Protein folding quality control in the endoplasmic reticulum in

PROTEIN FOLDING QUALITY CONTROL IN THE ENDOPLASMIC RETICULUM IN BUDDING YEAST XIE WEI (B. Sc., USTC) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENT I would like to express my deepest thanks to my supervisor A/Prof. Davis Ng for his professional guidance, his valuable insight and his stimulating discussion. I am extremely grateful for his constant support and encouragement through the course of study. Many thanks to my graduate committee members, Drs. Gregory Jedd, Naweed Naqvi and Yeong Foong May, for their helpful discussions and suggestions on this work. I also thank all current and previous members of Cell Stress and Homeostasis Group. Special thanks to Dr. Kazue Kanehara, for her help and contribution in the work in Chapter 3, and for the opportunity to participate in her exciting work in Chapter 4. I thank Ms. Wang Songyu and Dr. Ng Kian Hong for their critical readings of this thesis. I acknowledge Temasek Holdings for the financial support to my work. Finally, I would like to thank my family: my father, my mother, and my fiancée, Ms Yau Wing Tak, for their selfless support, for always being there for me through all these years. ii TABLE OF CONTENTS Title page i Acknowledgements ii Table of contents iii Summary vi List of figures ix List of tables xii List of abbreviations xiii List of publications xvi CHAPTER 1: Introduction 1.1 General introduction 1.1.1 Quality control in the cell 1.1.2 The secretory pathway 1.1.3 Quality control in the ER 1.1.4 Advantages for studying quality control in yeast 1.2 ER quality control machinery 1.2.1 Role of N-linked glycosylation in ERQC 6 1.2.1.1 The calnexin/calreticulin cycle 1.2.1.2 “Mannose timer” hypothesis 12 1.2.2 ER molecular chaperones 16 1.2.2.1 BiP/Kar2p 16 1.2.2.2 PDI 18 1.3 ER-associated protein degradation 19 1.3.1 ERAD depends on ubiquitin-proteasome system 20 1.3.2 Distinct ERAD complexes 21 1.3.2.1 The Hrd1p complex 22 1.3.2.2 The Doa10p complex 32 iii 1.3.2.3 Mammalian ERAD complexes 35 1.4 Objectives of the thesis 36 CHAPTER 2: Materials and methods 38 2.1 38 2.2 2.3 2.4 S. cerevisiae strains and genetic methods 2.1.1 List of strains used in this study 38 2.1.2 Media for culturing S. cerevisiae 38 2.1.3 Mating and sporulation of S. cerevisiae 38 2.1.4 Transformation of S. cerevisiae 46 2.1.4.1 Low efficiency plasmid transformation 46 2.1.4.2 Preparation of yeast competent cells 47 2.1.4.3 High efficiency DNA fragment transformation 47 Molecular biology methods 48 2.2.1 List of plasmids used in this study 48 2.2.2 List of oligonucleotide primers used in this study 48 2.2.3 Plasmid construction 48 2.2.4 Yeast genomic DNA extraction 66 Biochemistry methods 67 2.3.1 Antibody used in this study 67 2.3.2 TCA precipitation of yeast whole cell lysate 67 2.3.3 Western blot of yeast proteins 68 2.3.4 Cycloheximide-chase analysis 69 2.3.5 Cell labeling and immunoprecipitation 69 2.3.6 Yeast microsome preparation and native co-immunoprecipitation 70 2.3.7 Protease sensitivity assay 71 2.3.8 Preparation of yeast proteins for mass spectrometry 72 Cell biology and microscopy methods 73 2.4.1 Indirect immunofluorescence 73 2.4.2 Confocal microscopy 74 iv CHAPTER 3: Quality control of glycoproteins in the ER 76 3.1 Introduction 76 3.2 Results 78 3.2.1 A bipartite signal targets misfolded glycoproteins to ERAD 78 3.2.2 Local conformational perturbations activate non-signal glycans 89 for ERAD 3.2.3 The CPY ERAD determinant is recognized by the BiP/Kar2p 97 chaperon 3.2.4 Substrate signaling domains act as reporters of protein misfolding 3.3 Discussion 102 111 CHAPTER 4: Quality control of non-glycosylated proteins in the ER 118 4.1 Introduction 118 4.2 Results 120 4.2.1 Novel PrA variants reveal a third substrate class of the yeast 120 Hrd1p complex 4.2.2 The glycan-independent ERAD requires most but not all factors 125 of the Hrd1p complex 4.2.3 ngPrA∆295-331 competes with the glycan-dependent substrate 132 CPY* for degradation 4.2.4 The glycan-independent mode of Hrd1p pathway recognizes 132 distinct degradation signals 4.3 Discussion 138 CHAPTER 5: Conclusions and future directions 143 References 147 v SUMMARY Endoplasmic reticulum (ER) is the first membrane compartment of secretory pathway in eukaryotic cells. Newly synthesized proteins are translocated into ER lumen, and they are screened by endoplasmic reticulum quality control (ERQC) system. Only correctly folded and functional proteins can be sorted out to Golgi and later membrane compartments. Misfolded proteins are retained in the ER and turned over by a mechanism conserved from yeast to human known as endoplasmic reticulum-associated protein degradation (ERAD). While the mammalian system is less understood, the ERAD mechanism in yeast is explained in more detail, and it is shown to be centered on two membrane associated E3 ubiquitin ligases: Hrd1p and Doa10p. Previous studies suggested that Hrd1p ubiquitinates misfolded luminal proteins and membrane proteins with luminal lesions, while Doa10p targets membrane proteins with misfolded cytosolic domain. But how exactly the two ERAD E3s detects these lesions remains elusive. In this thesis, I have used Saccharomyces cerevisiae as a model organism to study the quality control of two classes of ER luminal proteins – N-linked glycoproteins and non-glycosylated proteins, both of which are ERAD substrates and degraded by Hrd1p when misfolded. In Chapter of this thesis, to study how misfolded N-linked glycoproteins are recognized by ERQC and ERAD, I started with analyzing two model substrates CPY* vi and PrA*. Both of these misfolded ER luminal proteins contain multiple N-linked glycans, but only one of them is necessary and sufficient for ERAD. Serial deletion analyses in neither CPY* nor PrA* identified ERAD determinant in the polypeptide primary sequences, suggesting the determinant might exist in higher order structures. I inspected the tertiary structure of wild type CPY and found the specific ERAD signal glycan is positioned on an 11-stranded β-sheet that is arranged mostly in parallel. This suggests that formation of the local structure adjacent the glycan is dependent on the overall folding of the polypeptide. Biochemical analysis of CPY* showed that the polypeptide region adjacent the ERAD signal glycan – termed bipartite ERAD signal, is tightly bound to Kar2p, a molecular chaperone in the ER lumen and essential component of the Hrd1p ERAD complex. Indeed the bipartite signal is as simple as a glycan attached to an unfolded/disordered structure. Consistent with this hypothesis, lesions introduced throughout CPY to specifically disrupt local structures surrounding non-ERAD glycans could efficiently report to ERAD through that designated glycan. Moreover, the position of the bipartite signal on a glycoprotein suggests a possible role in sensing the overall folding of the polypeptide. Normally the bipartite signal exists in a stable conformation buried into the tertiary structure of a folded glycoprotein to pass quality control. However should the protein misfold, the bipartite signal will remain disordered and exposed to ERQC and ERAD. In Chapter of this thesis, I described the study in collaboration with Dr. Kazue Kanehara (experiments done by Dr. Kazue Kanehara are indicated in respective figure vii legends) to decipher the mechanism for quality control of non-glycosylated proteins in the ER lumen. Similar to N-linked glycoproteins, non-glycosylated proteins also subject to ERQC, but the exact machinery responsible is largely unknown. In this chapter, Dr. Kazue Kanehara performed a comprehensive analysis to reveal the genetic requirements for ERAD of misfolded glycoprotein as well as non-glycosylated proteins. Although both depend on Hrd1p, glycoproteins require additional luminal factors for their degradation compared to non-glycosylated proteins. By systematic deleting primary sequence of non-glycosylated PrA* variant, I discovered a signal in the polypeptide chain both necessary and sufficient for its degradation, suggesting the glycan-independent route of Hrd1p ERAD pathway also operates in a signal-receptor based mechanism. viii LIST OF FIGURES Figures Pages Figure 1.1 Saccharomyces cerevisiae under microscopy Figure 1.2 Synthesis of N-linked oligosaccharide and its transfer to a polypeptide Figure 1.3 Regulation of calnexin/calreticulin cycle by 10 de-glucosylation and re-glucosylation enzymes Figure 1.4 Mannosidase-lectin signal-receptor system 13 Figure 1.5 Organization of the Hrd1p and Doa10p E3 complexes 24 for ERAD Figure 1.6 ERAD of luminal substrates by the Hrd1p complex 27 Figure 1.7 ERAD of membrane substrates by the Hrd1p complex 30 Figure 1.8 ERAD of membrane substrates by the Doa10p complex 33 Figure 3.1 Deletion variants of CPY* and PrA* are degraded 79 efficiently in wild type cells Figure 3.2 Ribbon diagram of mature CPY 82 Figure 3.3 Signal glycans and adjacent peptide segments are 84 sufficient to signal ERAD Figure 3.4 Glycan structure alone is not sufficient for ERAD 87 substrate recognition Figure 3.5 Glycan-proximal lesions are structural disruptive 90 ix Figures Figure 3.6 Pages Glycan-proximal lesions can generate artificial 92 ERAD determinants Figure 3.7 Glycan proximity is not a major determinant of 95 substrate recognition Figure 3.8 The peptide segments adjacent the CPY* signal 98 glycan are recognized by the chaperone BiP/Kar2p Figure 3.9 The CPY ERAD determinant can detect lesions 103 throughout the polypeptide Figure 3.10 Intracellular processing of CPY and PrA point mutants 106 Figure 3.11 The CPY and PrA signal glycans mark domains 108 broadly sensitive to structural defects Figure 3.12 Model of glycoprotein substrate recognition by the 115 Hrd1p complex Figure 4.1 Specific PrA* variants bypass the Htm1p requirement 121 for degradation Figure 4.2 ngPrA variants are substrates of the Hrd1p complex 123 Figure 4.3 The Kar2 chaperone is required for glycan-independent 126 ERAD Figure 4.4 ngPrA∆295-331 degradation requires multiple 130 components of the Hrd1 complex x endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure. 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The logic behind this seemingly energy-wasting effort is that each trimming product reports to the ER quality control system about the folding state of the nascent polypeptide chain 1.2.1.1 The calnexin/calreticulin cycle As soon as the core Glc3Man9GlcNAc2 oligosaccharide is attached to the emerging polypeptide in the ER lumen, the protein enters the calnexin/calreticulin... compartments of the secretory pathway (Barlowe, 2003) 1.1.3 Quality control in the ER The high throughput assembly line of ER protein synthesis will inevitably encounter a population of proteins that fail to acquire their native structure The ER must employ 3 a censoring system to search and detain these misfolded ones, otherwise allowing the malfunctioning proteins to slip through would be detrimental to the. .. Moreover, the advanced techniques in yeast genetics, as well as the availability of many mutant strains, makes the study in all much easier 1.2 ER quality control machinery 1.2.1 Role of N-linked glycosylation in ERQC N-linked glycoproteins constitute majority of secretory proteins among all eukaryotes The N-linked oligosaccharides are presynthesized on the ER membrane and then added to proteins all... calnexin/calreticulin cycle Calnexin (CNX) is a type I transmembrane protein, while calreticulin (CRT) is a luminal protein, and together these two lectins act as the first stage of the ER quality control system (Caramelo and Parodi, 2008; Williams, 2006) Association of the nascent polypeptide chain with the CNX/CRT requires the sequential trimming of the outmost two glucose residues on branch A of the glycan... problematic to the ER itself, making the removal of them critical Therefore the cells introduced another set of mechanism to deal with proteins deemed terminally misfolded by ERQC The misfolded proteins are first extracted out of the ER by a transmembrane complex - a process called retro-translocation On the cytosolic face of the ER membrane, the misfolded protein is poly-ubiquitinated and degraded by the 26S... further strengthens the idea that ERQC is a highly regulated mechanism dependent on not only a variety of components but also the interplay among them 1.3 ER-associated protein degradation All the efforts ERQC puts in to retain misfolded and malfunctioning proteins is to prevent them from trafficking out of the ER and messing up normal functions 19 elsewhere But the accumulation of misfolded proteins... reported to cooperate in assisting the folding of nascent polypeptides (Mayer et al., 2000a) In an in vitro folding of antibody Fab fragment, BiP can bind the antibody chain and expose it so that PDI is 18 able to access the cysteine residue side chains Without BiP, the unfolded polypeptide chain aggregates rapidly therefore PDI is unable to rearrange the disulfide bond necessary for its folding Althought... assembled into oligomers can exit the ER (Gething et al., 1986), whereas the misfolded species are bound to ER resident chaperon BiP (immunoglobulin heavy chain binding protein) and retained (Hurtley et al., 1989) Similar results were found in the study on vesicular stomatitis virus G protein, and it is during that time de Silva and coworkers first gave this system its name: endoplasmic reticulum quality control. .. allowing the polypeptide for another round of folding attempt in the cycle 11 1.2.1.2 “Mannose timer” hypothesis After the glucose residues are removed from the core oligosaccharide, the nascent polypeptide with Man9GlcNAc2 glycans emerging from the translocon in yeast (or after the polypeptide chain is released from CNX/CRT cycle in mammalian cells), it is subject to another important ER quality control. .. Mns1p The folding state of the glycoprotein is then examined by ER quality control mechanism Those deemed as irreversibly misfolded, will have another mannose residue cleaved by the Htm1p/PDI complex The end product, Man7GlcNAc2, exposes an α1,6-linked mannose residue in its structure Lectin receptor Yos9p recognizes this mannose residue with the specific α1,6-linkage, and commits the misfolded glycoprotein . PROTEIN FOLDING QUALITY CONTROL IN THE ENDOPLASMIC RETICULUM IN BUDDING YEAST XIE WEI (B. Sc., USTC) A THESIS SUBMITTED FOR THE DEGREE. 1: Introduction 1 1.1 General introduction 1 1.1.1 Quality control in the cell 1 1.1.2 The secretory pathway 2 1.1.3 Quality control in the ER 3 1.1.4 Advantages for studying quality control. mechanism for quality control of non-glycosylated proteins in the ER lumen. Similar to N-linked glycoproteins, non-glycosylated proteins also subject to ERQC, but the exact machinery responsible