Characterization and function studies of ncr1p a yeast ortholog of mammalian niemann pick c1 protein (NPC1)

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 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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