the PM 44 1.2.3.6 Intracellular Cholesterol Transport 51 1.3 A Putative Cholesterol Transporter, NPC1 60 1.3.1 Molecular and Cell Biology of NPC1 60 1.3.2 The Pathogenicityof NPC Disease .62 1.3.3 The Gene and Localization of NPC1 Protein .63 1.3.4 The Topology of NPC1 .64 1.3.5 Function of NPC1 .65 1.3.6 The Effect of NPC1 in Brain .69 1.3.7 Mutant Proteins which Produce NPC-like Phenotypes .70 1.4 The Advantages of Studying Human Disease Using S cerevisiae as a Model .70 1.5 Sterol Homeostasis in Yeast .72 1.6 Ncr1p, the Ortholog of NPC1 in S.cerevisiae 73 1.6 Objectives and Significance of this Study 73 Chapter Materials and Methods 77 2.1 Strains, Media and Materials 77 2.2 Production of Antisera Against Ncr1p 79 2.2.1 Expression of an 189 aa Ncr1p Polypeptide 79 2.2.2 Purification of 189aa Ncr1p Peptides Using Talon® Beads 79 2.2.3 Electro-elution to Purify the Peptide 80 -5- 2.3 Characterization of Ncr1p 81 2.3.1 Mini Yeast Chromosomal DNA Preparation 81 2.3.2 Transformation of S cerevisiae 82 2.3.3 Construction of Plasmids and Gene Disruption 82 2.3.4 Protein Extraction from Yeast Cells 84 2.3.5 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting Analysis .85 2.3.6 Visualization of Ncr1p-GFP .85 2.3.7 FM4-64 Internalization 86 2.3.8 4', 6'-Diamidino-2-phenylindole (DAPI) Staining of Nuclei .86 2.3.9 Subcellular Fractionation .87 2.3.10 Detergent Resistant Membrane (DRM) Isolation 88 2.3.11 Sucrose Density Gradient Fractionation 88 2.4 Functional Study .90 2.4.1 In vivo Assays for Sterol Esterification .90 2.4.1.1 Labeling and Drying 90 2.4.1.2 Cell Lysis and Neutral Lipid Extraction 90 2.4.2 In vivo Assays for Sterol Esterification Under Acute Glucose Starvation Condition .92 2.4.3 Acetate Incorporation Assay 92 2.4.4 In vitro (Microsomal) Assay of Sterol Esterification 93 2.4.4.1 Isolation of Microsomes .93 -6- 2.4.4.2 Reaction System 94 2.4.4.3 Measure of Sterol Esterification Activity 94 2.4.5 GST Pull-down Assay: .95 2.4.6 β-galactosidase Assay with ONPG as Substrate in Liquid Culture .97 2.4.7 Isolation of Intact Vacuoles from Yeast .98 2.4.8 Isolation of Vacuolar Lipids .100 Chapter Localization and Transport of Ncr1p 101 3.1 Introduction 101 3.2 Results 102 3.2.1 Purification of Ncr1 189 aa peptide with Talon® beads 102 3.2.2 Purification of the Ncr1p 189aa Peptide by Electroelution 103 3.2.3 Ncr1p Antibody Preparation and Detection of Ncr1p in S cerevisiae Cells 104 3.2.4 Ncr1p is Predominantly Located in the P13 Membrane Fraction 105 3.2.5 Ncr1p Localizes to the Limiting Membrane of Yeast Vacuole 107 3.2.6 Localization of Ncr1p-GFP 107 3.2.7 Ncr1p is not Associated with Detergent-resistant Membranes (DRMs) .108 3.2.8 Vacuolar Localization of Ncr1p is Impaired in Mutants Affecting Early but notLate Steps of the Secretory Pathway 108 3.2.9 Vacuolar Localization of Ncr1p is Impaired by the Defect in the Vacuolar Protein Sorting (VPS) Pathway and Vacuole Morphology, but -7- not in Endocytosis or ALP Pathway 110 3.2.10 Deletion of 11 Amino Acids at the Carboxyl Terminus of Ncr1p does not Perturb its Localization .115 3.2.11 Loss of Ncr1p does not Affect ALP Transport to the Vacuole 115 3.3 Discussion 117 Chapter Functional Studies on Ncr1p .123 4.1 Introduction 123 4.2 Results 124 4.2.1 The Effect of Ncr1p on Ergosterol Homeostasis .124 4.2.2 Two Yeast Orthologs of Insigs, Nsg1p and Nsg2p 128 4.2.3 The Localization of Nsgs-GFP 130 4.2.4 Interaction Between Nsgs and Hmg2p-GFP 130 4.2.5 Interaction between Ncr1p and Nsgs 132 4.2.6 The Role of Ncr1p in the Unfolded Protein Response (UPR) Incluced by overloaded sterol .134 4.3 Disscusion 137 Chapter Role of Ncr1p in Subcellular Sterol Transport in Saccharomyces cerevisiae 143 5.1 Introduction 143 5.2 Results 145 5.2.1 Acute Glucose Starvation Induces Increase a Sterol Esterification 145 5.2.2 Deletion of NCR1 Decreases Sterol Eesterification in are2Δ but -8- not in are1Δ Mutant Cells During Acute Glucose Starvation 146 5.2.3 The Effect of ncr1Δ Deletion During Acute Glucose Starvation is Time Dependant 148 5.2.4 The Enzyme Activity of Are2p does not Change Upon Glucose Withdrawal 150 5.2.5 Free Sterol is Accumulated in the Vacuole of are2Δncr1Δ and are1Δare2Δncr1Δ 150 5.3 Discussion 155 Chapter Conclusions and Prospects 162 6.1 Conclusions 162 6.2 Prospects 163 Reference 166 Bibliography 204 -9- Summary Niemann-Pick disease type C (NPC) is a neurodegenerative lipid storage disorder This disease is characterized by the accumulation of free cholesterol within the endosomal/lysosomal system Mutations of the NPC1 gene induce over 95% of NPC cases The NPC1 protein predominantly localizes to late endosomes, and transiently associates with lysosomes Although it is believed that the NPC1 protein modulates the transport of lipids in the endosomal system, the exact molecular function of NPC1 remains elusive Ncr1p is a yeast ortholog of NPC1 Little is known about Ncr1p in yeast The purposes of this study were to confirm the localization of Ncr1p and examine the synergistic effect of Ncr1p on ergosterol homeostasis with other proteins The other important goal of this study was to identify other proteins involved in ergosterol transport pathways and to determine their relationship with Ncr1p Sucrose density gradient centrifugation and fluorescence microscopy were used to observe the localization of Ncr1p Oleate incorporation assay was performed to examine the effect of Ncr1p on sterol esterification β-galactosidase assay was used to detect unfolded protein response (UPR) caused by loaded ergosterol in the ER Acute glucose starvation provided a distinct intracellular ergosterol retranslocation condition Vacuoles were purified and their content of free ergosterol was determined and compared with cellular ergosterol composition The centrifugation and microscopy results showed that Ncr1p was localized on vacuole limiting membranes but not on detergent resistant membrane (DRM) domains This protein was transported to its destination via the vacuole protein sorting (VPS) pathway Biochemistry assays showed that there was no significant difference in ergosterol esterification, biosynthesis and total ergosterol between wild type and ncr1Δ cells Therefore, this suggests that there may be a synergistic effect between Ncr1p and other ergosterol metabolism and transport related proteins, such as Nsgs, - 10 - Blanchette-Mackie JE, Pentchev PG in The Metabolic and Molecular Bases of Inherited Disease (Scriver CR, Beaudet AL, Sly WS, Valle D) Vol III, 8th Ed., pp McGraw-Hill, New York 2001; 3611–3633 Patterson MC, Vanier MT, Suzuki K, Morris JA, Carstea ED, Neufeld EB, Blanchette-Mackie EJ, Pentchev PG, in: Schriver CR, Beaudet AL, Sly WS, Valle D (Eds.) McGraw-Hill, New York Metabolic and Molecular Bases of Inherited Disease, 1999 Pentchev PG, Blanchette-Mackie EJ, Dawidowicz EA The NP-C gene: a key to pathways of intracellular cholesterol transport Trends Cell Biol 1994; (10): 365-9 Pentchev PG, Blanchette-Mackie EJ, Liscum L Biological implications of the Niemann-Pick C mutation Subcell Biochem 1997; 28: 437–51 Pentchev PG, Comly ME, Kruth HS, Tokoro T, Butler J, Sokol J, Filling-Katz M, Quirk JM, Marshall DC, Patel S, et al Group C Niemann-Pick disease: faulty regulation of low-density lipoprotein uptake and cholesterol storage in cultured fibroblasts FASEB J 1987; (1): 40–5 Pentchev PG, Comly ME, Kruth HS, Tokoro T, Butler JD, Sokol J, Filling-Katz M, Quirk JM, Marshall DC, Patel S, Vanier MT, Brady RO Group C Niemann-Pick disease: faulty regulation of low-density lipoprotein uptake and cholesterol storage in cultured fibroblasts FASEB J 1987; 1: 40–-45 Pentchev PG, Comly ME, Kruth HS, Vanier MT, Wenger DA, Patel S, Brady RO A defect in cholesterol esterification in Niemann-Pick disease (type C) patients Proc Nat Acad Sci 1985; 82: 8247–8251 Pentchev PG, Comly ME, Vanier MT, Kruth HS, Patel S, Brady RO Type C Niemann-Pick disease: a defect in intracellular esterification Am J Hum Genet 1985; 37: A15 Pfeffer S, Aivazian D Targeting Rab GTPases to distinct membrane compartments Nat Rev Mol Cell Biol 2004; (11): 886–96 Phillips MC, Johnson WJ, Rothblat GH Mechanism and consequence of cellular cholesterol exchange and transfer Biochim Biophys Acta 1987; 906: 223–276 Phillips MC, Johnson WJ, Rothblat GH Mechanisms and consequences of cellular cholesterol exchange and transfer Biochim Biophys Acta 1987; 906: 223–276 - 191 - Pike LJ, Casey L, Localization and turnover of phosphatidylinositol 4,5-biphosphate in caveolin-enriched membrane domains J Biol Chem 1996; 271: 26453–26456 Pike LJ Lipid rafts: bringing order to chaos J Lipid Res 2003; 44: 655–667 Prinz W, Cholesterol trafficking in the secretory and endocytic systems Semin Cell Dev Biol 2002; 13 (3): 197–203 Puglielli L, Rigotti A, Amigo L, Nunez L, Greco AV, Santos MJ, Nervi F Modulation of intrahepatic cholesterol trafficking: evidence by in vivo antisense treatment for the involvement of sterol carrier protein-2 in newly synthesized cholesterol transport into rat bile Biochem J 1996; 317(Pt 3): 681–687 Puglielli L, Rigotti A, Greco AV, Santos MJ, Nervi F, Sterol carrier protein-2 is involved in cholesterol transfer from the endoplasmic-reticulum to the plasma-membrane in human fibroblasts J Biol Chem 1995; 270: 18723–18726 Puri V, Jefferson JR, Singh RD, Wheatley CL, Marks DL, Pagano RE J Biol Chem 2003; 278, 20961–20970 Puri V, Watanabe R, Dominguez M, Sun X, Wheatley CL, Marks DL, Pagano RE Nat Cell Biol 1999; 1, 386–388 Puri V,Watanabe R, Dominguez M, Sun X, Wheatley CL, Marks DL, Pagano RE Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases Nat Cell Biol 1999; 1: 386–388 Putney JW Jr “Kissin’ cousins”: intimate plasma membrane-ER interactions underlie capacitative calcium entry Cell 1999; 99: 5–8 Radhakrishnan A, Sun LP, Kwon HJ, Brown MS, Goldstein JL Direct binding of cholesterol to the purified membrane region of SCAP: mechanism for a sterol-sensing domain Mol Cell 2004; 15 (2): 259–68 Ramsdale EE, Kingsman SM, Kingsman AJ The "putative" leucine zipper region of murine leukemia virus transmembrane protein (P15e) is essential for viral infectivity Virology 1996; 220 (1): 100–8 Ravid T, Doolman R, Avner R, Harats D, Roitelman J The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase J Biol Chem 2000; 275, 35840–35847 - 192 - Raymond CK, Howald-Stevenson I, Vater CA, Stevens TH Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants Mol Biol Cell 1992; 3: 1389–1402 Razani B, Engelman JA, Wang XB, Schubert W, Zhang XL, Marks CB, Macaluso F, Russell RG, Li M, Pestell RG, Di Vizio D, Hou Jr H, Kneitz B, Lagaud G, Christ GJ, Edelmann W, Lisanti MP Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities J Biol Chem 2001; 276: 38121–38138 Reid PC, Sakashita N, Sugii S, Ohno-Iwashita Y, Shimada Y, Hickey WF, Chang TY A novel cholesterol stain reveals early neuronal cholesterol accumulation in the Niemann-Pick type C1 mouse brain J Lipid Res 2004; 45 (3): 582–91 Reid PC, Sugii S, Chang TY Trafficking defects in endogenously synthesized cholesterol in fibroblasts, macrophages, hepatocytes, and glial cells from Niemann-Pick type C1 mice J Lipid Res 2003; 44 (5): 1010–9 Repa JJ, Berge KE, Pomajzl C, Richardson JA, Hobbs H, Biol Chem 2002; 277, 18793–18800 Mangelsdorf DJ J Ridgway ND, Dawson PA, Ho YK, Brown MS, Goldstein JL Translocation of oxysterol binding protein to Golgi apparatus triggered by ligand binding J Cell Biol 1992; 116: 307–319 Rinninger F, Pittman RC Regulation of the selective uptake of high density lipoprotein-associated cholesteryl esters J Lipid Res 1987; 28: 1313–1325 Roff CF, Goldin E, Comly ME, Cooney A, Brown A, Vanier MT Miller SPF, Brady RO, Pentchev PG Type C Niemann-Pick disease: use of hydrophobic amines to study defective cholesterol transport Dev Neurosci 1991; 13: 315–319 Roitelman J, Olender EH, Bar-Nun S, Dunn WA Jr, Simoni RD Immunological evidence for eight spans in the membrane domain of 3-hydroxy-3-methylglutaryl Coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum J Cell Biol 1992; 117, 959–973 Rothberg K, Heuser JE, Donzell WC, Ying Y-S, Glenney JR, Anderson RGW Caveolin, a protein component of caveolae membranes Cell 1992; 68: 673–682 Rumsey SC, Galeano NF, Lipschitz B, Deckelbaum RJ Oleate and other long chain fatty acids stimulate low density lipoprotein receptor activity by enhancing - 193 - acyl coenzyme A:cholesterol acyltransferase activity and altering intracellular regulatory cholesterol pools in cultured cells J Biol Chem 1995; 270 (17): 10008–16 Russell D Oxysterol biosynthetic enzymes Biochim Biophys Acta 2000; 1529: 126–135 Russell DW, Yamamoto T, Schneider WJ, Slaughter CJ, Brown MS, Goldstein JL cDNA cloning of the bovine low density lipoprotein receptor: feedback regulation of a receptor mRNA Proc Natl Acad Sci USA 1983; 80 (24): 7501–5 Russell DW The enzymes, regulation and genetics of bile acid synthesis Annu Rev Biochem 2003; 72: 137–174 Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, Deleuze JF, Brewer HB, Duverger N, Denefle P, Assmann G Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter Nat Genet 1999; 22 (4): 352–5 Rustow B, Risse S, Kunze D Endogenous lipid pattern, organ distribution and effect of diet on a fatty acid binding protein fraction of rat liver cytosol German Acta Biolog Med Germ 1982; 41: 439–45 Saito Y, Suzuki K, Nanba E, Yamamoto T, Ohno K, Murayama S Niemann-Pick type C disease: accelerated neurofibrillary tangle formation and amyloid beta deposition associated with apolipoprotein E epsilon-4 homozygosity Ann Neurol 2002; 52: 351–355 Sakai J, Duncan EA, Rawson RB, Hua X, Brown MS, Goldstein J: Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembrane segment Cell 1996; 85:1037–1046 Sakai J, Nohturfft A, Cheng D, Ho YK, Brown MS, Goldstein J: Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavage-activating protein J Biol Chem 1997; 272: 20213–20221 Sakai J, Nohturfft A, Goldstein JL, Brown MS: Cleavage of sterol regulatory element-binding proteins (SREBPs) at site-1 requires interaction with SREBP cleavage-activating protein Evidence from in vivo competition studies J Biol Chem 1998; 273: 5765–5793 Salvioli R, Scarpa S, Ciaffoni F, Tatti M, Ramoni C, Vanier MT, Vaccaro AM Glucosylceramidase mass and subcellular localization are modulated by - 194 - cholesterol in Niemann-Pick disease type C J Biol Chem 2004; 279 (17): 17674–80 Sandager L, Dahlqvist A, Banas A, Stahl U, Lenman M, Gustavsson M, Stymne S An acyl-CoA:cholesterol acyltransferase (ACAT)-related gene is involved in the accumulation of triacylglycerols in Saccharomyces cerevisiae Biochem Soc Trans 2000; 28 (6): 700–2 Sato R, Goldstein JL, Brown MS Replacement of serine-871 of hamster 3-hydroxy-3-methylglutaryl-CoA reductase prevents phosphorylation by AMP-activated kinase and blocks inhibition of sterol synthesis induced by ATP depletion Proc Natl Acad Sci USA 1993; 90 (20): 9261–5 Scheek S, Brown MS, Goldstein J Sphingomyelin depletion in cultured cells blocks proteolysis of sterol regulatory element binding proteins at site Proc Natl Acad Sci USA 1997; 94: 11179–11183 Scheel J, Srinivasan J, Honnert U, Henske A, Kurzchalia TV Involvement of caveolin-1 in meiotic cell-cycle progression in Caenorhabditis elegans Nat Cell Biol 1999; 1: 127–129 Schmitz G, Langmann T, Heimerl S Role of ABCG1 and other ABCG family members in lipid metabolism J Lipid Res 2001; 42 (10): 1513–20 Schmitz G, Muller G Structure and function of lamellar bodies, lipid-protein complexes involved in storage and secretion of cellular lipids J Lipid Res 1991; 32: 1539–1570 Schneider WJ, Beisiegel U, Goldstein JL, Brown MS Purification of the low density lipoprotein receptor, an acidic glycoprotein of 164,000 molecular weight J Biol Chem 1982; 257 (5): 2664–73 Schroeder F, Frolov A, Schoer J, Gallegos A, Atshaves BP, J StolowichN, Scott AI, Kier AB Intracellular cholesterol binding proteins, cholesterol transport and membrane domains In Chang TY, Freeman DA, eds Intracellular Cholesterol Trafficking Boston, Kluwer Academic Publishers 1998; 213–234 Schroeder F, Gallegos AM, Atshaves BP, Storey SM, McIntosh AL, Petrescu AD, Huang H, Starodub O, Chao H, Yang H, Frolov A, Kier AB Recent advances in membrane microdomains: rafts, caveolae, and intracellular cholesterol trafficking Exp Biol Med 2001; 226 (10): 873–90 Scott C, Ioannou YA The NPC1 protein: structure implies function Biochim Biophys Acta 2004; 1685 (1-3): 8–13 - 195 - Seedorf U, Brysch P, Engel T, Schrage K, Assmann G Sterol carrier protein-x is peroxisomal 3-oxoacyl coenzyme-A thiolase with intrinsic sterol carrier and lipid transfer activity J Biol Chem 1994; 269: 21277–21283 Seedorf U, Engel T, Assmann G, Leenders F, Adamski J intrinsic sterol- and phosphatidylcholine transfer activities of 17 L-hydroxysteroid dehydrogenase type IV, J Steroid Biochem Mol Biol 1995; 55: 549–553 Sever N, Yang T, Brown MS, Goldstein JL, DeBose-Boyd RA Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain Mol Cell 2003; 11 (1): 25–33 Shamburek RD, Pentchev PG, Zech LA, Blanchette-Mackie EJ, Castea ED, VandenBrock JM, Cooper PS, Neufeld EB, Phair RD, Brewer HB Jr, Brady RO, Schwartz CC Intracellular traÔcking of the free cholesterol derived from LDL cholesteryl ester is defective in vivo in Niemann-Pick C disease: insights on normal metabolism of HDL and LDL gained from the NP-C mutation J Lipid Res 1997; 38: 2422–2435 Shen WJ, Patel S, Natu V, Hong R, Azhar S, Kraemer FB Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein: Facilitation of cholesterol transfer in adrenal J Biol Chem 2003; 278 (44): 43870–6 Shiao YJ, Balcerzak B, Vance JE A mitochondrial membrane protein is required for translocation of phosphatidylserine from mitochondria-associated membranes to mitochondria Biochem J 1998; 331: 217–223 Simons K, Ikonen E Functional rafts in cell membranes Nature 1997; 387: 569–572 Simons K, Toomre D Lipid rafts and signal transduction Nat Rev Mol Cell Biol 2000; 1: 31–39 Simons K, Van Meer G Lipid sorting in epithelial cells Biochemistry 1988; 27: 6197–6202 Slotte JP Cholesterol–sphingomyelin interactions in cells-effects on lipid metabolism Subcell Biochem 1997; 28: 277–293 Small DM George Lyman Duff memorial lecture Progression and regression of atherosclerotic lesions Insights from lipid physical biochemistry Arteriosclerosis 1988; (2): 103–29 - 196 - Smart EJ, Ying Y, Donzell WC, Anderson RG A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membrane J Biol Chem 1996; 271: 29427-29435 Smith LL Review of progress in sterol oxidations: 1987–1995 Lipids 1996; 31: 453–487 Soccio RE, Breslow JL Intracellular cholesterol transport Arterioscler Thromb Vasc Biol 2004; 24 (7): 1150–60 Sokol J, Blanchette-Mackie EJ, Kruth HS, Dwyer NK, Amende LM, Butler JD, Robinson E, Patel S, Brady RO, Comly ME, Vanier MT, Pentchev PG Type C Niemann-Pick disease: lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol J Biol Chem 1988; 263: 3411–3417 Song BL, Sever N, Debose-Boyd RA Gp78, a Membrane-Anchored Ubiquitin Ligase, Associates with Insig-1 and Couples Sterol-Regulated Ubiquitination to Degradation of HMG CoA Reductase Mol Cell 2005; 19 (6): 829–40 Spady DK, Dietschy JM Sterol synthesis in vivo in 18 tissues of the squirrel monkey, guinea pig, rabbit, hamster and rat J Lipid Res 1983; 24: 303–315 Sparks CE, Rader DJ, Marsh JB Metabolism of two forms of apolipoprotein B of VLDL by rat liver J Lipid Res 1983; 24 (2): 156–66 Steck TL, Kezdy FJ, Lange Y Anactivation-collision mechanism for cholesterol transfer between membranes J Biol Chem 1988; 263: 13023–13031 Stocco D A review of the characteristics of the protein required for the acute regulation of steroid hormone biosynthesis: the case for the steroidogenic acute regulatory (STAR) protein Proc Soc Exp Biol Med 1998; 217: 123–129 Stocco DM, Clark BJ Regulation of the acute production of steroids in steroidogenic cells Endocr Rev 1996; 17, 221–244 Sturley SL A molecular approachto understanding human sterol metabolism using yeast genetics Curr Opin Lipidol 1998; 9: 85–91 Sturley SL Conservation of eukaryotic sterol homeostasis: new insights from studies in budding yeast Biochim Biophys Acta 2000; 1529: 155–163 Suckling KE, Stange EF Role of acyl-CoA: cholesterol acyltransferase in cellular cholesterol metabolism J Lipid Res 1985; 26 (6): 647–71 - 197 - Sugawara T, Fujimoto S The potential function of steroid sulphatase activity in steroid production and steroidogenic acute regulatory protein expression Biochem J 2004; 380 (Pt 1): 153–60 Sun X, Marks DL, Park WD, Wheatley CL, Puri V, O’Brien JF, Kraft DL, Lundquist PA, Patterson MC, Pagano RE, Snow K Am J Hum Genet 2001; 68: 1361–1372 Swain E, Stukey J, McDonough V, Germann M, Liu Y, Sturley SL, Nickels JT Jr Yeast cells lacking the ARV1 gene harbor defects in sphingolipid metabolism Complementation by human ARV1 J Biol Chem 2002; 277 (39): 36152–60 Tang Z, Scherer PE, Okamoto T, Song K, Chu C, Kohtz DS, Nishimoto I, Lodish HF, Lisanti MP Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle J Biol Chem 1996; 271: 2255–2261 Thomas GH, Tuck-Muller CM, Miller CS, Reynolds LW Correction of sphingomyelinase deficiency in Niemann-Pick type C fibroblasts by removal of lipoprotein fraction from culture media J Inherit Metab Dis 1989; 12: 139-151 Thumm M Structure and function of the yeast vacuole and its role in autophagy Microsc Res Tech 2000; 51 (6): 563–72 Thumser AE, DC Wilton The binding of cholesterol and bile salts to recombinant rat liver fatty acid-binding protein Biochem J 1996; 320: 729-733 Tinkelenberg AH, Liu Y, Alcantara F, Khan S, Guo Z, Bard M, Sturley SL Mutations in yeast ARV1 alter intracellular sterol distribution and are complemented by human ARV1 J Biol Chem 2000; 275 (52): 40667–70 Tolleshaug H, Goldstein JL, Schneider WJ, Brown MS Posttranslational processing of the LDL receptor and its genetic disruption in familial hypercholesterolemia Cell 1982; 30 (3): 715–24 Tong Q, Xing S, Jhiang SM Leucine zipper-mediated dimerization is essential for the PTC1 oncogenic activity J Biol Chem 1997; 272 (14): 9043–7 Tsuneoka M, Yamamoto A, Fujiki Y, Tashiro Y Nonspecific lipid transfer protein (sterol carrier protein-2) is located in rat liver peroxisomes J Biochem (Tokyo) 1988; 104 (4): 560–4 Uelmen PJ, Oka K, Sullivan M, Chang CC, Chang TY, Chan L Tissue-specific - 198 - expression and cholesterol regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) in mice Molecular cloning of mouse ACAT cDNA, chromosomal localization, and regulation of ACAT in vivo and in vitro J Biol Chem 1995; 270 (44): 26192–201 Uittenbogaard A, Smart EJ Palmitoylation of caveolin-1 is required for cholesterol binding, chaperone complex formation, and rapid transport of cholesterol to caveolae J Biol Chem 2000; 275: 25595–25599 Uittenbogaard A, Ying Y, Smart EJ Characterization of a cytosolic heat-shock protein-caveolin chaperone complex: involvement in cholesterol trafficking J Biol Chem 1998; 273: 6525–6532 Underwood KW, Jacobs NL, Howley A, Liscum L: Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane J Biol Chem 1998; 273: 4266–4274 Urbani L, Simoni RD Cholesterol and vesicular stomatitis virus G protein take separate routes from the endoplasmic reticulum to the plasma membrane J Biol Chem 1990; 265: 1919–1923 van Meer G Caveolin, cholesterol, and lipid droplets? J Cell Biol 2001; 152: F29–34 van Meer G Cell biology The different hues of lipid rafts Science 2002; 296: 855–857 van Meer G Lipid traffic in animal cells Annu Rev Cell Biol 1989; 5: 247–275 Vance JE, Vance DE Lipoprotein assembly and secretion by hepatocytes Annu Rev Nutr 1990; 10: 37–56 Vanier MT, Wenger DA, Comly ME, Rousson R, Brady RO, Pentchev PG Niemann-Pick disease group C: clinical variability and diagnosis based on defective cholesterol esterification: a collaborative study on 70 patients Clin Genet 1988; 33: 331–348 Virkkunen M, Penttinen H Serum cholesterol in aggressive conduct disorder: a preliminary study Biol Psychiatry 1984; 19: 435–439 Vlahcevic ZR, Hylemon PB, Chiang JYL Hepatic cholesterol metabolism See Arias et al, pp 1994; 379–89 Volchuk A, Amherdt M, Ravazzola M, Brugger B, Rivera VM, Clackson T, - 199 - Perrelet A, Sollner TH, Rothman JE, Orci L Megavesicles implicated in the rapid transport of intracisternal aggregates across the Golgi stack Cell 2000; 102: 335–348 Vuoristo M, Miettinen TA Increased biliary lipid secretion in celiac disease Gastroenterology 1985; 88:134–42 Wach A, Brachat A, Alberti-Segui C, Rebischung C, Philippsen P Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae Yeast 1997; 13: 1065–1075 Walter M, Davies JP, Ioannou YA Telomerase immortalization upregulates Rab9 expression and restores LDL cholesterol egress from Niemann-Pick C1 late endosomes J Lipid Res 2003; 44 (2): 243–53 Wang H, Germain SJ, Benfield PP, Gillies PJ Gene expression of acyl-coenzyme-A:cholesterol-acyltransferase is upregulated in human monocytes during differentiation and foam cell formation Arterioscler Thromb Vasc Biol 1996; 16 (6): 809–14 Wang X, Sato R, Brown MS, Hua X, Goldstein JL SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis Cell 1994; 77 (1): 53-62 Warner GJ, Stoudt G, Bamberger M, Johnson WJ, Rothblat GH Cell toxicity induced by inhibition of acyl coenzyme A:cholesterol acyltransferase and accumulation of unesterified cholesterol J Biol Chem 1995; 270 (11): 5772–8 Warnock DE, Roberts C, Lutz MS, Blackburn WA, Young WW, Baenziger JU Determination of plasma membrane lipid mass and composition in cultured Chinese hamster ovary cells using high gradient magnetic affinity chromatography J Biol Chem 1993; 268 (14): 10145–53 Wassif CA, Vied D, Tsokos M, Connor WE, Steiner RD, Porter FD Cholesterol storage defect in RSH/Smith-Lemli-Opitz syndrome fibroblasts Mol Genet Metab 2002; 75 (4): 325–34 Watari H, Blanchette-Mackie EJ, Dwyer NK, Glick JM, Patel S, Neufeld EB, Brady RO, Pentchev PG, Strauss JF 3rd Niemann-Pick C1 protein: obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization Proc Natl Acad Sci USA 1999; 96: 805–810 Watari H, Blanchette-Mackie EJ, Dwyer NK, Watari M, Neufeld EB, Patel S, Pentchev PG, Strauss JF 3rd Mutations in the leucine zipper motif and - 200 - sterol-sensing domain inactivate the Niemann-Pick C1 glycoprotein J Biol Chem 1999; 274: 21861–21866 Wesp A, Hicke L, Palecek J, Lombardi R, Aust T, Munn AL, Riezman H End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae Mol Biol Cell 1997; 8: 2291–2306 Will Prinz Cholesterol trafficking in the secretory and endocytic systems Semin Cell Dev Biol 2002; 13 (3): 197–203 Witsch-Baumgartner M, Loffler J, Utermann G (2001) Mutations in the human DHCR7 gene Hum Mutat 2001; 17 (3): 172–82 Wyles JP, McMaster CR, Ridgway ND Vesicle associated Membrane Protein-associated Protein-A (VAP-A) Interacts with the Oxysterol-binding Protein to Modify Export from the Endoplasmic Reticulum J Biol Chem 2002; 277: 29908–29918 Xie C, Burns DK, Turley SD, Dietschy JM Cholesterol is sequestered in the brains of mice with Niemann-Pick type C disease but turnover is increased J Neuropathol Exp Neurol 2000; 59 (12): 1106–17 Xie C, Lund EG, Turley SD, Russell DW, Dietschy JM Quantitation of two pathways for cholesterol excretion from the brain in normal mice and mice with neurodegeneration J Lipid Res 2003; 44: 1780–1789 Xie C, Turley SD, Dietschy JM Centripetal cholesterol flow from the extrahepatic organs through the liver is normal in mice with mutated Niemann-Pick type C protein (NPC1) J Lipid Res 2000; 41: 1278–1289 Xie C, Turley SD, Pentchev PG, Dietschy JM Cholesterol balance and metabolism in mice with loss of function of Niemann-Pick C protein Am J Physiol 1999; 276: E336–E344 Yabe D, Brown MS, Goldstein JL Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins Proc Natl Acad Sci USA 2002; 99 (20): 12753–8 Yamamoto T, Davis CG, Brown MS, Schneider WJ, Casey ML, Goldstein JL, Russell DW The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA Cell 1984; 39 (1): 27–38 Yamamoto T, Nanba E, Ninomiya H, Higaki K, Taniguchi M, Zhang H, Akaboshi S, Watanabe Y, Takeshima T, Inui K, Okada S, Tanaka A, Sakuragawa N, Millat - 201 - G, Vanier MT, Morris JA, Pentchev PG, Ohno K Hum Genet 1999; 105: 10–16 Yamamoto T, Ninomiya H, Matsumoto M, Ohta Y, Nanba E, Tsutsumi Y, Yamakawa K, Millat G, Vanier MT, Pentchev PG, Ohno K J Med Genet 2000; 37: 707– 712 Yang H, Bard M, Bruner DA, Gleeson A, Deckelbaum RJ, Aljinovic G, Pohl TM, Rothstein R, Sturley SL Science 1996; 272: 1353–6 Yang H, Cromley D, Wang H, Billheimer JT, Sturley S J Biol Chem 1997; 272: 3980–5 Yang T, Espenshade PJ, Wright ME, Yabe D, Gong Y, Aebersold R, Goldstein JL, Brown MS Crucial step in cholesterol homeostasis: Sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER Cell 2002; 110: 489–500 Yang T, Goldstein JL, Brown MS Overexpression of membrane domain of SCAP prevents sterols from inhibiting SCAP-SREBP exit from endoplasmic reticulum J Biol Chem 2000; 275: 29881–29886 Yeo SC, Xu L, Ren J, Boulton VJ, Wagle MD, Liu C, Ren G, Wong P, Zahn R, Sasajala P, Yang H, Piper RC, Munn AL Vps20p and Vta1p interact with Vps4p and function in multivesicular body sorting and endosomal transport in Saccharomyces cerevisiae J Cell Sci 2003; 116: 3957–3970 Yokoyama S Apolipoprotein-mediated cellular cholesterol efflux Biochim Biophys Acta 1998; 1392: 1–15 Yokoyama S Release of cellular cholesterol: molecular mechanism for cholesterol homeostasis in cells and in the body Biochim Biophys Acta 2000; 1529 (1-3): 231–44 Yokoyama S Release of cellular cholesterol: molecular mechanism for cholesterol homeostasis in cells and in the body Biochim Biophys Acta 2000; 1529: 231–244 Yu L, Hammer RE, Li-Hawkins J, von Bergmann K, Lutjohann D, Cohen JC, Hobbs HH Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion Proc Natl Acad Sci USA 2002; 99: 16237–16242 Zhang M, Dwyer NK, Love DC, Cooney A, Comly M, Neufeld E, Pentchev PG, Blanchette-Mackie EJ, Hanover JA Cessation of rapid late endosomal tubulovesicular trafficking in Niemann–Pick type C1 disease Proc Natl Acad Sci - 202 - USA 2001; 98:4466–4471 Zhang M, Dwyer NK, Neufeld EB, Love DC, Cooney A, Comly M, Patel S, Watari H, Strauss JF 3rd, Pentchev PG, Hanover JA, Blanchette-Mackie EJ Sterol-modulated glycolipid sorting occurs in Niemann-Pick C1 late endosomes J Biol Chem 2000; 276: 3417–3425 Zhang M, Liu P, Dwyer NK, Christenson LK, Fujimoto T, Martinez F, Comly M, Hanover JA, Blanchette-Mackie EJ, Strauss JF, 3rd MLN64 mediates mobilization of lysosomal cholesterol to steroidogenic mitochondria J Biol Chem 2002; 277: 33300–33310 Zhang SC, Ren J, Li H, Zhang Q, Armstrong JS, Munn AL, Yang H Ncr1p, the yeast ortholog of mammalian Niemann Pick C1 protein, is dispensable for endocytic transport Traffic 2004; (12): 1017–30 Zhao Y, Marcel YL Serum albumin is a significant intermediate in cholesterol transfer between cells and lipoproteins Biochemistry 1996; 35: 7174–7180 Zinser E, Daum G Isolation and biochemical characterization of organelles from the yeast, Saccharomyces cerevisiae Yeast 1995; 11:493–536 Zinser E, Paltauf F, Daum G Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism J Bacteriol 1993; 175: 2853–2858 Zweytick D, Leitner E, Kohlwein SD, Yu C, Rothblatt J, Daum G Contribution of Are1p and Are2p to steryl ester synthesis in the yeast Saccharomyces cerevisiae Eur J Biochem 2000; 267 (4): 1075–82 - 203 - Bibliography Name: Date of Birth: Zhang ShaoChong Gender: Male April 1974 Nationality: P.R.China Current position: Fellow, Department of Molecular and Cellular Biology, Harvard University, MA, USA Education Experience 2000-2006 PhD, Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore 1996-1999 Master of Science, Huazhong Agricultural Universityinese, Wuhan, China 1992-1996 Bachelor of Science, Wuhan University, Wuhan, China Working Experience 1999-2000 Research Fellow, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences Publication Shaochong Zhang, Jihui Ren, Hongzhe Li, Qian Zhang, Jeffery S Armstrong, Alan L Munn and Hongyuan Yang Ncr1p, the Yeast Ortholog of Mammalian Niemann Pick C1 Protein, is Dispensable for Endocytic Transport Traffic 2004; 5: 1017–1030 Zhang Q, Chieu HK, Low CP, Zhang SC, HengCK, Yang H Schizosaccharomyces pombe cells deficient in triacylglycerols synthesis undergo apoptosis upon entry into stationary phase J Biol Chem 2003; 278 (47): 47145–47155 Shaochong Zhang, Qian Zhang, Li Zheng, Jeffrey S Armstrong, Penghua Wang and Hongyuan Yang Acute glucose starvation reveals a critical role of Ncr1p in subcellular sterol transport in the yeast Saccharomyces cerevisiae Mol Biol Cell In preparation Stockinger W, Zhang SC, Trivedi V, Jarzylo LA, Shieh EC, Lane WS, Castoreno AB, Nohturfft A Differential Requirements for Actin Polymerization, Calmodulin, and Ca2+ Define Distinct Stages of Lysosome/Phagosome Targeting Mol Biol Cell 2006; 17(4): 1697–710 Trivedi V, Zhang SC, Castoreno AB, Stockinger W, Shieh EC, Vyas JM, Frickel EM, Nohturfft A Immunoglobulin G Signaling Activates Lysosome/Phagosome Docking Proc - 204 - Natl Acad Sci USA 2006; 103: 18226–18231 Trivedi V, Zhang SC, Stockinger W, Nohturfft A A Cell-Free Scintillation Proximity Assay for Studies on Lysosome/Phagosome Targeting Science STKE In preparation Invited paper, Co-first author - 205 - ... Abbreviations and Symbols Used ABCG ATP-binding cassette protein G gene subfamily ACAT acyl-CoA: cholesterol acyltransferase ALP alkaline phosphatase AMPK AMP-activated protein kinase APOE4 apolipoprotein... 35 - associates with ER membranes by interacting with VAMP associated protein (VAP, VAP -A and VAP-B) (Wyles et al., 2002) Both VAP -A and VAP-B (INSIG1 and 2) associate with SCAP, which interacts... CoA Acetyl CoA CoA-SH CoA-SH Acetyl CoA Acetoacetyl CoA Hydroxymethylglutaryl CoA NADPH+2 H+ NADP++CoA-SH ATP ATP ADP ADP 5-Pyrophosphomevalonate 5-Phosphomevalonate Mevalonate ATP ADP+Pi+CO2 Isopentenyl