THE SECRETORY PATHWAY USES MULTIPLE MECHANISMS FOR PROTEIN QUALITY CONTROL WANG SONGYU (B.Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I am deeply grateful for my supervisor, Dr Davis Ng, for his valuable guidance and support throughout the course of this work. His wide knowledge and interesting ideas have never failed to impress me. His constant encouragement has always given me full confidence and is one of the major driving forces for me to complete my PhD. I would like to thank my thesis committee, Dr Wanjin Hong, Dr Snezhana Oliferenko and Dr Cynthia He for their valuable comments and suggestions. Special thanks are also given to Dr Graham Wright and Cristiana Barzaghi for their great help on confocal microscopy and for their patience to answer my numerous questions. Many thanks to all the past and current members of Davis’ lab, especially Dr Guillaume Thibault, Dr Kazue Kanehara, Dr Nurzian Ismail and Dr Chia Ling Hsu. All of them have taught me how to be a good scientist and they are always there when I need help. I would like to express my appreciation to Rupali, Chengchao, Alisha, Dr Shinichi Kawaguchi, Liu Ying, Sylvia, Gerard, Yu Jun, Sandy and Jeremy for stimulating scientific discussions and friendship. Thanks also go to Hong Xin, Jing Jing, Xue Jing, Lu Song, Anbu and Sook Keat for our happy friendships. Last but not least, I dedicated this thesis to my beloved husband Seng Kah and parents for their love, support, help and encouragement throughout these years. Without them, I could not have completed my PhD smoothly. i TABLE OF CONTENTS SUMMARY . v LIST OF TABLES vii LIST OF FIGURES . viii LIST OF VIDEOS viii LIST OF SYMBOLS AND ABBREVIATIONS . xi LIST OF PUBLICATIONS xiii Chapter Introduction . 1.1 Quality control in the ER 1.1.1 ER retention of misfolded and unassembled proteins 1.1.1.1 By BiP and other molecular chaperones . 1.1.1.2 Thiol-mediated retention . 1.1.1.3 By chaperone-like molecule Rer1p . 1.1.2 Substrate recognition during ERAD 1.1.3 Classification of ERAD pathways . 1.1.4 Retrotranslocation and degradation by the ubiquitin-proteasome system 11 1.1.5 Degradation of endogenous proteins . 12 1.2 Balance among folding, ER export and quality control 13 1.2.1 ER export . 13 1.2.1.1 COPII coat formation 14 1.2.1.2 Signals in transmembrane cargoes 16 1.2.1.3 Export of soluble cargoes from the ER and transmembrane sorting receptors 17 1.2.1.4 Packaging chaperones that modulate ER exit . 19 1.2.1.5 Oligomeric assembly 20 1.2.2 Competition between ER export and ER retention for misfolded proteins . 21 1.3 Post-ER quality control . 23 1.3.1 Substrate recognition in the Golgi apparatus . 24 1.3.1.1 Receptor-mediated mechanism . 24 1.3.1.2 A Golgi environment-specific recognition . 27 1.3.1.3 Golgi modifications mark mutant proteins abnormal . 28 1.3.1.4 Aggregation in the Golgi lumen . 29 1.3.2 Plasma membrane quality control 29 1.4 The ESCRT machinery and the multi-vesicular bodies 31 1.4.1 Function of MVBs in the biosynthetic and endocytic pathway . 31 1.4.2 The multivesicular body biogenesis requires ESCRT complexes . 34 1.5 Ubiquitin signals in the biosynthetic and endocytic pathways . 39 ii 1.5.1 Ubiquitin-dependent endocytosis . 40 1.5.2 Ubiquitin as a signal for MVB sorting . 42 1.5.3 Ubiquitin-dependent sorting at the trans-Golgi network . 42 1.5.4 Deubiquitinating enzymes . 43 1.6 Wsc1p as a model substrate 43 1.7 Thesis objectives . 47 Chapter Materials and methods . 50 2.1 S. cerevisiae strains and growth media . 50 2.1.1 List of strains 50 2.1.2 Growth media . 50 2.2 Genetic and molecular methods 50 2.2.1 Yeast transformation 50 2.2.1.1 Plasmid transformation via a simple and rapid way . 50 2.2.1.2 High efficiency DNA fragment transformation 51 2.2.2 Strain construction via mating, sporulation and tetrad dissection . 51 2.2.3 Yeast genomic DNA extraction . 52 2.3 Plasmid construction . 52 2.3.1 Site-directed mutagenesis 55 2.3.2 Oligonucleotide primers used in this study 55 2.4 Protein biochemistry and cell biology 55 2.4.1 Antibodies 55 2.4.2 SDS-PAGE and immunoblot analysis . 56 2.4.3 Preparation of yeast extracts 56 2.4.4 Co-immunoprecipitation 57 2.4.5 Cell labeling and Immunoprecipitation analysis 58 2.4.5.1 Metabolic pulse-chase analysis and denaturing immunoprecipitation . 58 2.4.5.2 PEGylation-based protein-folding assay . 59 2.5 Microscopy . 60 2.5.1 Indirect immunofluorescence . 60 2.5.2 Live cell imaging . 61 Chapter Golgi quality control captures misfolded Wsc1 proteins that evade ERQC . 69 3.1 Introduction . 69 3.2 Wsc1p variants are misfolded . 71 3.2.1 Wsc1p variants are transported from the ER to the Golgi via COPII vesicles 71 3.2.2 All Wsc1p variants are grossly misfolded . 74 3.3 Misfolded Wsc1p is an obligate substrate of Golgi quality control 79 3.3.1 The variants are subject to protein quality control . 79 3.3.2 Wsc1p variants are degraded independent of ERAD 80 3.3.3 Misfolded Wsc1p traffics to the vacuole for degradation 84 3.3.4 The degradation is autophagy independent 87 3.3.5 Golgi quality control recognizes misfolded Wsc1p . 88 iii 3.4 Misfolded Wsc1p evades ER surveillance 91 3.4.1 ERQC does not recognize Wsc1p variants when they are retained in the ER . 91 3.4.2 Wsc1p lacks an ERAD determinant in its luminal domain . 95 3.4.3 ER chaperone Kar2p does not recognize misfolded Wsc1p 100 3.5 Discussion . 105 3.5.1 Reported substrates involved in Golgi QC 105 3.5.2 The machinery of Golgi QC 107 3.5.3 ER retention of soluble misfolded Wsc1p . 110 3.5.4 ER export of misfolded proteins 111 3.5.5 Poor recognition of misfolded Wsc1p by Kar2p . 112 Chapter The multi-vesicular body pathway is essential in the complete degradation of misfolded membrane proteins in Golgi quality control . 113 4.1 Introduction . 113 4.2 Misfolded Wsc1 proteins are degraded in the vacuolar lumen . 116 4.3 ESCRT mutants alter the vacuolar localization pattern of misfolded Wsc1p 122 4.4 The MVB pathway is essential for complete degradation of misfolded Wsc1p128 4.5 Re-routing of misfolded Wsc1p to the plasma membrane in ESCRT mutants 131 4.6 Entry of misfolded Wsc1p into the MVB pathway is ubiquitination dependent . 133 4.7 Discussion . 141 4.7.1 The dual functions of the cytoplasmic domain of Wsc1p 143 4.7.2 The cell surface re-routing pathway 143 4.7.3 The ubiquitination of misfolded Wsc1p 144 4.7.4 The importance and physiological relevance of the MVB pathway in protein quality control . 145 Chapter Conclusions and future perspectives . 148 REFERENCES 151 iv SUMMARY Quality control (QC) mechanisms monitor the folding and assembly of newly synthesized proteins. The most well characterized QC pathway occurs in the ER and is termed ER quality control (ERQC) which targets misfolded proteins to be degraded via ERassociated degradation (ERAD). Post-ER QC pathways, albeit poorly understood, function to capture proteins that exit the ER prematurely. In our study, we reported a yeast plasma membrane protein Wsc1p to be a substrate that demonstrates the fundamental role of the Golgi in protein QC. A panel of Wsc1p variants misfolded in the extracellular/luminal domain was generated. The variants are degraded in an ERADindependent pathway. Instead, they traffic to the Golgi from where they are delivered to the vacuole for degradation. Two reasons can account for the ERQC evasion of Wsc1p. First, a strong export signal in the cytoplasmic domain renders its efficient ER exit whether it is folded or not and whether it contains an ERAD determinant. Second, the luminal domain of Wsc1p lacks functional ERAD signals and a chaperone binding site. The identification and characterization of Wsc1p as an endogenous and obligate substrate reinforces the importance of the Golgi QC as a primary surveillance mechanism in the secretory pathway and provides a physiological basis for its existence. Golgi QC generally recognizes misfolded proteins in the Golgi apparatus and targets them to the vacuole/lysosome for degradation. For misfolded membrane proteins, there are two fates. They can be localized to either the limiting vacuolar/lysosomal membrane or the lumen. To understand how Golgi QC delivers its misfolded membrane proteins to v the vacuole, we examined Wsc1p variants with a misfolded luminal domain that are bona fide substrate of Golgi QC. We found that the mutants are transported from the Golgi to the vacuolar lumen via the multi-vesicular body (MVB) pathway. MVB sorting requires ubiquitination at the lysine residue(s) in the cytoplasmic domain of misfolded Wsc1p and the endosomal sorting complex required for transport (ESCRT) machinery. Most importantly, mislocalization of the variants at the limiting vacuolar membrane results in a series of degradation fragments suggesting incomplete elimination. This provides a physiological basis for the vacuolar lumen targeting of misfolded membrane substrates in Golgi QC. It ensures efficient degradation of the entire molecules and prevents the accumulation of potentially toxic fragments. vi LIST OF TABLES Table 1.1 ER export signals in transmembrane and soluble cargoes 17 Table 1.2 Transmembrane cargoes of the MVB pathway. . 33 Table 2.1 Strains used in chapter . 62 Table 2.2 Strains used in chapter . 64 Table 2.3 Plasmids modified by site-directed mutagenesis in chapter 66 Table 2.4 Plasmids used in chapter 67 Table 2.5 Oligonucleotide primers used in chapter . 68 Table 2.6 Oligonucleotide primers used in chapter . 68 Table 4.1 Effect of ESCRT mutants on the localization of Wsc1-L63R-GFP . 127 vii LIST OF FIGURES Figure 1.1 ERAD recognition of misfolded N-glycosylated proteins. . Figure 1.2 ERAD pathways in yeast . 10 Figure 1.3 Bidirectional transport between the ER and Golgi apparatus. 14 Figure 1.4 COPII vesicle formation 15 Figure 1.5 Model for post-ER quality control. . 23 Figure 1.6 The ubiquitination pathway . 27 Figure 1.7 The ESCRT machinery 38 Figure 1.8 Molecular mechanism of MVB biogenesis. 38 Figure 1.9 A cartoon depicting Wsc1p. 46 Figure 1.10 O-mannosylation in yeast and mammalian cells. 47 Figure 3.1 Generation of Wsc1p variants. 72 Figure 3.2 Wsc1p and its variants show similar mobility . 73 Figure 3.3 The principle of the PEGylation-based protein folding assay . 76 Figure 3.4 CPY* is grossly misfolded. . 77 Figure 3.5 Wsc1p variants are misfolded. 78 Figure 3.6 Wsc1-L63R is degraded rapidly 79 Figure 3.7 Wsc1-L63R is degraded independent of ERAD. 81 Figure 3.8 Degradation of Wsc1p variants does not require ERAD . 82 Figure 3.9 Wsc1-L63R is degraded independent of the proteasome. . 83 Figure 3.10 Wsc1-L63R is transported to the vacuole for degradation 84 Figure 3.11 Misfolded Wsc1p degrades in the vacuole 85 Figure 3.12 The stabilization results in strong vacuolar staining of misfolded Wsc1p in ∆pep4 cells. . 86 Figure 3.13 Visualization of the vacuolar ATPase by indirect immunofluorescence. . 86 Figure 3.14 Wsc1-L63R is not degraded via the autophagy pathway. . 87 Figure 3.15 Wsc1-L63R is transported to the vacuole via the Golgi 89 Figure 3.16 Misfolded Wsc1p is degraded by Golgi QC 90 Figure 3.17 Wsc1p mutants are stabilized when the transport from the ER to the Golgi is blocked 92 Figure 3.18 Generation of the soluble version of misfolded Wsc1p. . 93 Figure 3.19 The soluble forms of Wsc1p variants are retained in the ER. . 93 Figure 3.20 Wsc1-L63RLuminal and Wsc1-68-80Luminal are stable in the ER 94 viii Figure 3.21 The slight mobility shift of Wsc1-L63RLuminal and Wsc1-∆68-80Luminal is due to ER modifications. . 94 Figure 3.22 An ERAD determinant is appended to Wsc1p variants. . 97 Figure 3.23 ED-Wsc1-L63R and ED-Wsc1-∆68-80 are ERAD substrates 98 Figure 3.24 ED-Wsc1-L63RLuminal and ED-Wsc1-∆68-80Luminal are completely dependent on ERAD for degradation. 99 Figure 3.25 Misfolded Wsc1p is not recognized by the major ER chaperone BiP/Kar2p. . 103 Figure 3.26 Misfolded Wsc1p fused with an ERAD determinant binds Kar2p efficiently. . 104 Figure 3.27 Degradation of misfolded Wsc1p is partially Vps10p dependent. 109 Figure 4.1 Wsc1-L63R is localized to the vacuolar lumen in the ∆pep4 strain 118 Figure 4.2 (PGAS1)Wsc1-L63R behaves similarly to (PPRC1)Wsc1-L63R. 120 Figure 4.3 Wsc1-∆68-80 is localized to the vacuolar lumen in ∆pep4 cells. . 121 Figure 4.4 ESCRT proteins are essential in transporting Wsc1-L63R-GFP to the vacuolar lumen . 124 Figure 4.5 Wsc1-L63R-GFP is degraded in a Pep4p-dependent manner . 124 Figure 4.6 ESCRT mutants alter the localization of Wsc1-L63R. . 125 Figure 4.7 The ESCRT mutants affect the localization of Wsc1-∆68-80 . 126 Figure 4.8 Misfolded Wsc1p is degraded into multiple fragments in ESCRT mutants. 129 Figure 4.9 ESCRT mutants affect the degradation of (PGAS1)Wsc1-∆68-80. . 130 Figure 4.10 Misfolded Wsc1p is re-routed to the plasma membrane in ESCRT mutants. . 132 Figure 4.11 Wsc1-L63R is ubiquitinated by Rsp5p before entry into the MVB pathway. . 136 Figure 4.12 The entry of Wsc1-∆68-80 into the MVB pathway requires Rsp5p. 137 Figure 4.13 Ubiquitination at the lysine residue(s) of Wsc1-L63R provides the MVB sorting signal. 139 Figure 4.14 Wsc1-∆68-80-3R is not sorted to the vacuolar lumen via the MVB pathway. . 140 Figure 4.15 Model of the MVB-dependent pathway for the transport of misfolded Wsc1p. . 142 Figure 5.1 The basis of the genetic screen using invertase-misfolded Wsc1p fusion protein. 150 ix Bue, C. 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J Cell Biol 153, 1187-1198. 168 [...]... of millions annually 1.1 Quality control in the ER In eukaryotic cells, secretory and transmembrane (TM) proteins enter the endoplasmic reticulum (ER) for maturation To ensure that only properly folded and assembled proteins are exported, mechanisms collectively termed ER quality control (ERQC) allow only properly folded proteins to be transported to their sites of functions Misfolded or unassembled... proteins in the ER Once the protein is folded, it is released from BiP/Kar2p and transported to its destination Prolonged interaction with BiP/Kar2p leads to ERAD After discovery of the interaction between BiP and unassembled HCs, it was subsequently found that other chaperones interact with misfolded or unassembled proteins after BiP interaction One example is GRP94, an Hsp90 chaperone BiP first associates... in QC Althernative, it is possible that their conformation is somehow not “seen” as misfolded by the QC system In either case, this results in poor degradation and the onset of neurodegenerative diseases 1.2 Balance among folding, ER export and quality control 1.2.1 ER export Properly folded secretory proteins are ready for ER export in order to be delivered to sites of function From the ER, the coat... recycles between ER and post- ER compartments (the ER- Golgi intermediate compartment [ERGIC] and cis-Golgi) (Anelli et al., 2007; Gilchrist et al., 2006; Wang et al., 2007) In the cis-Golgi, ERp44 captures unpolymerized IgM subunits which are capable of ER exit and retrieves them back to the ER in an RDEL-dependent manner (Anelli et al., 2002; Anelli et al., 2003; Anelli et al., 2007) 4 Another substrate... adipocytes (Scherer et al., 1995) Plasma adiponectin can form trimers, hexamers and oligomers (Bobbert et al., 2005; Lara-Castro et al., 2006; Tonelli et al., 2004) ERp44 retains folded adiponectin trimers by forming mixed disulfides with Cys39 in one of the subunits Ero1-Lα, an oxidoreductase in the ER lumen releases adiponectin from ERp44 and facilitates secretion of adiponectin oligomers (Qiang et... thyroglobulin interacts with calnexin first, followed by BiP (Kim and Arvan, 1995) The difference in the interaction order could be due to specific structural features of substrates In addition to the sequential action between BiP and other chaperones, BiP is able to act synergistically with chaperones like PDI (protein disulfide isomerase) in retaining misfolded ERAD substrates in the ER BACE457 is a... Another similar example is the complex of Erv41p and Erv46p ensures ER export despite the presence of ER exit motifs in both proteins (Otte and Barlowe, 2002) Together, these studies suggest that assembly acts together with the ER retention machinery to exclude unassembled proteins from COPII vesicles This in turn explains why proper assembly is an important element in ERQC 1.2.2 Competition between ER. .. export and ER retention for misfolded proteins The fundamental principle of ERQC is to ensure only properly folded and assembled proteins are allowed to traffic to their final destinations However, some misfolded proteins are capable of ER exit with a functional ER export signal despite active ER retention mechanisms imposed on them The classical ERAD substrate CPY* contains a Kar2p binding site where active... acid permeases like Gap1p The interaction between Shr3p and the COPII coat delivers permeases into transport vesicles and Shr3p itself is not packaged into (Gilstring et al., 1999) In ∆shr3 cells, amino acid permeases aggregate, accumulate in the ER and are degraded by ERAD (Kota and Ljungdahl, 2005; Kota et al., 2007) Another ER membrane protein Gsf2p is required for export of hexose transporters from... degraded by ERAD (Hill and Cooper, 2000) Similar examples are observed in higher eukaryotes A Drosophila protein, NinaA, seems to function as an isomerase and regulates ER export of rhodopsin (Baker et al., 1994; Colley et al., 1991) The ER membrane proteins BAP31 and BAP29 promote ER export of secretory proteins such as MHCI (major histocompatibility complex class I) and cellubrevin (Annaert et al., . Packaging chaperones that modulate ER exit 19 1.2.1.5 Oligomeric assembly 20 1.2.2 Competition between ER export and ER retention for misfolded proteins 21 1.3 Post- ER quality control 23 1.3.1. Chapter 3 Golgi quality control captures misfolded Wsc1 proteins that evade ERQC 69 3.1 Introduction 69 3.2 Wsc1p variants are misfolded 71 3.2.1 Wsc1p variants are transported from the ER. All Wsc1p variants are grossly misfolded 74 3.3 Misfolded Wsc1p is an obligate substrate of Golgi quality control 79 3.3.1 The variants are subject to protein quality control 79 3.3.2 Wsc1p