Mechanism of protein quality control in the cytosol in budding yeast

MECHANISM OF PROTEIN QUALITY CONTROL IN THE CYTOSOL IN BUDDING YEAST RUPALI PRASAD (M.Sc, IIT Bombay) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENTS This research work is by far one of the most significant scientific accomplishments in my life and it would have been impossible without the following people, who supported me and had belief in me. First and foremost, I want to express my wholehearted gratitude to my mentor and research advisor Associate Professor Davis Ng, for his expert guidance and motivation throughout my research work. I am grateful to him for his invaluable support and also for introducing me to the wonderful and interesting field of protein quality control. I would also like to express my sincere thanks to Dr. Shinichi Kawaguchi and Ms Alisha for assisting me and being a part in the projects. I am also thankful to Dr Kazue Kanehara, Dr Guillaume Thibault, and Songyu Wang for fruitful discussions and suggestions. I owe very special thanks to all current and previous members of Cell Stress and Homeostasis Group, especially Dr Nurzian Ismail, Dr. Chia Ling Hsu and Dr. Xie Wei and to all my friends at TLL. I want to thank them for all their help, support, interest and valuable hints. I gratefully acknowledge the financial support rendered by the National University of Singapore in the form of Research Scholarship. I am also grateful to the academic and technical staffs at the Temasek Life Sciences Laboratory who have helped me in one way or the other in my research work. i Above all, I want to thank my family, which continuously supported me at all times. I thank my parents for teaching me the value of education at a young age and instilled in me a desire for higher education. I wish to thank my sisters for their love and support. Words cannot express the love, encouragement and unequivocal support I received from my beloved husband Anil without whose constant help and support, my PhD. research work would have remained a daydream. The loving family environment and support I enjoyed from all my family members was greatly instrumental in providing me the tranquility and enthusiasm to pursue my research with a piece of mind. ii TABLE OF CONTENTS Acknowledgements i Table of contents iii Summary vii List of tables ix List of figures x List of abbreviations xii List of publications xiv CHAPTER 1: Introduction 1.1 Protein quality control (PQC) 1.2 ER quality control 1.2.1 Role of ER-lumenal chaperones 1.2.2 Recognition of ERAD substrate 1.2.3 ERAD Complex 1.2.4 Retrotranslocation and the Cdc48 complex 1.3 Cytosolic protein quality control 10 1.3.1 Recognition of damaged proteins and repair mechanism 10 1.3.2 Transcriptional regulation 13 1.3.3 Autophagy-lysosome system 14 1.3.4 Sequestration into large aggregates 15 1.3.5 Degradation of misfolded proteins by UPS 16 1.4 Ubiquitin-proteasome system 18 1.5 26S proteasome 20 1.5.1 Substrate recognition by 26S proteasome 20 1.5.2 Substrate unfolding, translocation and proteolysis 21 1.5.3 Cellular localization of 26S proteasome 21 1.6 Structure and function of selected cytosolic chaperones and cochaperones 23 1.6.1 Hsp70/Hsc70 23 1.6.2 Hsp40 26 1.6.3 Nucleotide Exchange Factors (NEFs) 28 iii 1.6.4 Hsp90 29 1.7 Objectives of the thesis 31 CHAPTER 2: Materials and methods 33 2.1 S. cerevisiae strains, growth media and genetic techniques 33 2.1.1 List of S. cerevisiae strains 33 2.1.2 Media for culturing yeast 36 2.1.3 Mating, sporulation and tetrad dissection 36 2.1.4 Yeast transformation 37 2.1.4.1 Low efficiency plasmid transformation via simple and rapid way 2.1.4.2 High efficiency transformation using lithium acetate 2.1.5 Serial dilution-spotting growth assay 2.2 Molecular biology techniques 37 37 38 38 2.2.1 List of plasmids used in this study 38 2.2.2 List of oligonucleotide primers used in this study 39 2.2.3 Plasmids construction 40 2.2.4 Yeast genomic DNA extraction 43 2.2.5 Plasmid DNA extraction from yeast 44 2.3 Biochemical and immunological techniques 44 2.3.1 Antibodies used in this study 44 2.3.2 TCA precipitation of yeast whole Cell Lysate 44 2.3.3 SDS-PAGE and western blot analysis 45 2.3.4 Cycloheximide chase assay 46 2.3.5 Cytosol/membrane fractionation 46 2.3.6 Ubiquitination assay 46 2.3.7 Trypsin sensitivity assay 47 2.3.8 Co-immunoprecipitation Assays 47 2.3.9 TAP-tagged Hsp70 pulldown assay 48 2.3.10 Cell labeling and Immunoprecipitation analysis 48 2.3.10.1 Pulse chase assay 48 2.3.10.2 Immunoprecipitation 49 iv 2.4 Microscopy techniques 50 2.4.1 Indirect immunofluorescence 50 2.4.2 Live cell imaging 51 2.5 Genetic screening method used in this study 51 2.5.1 UV mutagenesis 51 2.5.2 Primary selection 52 2.5.3 Secondary screen for CytoQC defect 53 2.5.4 Cloning of QCC genes 54 CHAPTER 3: A nuclear-based quality control mechanism for cytosolic proteins 55 3.1 Introduction 55 3.2 The model cytosolic substrate ΔssCPY* is degraded by CytoQC and ERAD 57 3.3 DssPrA and D2GFP are misfolded proteins and degraded by proteasome 61 3.3.1 Novel CytoQC substrates are highly unstable 61 3.3.2 DssPrA and D2GFP are bona fide substrates for CytoQC 63 3.4 Misfolded cytosolic proteins traffic into the nucleus for degradation 65 3.5 San1p-dependent pathway is a general mechanism of CytoQC substrates 3.5.1 Nuclear E3 ligase San1p is required for degradation 67 67 3.5.2 CytoQC substrates polyubiquitination is dependent on E3 ligase San1p 69 3.6 San1p can interact with CytoQC substrate in vivo 70 3.7 San1p pathway is a constitutive mechanism of CytoQC 74 3.8 CytoQC substrates are polyubiquitinated and degraded inside the nucleus 76 3.8.1 Substrate degradation is independent of nuclear export 76 3.8.2 Nucleus is the site for CytoQC substrates degradation 77 3.9 E3 ligase Doa10p is not required for degradation of DssPrA and D2GFP 78 3.10 Ubr1p augments, but is not required for, DssPrA and D2GFP degradation 79 CHAPTER 4: Roles of molecular chaperones in the cytosolic quality control 85 4.1 Introduction 85 4.2 The Hsp70 chaperone machinery is essential for the degradation of cytosolic misfolded proteins 87 v 4.3 The Hsp70 chaperone can interact directly with cytosolic misfolded proteins in vivo 90 4.4 The Hsp70 chaperone system is required for efficient nuclear transport of cytosolic misfolded proteins 4.5 Effect of temperature on CytoQC substrate localization 92 93 4.6 The Hsp70 co-chaperone Ydj1p is directly involved in nuclear import of cytosolic misfolded proteins 4.7 Nucleotide exchange factor Sse1p is essential for CytoQC pathway 94 97 4.8 Lack of Hsp90 inhibit substrate degradation and has paltry effect on nuclear import 99 4.9 Discussion 101 CHAPTER 5: A genetic screen to identify genes required for cytosolic quality control 5.1 Introduction 106 106 5.2 Folding state of proteins in the cytosol or membrane tethered forms, are monitored by different PQC pathways 5.3 Ste6C requires all the factors of cytosolic quality control 107 112 5.4 Genetic screen to identify new components required for cytosolic quality control pathway 116 5.5 The qcc mutants are defective in cytosolic quality control pathway 121 5.6 Cloning of QCC genes by complementation 123 5.7 Identification of new candidates in CytoQC pathway 123 5.8 Discussion 128 CHAPTER 6: Conclusions and future directions 132 References 135 vi SUMMARY Intracellular quality control systems monitor protein conformational states. Irreversibly misfolded proteins are cleared through specialized degradation pathways. Their importance is underscored by numerous pathologies caused by aberrant proteins. In the cytosol, where most proteins are synthesized, quality control remains poorly understood. Stress-inducible chaperones and the 26S proteasome are known mediators but how their activities are linked is unclear. In this thesis, I have used Saccharomyces cerevisiae as a model organism to study the quality control of cytosolic misfolded proteins. To better understand quality control of cytosolic proteins in chapter and of this thesis, a panel of model misfolded substrates was analyzed in detail. Surprisingly, their degradation occurs not in the cytosol but in the nucleus (Prasad et al., 2010). Degradation is dependent on the E3 ubiquitin ligase San1p, known previously to direct the turnover of damaged nuclear proteins (Gardner et al., 2005). San1p, however, is not required for nuclear import of substrates. Two reasons can account for nuclear trafficking of misfolded cytosolic proteins. First, in S. cerevisie, nucleus accounts for over 80 % of proteasomes at steady state throughout the cell cycle, suggesting the requirement of nuclear import of misfolded cytosolic proteins. Second, by trafficking misfolded proteins in the nucleus, cells provide enough time for newly synthesized proteins to fold in proper conformation. One view asserts that a key strategy of protein quality control is the integration of timing devices to permit folding (Helenius and Aebi, 2004). As such, proteins failing to fold within a set window are targeted for degradation. Experimental precedence comes from ERAD studies where a sophisticated timing mechanism utilizes a series of glycosidases to set a time limit for folding (Clerc et al., 2009). Proteins still unfolded vii after the final trimming step by Htm1p are detected by the Yos9p ERAD factor (OS-9 in mammals), which binds the resulting glycan signal (Quan et al., 2008). In CytoQC, nuclear import of substrate can provide an analogous function. The detailed analyses of cytosolic substrates have provided a clue that the Hsp70 family proteins Ssa1p and Ssa2p and its co-chaperone Ydj1p are needed for efficient import and degradation. In chapter of this thesis, I have described a genome wide genetic screen to identify the genes involved and to decipher the mechanism for quality control of cytosolic protein. Among the genes identified, there are genes that encodes for proteasomal subunits (RPN7 and RPN11) and UMP1, a chaperone required for assembly of 26S proteasome. 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According to older model, chaperones would be primarily involved in the stabilizing and refolding of non-native polypeptides This means that, the primary role of chaperones in the quality control is just to maintain the solubility of misfolded intermediates and facilitate the sampling by the ubiquitination machinery However, recent analysis of the quality control mechanisms of mutant von Hippel-Lindau... remove the polyubiquitin chain from the substrates (Hirsch et al., 2009) 9 1.3 Cytosolic quality control Protein misfolding in cytosol is toxic to cells and the accumulated toxic proteins can lead to protein misfolding diseases (For examples, Parkinson s and Alzheimer s diseases) Misfolding of proteins can expose hydrophobic surfaces that result in unnecessary binding to normal proteins which disrupt the. .. folded proteins and sends them to their final destination Proteins which misfold or unfold, are recognized by the quality control system in the ER which retain and refold them (Ellgaard and Helenius, 2003) Accordingly, ER quality control mechanisms have the added responsibility to control trafficking, to prevent the premature exit of folding intermediates (Vembar and Brodsky, 2008) For proteins that... representing the strategy taken to obtain genes involved in cytosolic quality control 118 Figure 5.6 Stabilization of Ste6C-Ura3p fusion protein in qcc mutant strains 119 Figure 5.7 The qcc mutant strains can also stabilize other cytosolic quality control substrates Figure 5.8 Introduction of complementary gene restores the cytosolic quality control function Figure 5.9 122 125 Introduction of complementary... activated and translocate into the nucleus as trimers This induced HSF1 binds to a specific cis-acting element in the promoter region of the heat shock response element (HSE) of stress-responsive genes (Shi et al., 1998) The binding induces several factors; one of them being ubiquitin expression indicating its role in the regulation of the degradation system Under normal conditions, in the cytosol, HSF1 monomer... structure, in turn is recognized by glycan binding protein Yos9p (yeast osteosarcoma 9) (Quan et al., 2008) Recent studies have shown that Yos9p binds misfolded proteins but is unable to interact with their folded counterparts Yos9p is part of the Hrd1 complex, as it directly interacts with the large luminal domain of Hrd3p, indicating that it can links the recognition of misfolded glycoproteins to the ubiquitin-proteasome... essential interactions between cellular proteins To avoid this situation, quality control systems present in the cytosol monitor protein folding and remove misfolded proteins in the cytosol Hydrophobic patches of misfolded proteins are recognized by molecular chaperones that mask them and transfer the misfolded species to the ubiquitin-proteasome system and chaperone-mediated autophagy to eliminate them The. .. eliminate them The entire quality control systems in cytosol are regulated by stress-inducible transcription factors, molecular chaperones and other factors for the effective elimination of toxic proteins 1.3.1 Recognition of damaged proteins and repair mechanism Recognition of non-native cytosolic proteins is the first step towards their elimination, which rely mainly on the interaction between chaperones... interactions of Hsp70 with exposed hydrophobic patches (Zhu et al., 1996; Rüdiger et al., 1997) Binding of substrates with Hsp70, not only prevent protein aggregation but it also assist in the folding of proteins through one or several ATPase cycle of binding and release and also promote the disaggregation of proteins with the help of another chaperone family Hsp100 (Diamant et al., 2000; Goloubinoff... subunits 1-2) Multiubiquitin binding protein Rpn10 links the cap to base The proteolytic core 20S is composed of four heptameric rings ( 7 7 7 7) (Rechsteiner and Hill, 2005) The outer ring is composed of -subunits and the inner 2 rings are made up of -subunits -subunits are the sites for the binding of various regulatory factors, entry and exit of substrates, while -subunits harbour the catalytic site, . MECHANISM OF PROTEIN QUALITY CONTROL IN THE CYTOSOL IN BUDDING YEAST RUPALI PRASAD (M.Sc, IIT Bombay) A THESIS SUBMITTED FOR THE DEGREE OF. control of cytosolic proteins in chapter 3 and 4 of this thesis, a panel of model misfolded substrates was analyzed in detail. Surprisingly, their degradation occurs not in the cytosol but in the. is part of the Hrd1 complex, as it directly interacts with the large luminal domain of Hrd3p, indicating that it can links the recognition of misfolded glycoproteins to the ubiquitin-proteasome