Proteomics of the Endoplasmic Reticulum to Determine Cellular Organization and Protein Function
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Proteomics of the endoplasmic reticulum to determine cellular organization and protein function Michael D. Jain Degree of Doctorate in Philosophy Department of Anatomy and Cell Biology McGill University Montreal, Quebec, Canada May 2009 This thesis is submitted to McGill University in partial fulfillment of the requirements for the degree of Doctorate in Philosophy. ©Copyright 2009 All rights reserved i ACKNOWLEDGMENTS I would like to thank my supervisor Dr. John Bergeron for his enthusiasm and for enabling me to enjoy this work. I would like to thank the members of the Bergeron laboratory, particularly Hisao Nagaya, for their outstanding contributions to this project. I would like to thank the McGill M.D. – Ph.D. program for their confidence in me and continuous support. Most importantly, I would like to thank my family and friends for their affection and encouragement. ii Thesis Objectives This thesis has two overarching objectives: 1. To study the organization of the endoplasmic reticulum quality control pathway using a subcellular proteomics resource. 2. To identify novel proteins involved in endoplasmic reticulum quality control and to explore their function. iii Original contributions This body of work interrogates an organellar proteomics resource to make insights into the function of the endoplasmic reticulum in protein secretion. The following insights described in this thesis are original contributions to this scientific field: 1. Interrogation of the proteomics resource of Gilchrist et al. 2006, to determine the subdomain localization of over 100 proteins known to function in ER cargo processing. 2. Description of a spatiotemporal model of the ER: newly synthesized proteins traverse the ER from rough to smooth ER while undergoing synthesis, folding, and degradation. The resident proteins of the ER are segregated into physical and functional ER subdomains. 3. Identification of seven new targets of the mammalian unfolded protein response. 4. Identification of a new P-body protein (nudilin) and linking it to function at the ER. 5. Identification of a new constituent associated with the translocon (nicalin), the second protein suggested to regulate pre-emptive quality control, and a new constituent of the signal peptide peptidase complex. 6. Support for a novel process whereby the signal sequence of nicalin has a function separate from secretory pathway targeting, here involved in pre-emptive quality control. 7. Uncovering of four new proteins with chaperone-like characteristics (nicalin, stexin, erlin-1, and TMX2), with determination of the temporal ordering of their interaction with secretory cargo. 8. Domain studies on the short and long isoforms of TMX2 showing that the non- classical thioredoxin domain does not form mixed disulfides, yet is required for secretory cargo interaction. 9. Determining that the protein stexin interacts with diglucosylated forms of N- glycans. iv Contributions of Authors 1. FLAG tagged constructs shown throughout this thesis were subcloned from vectors originally cloned by Hisao Nagaya. 2. Localization of nudilin (Fig. 3.1c) using immunofluorescence was performed by Hisao Nagaya. 3. The structure of nudilin (Fig. 3.2) was determined by Zheng Ye, Rangarajan Erumbi, Irene Mazzoni, Christine Munger, John Wagner, Michael Sacher, and Miroslaw Cygler. 4. Overexpression of nudilin in Fig. 3.3a was performed by Hisao Nagaya. The nudilin DNA construct for overexpression experiments shown throughout was prepared by Hisao Nagaya. 5. Nicalin localization in Fig. 4.1c was performed by Hisao Nagaya. The nicalin YFP constructs used in Chapter 4 (including deletion mutants) were made by Hisao Nagaya. 6. Stexin, erlin, and TMX2 YFP localizations in Fig. 5.1b were performed by Hisao Nagaya. 7. FLAG-tagged TMX2 deletion mutant constructs used in Fig. 5.2c and the TMX2 SNDC to SNDA mutant construct used in Fig. 5.3c were made by Hisao Nagaya. 8. The abstract of this thesis was translated from English to French by Marie- Pascale Tremblay-Champagne. 9. All other attributions, including reagents obtained from other investigators, are indicated in the text. v TABLE OF CONTENTS Thesis Objectives ............................................................................................................................ iii Original contributions ..................................................................................................................... iv Contributions of Authors...................................................................................................................v TABLE OF CONTENTS ................................................................................................................ vi LIST OF FIGURES AND TABLES ............................................................................................. viii LIST OF ABBREVIATIONS ......................................................................................................... ix INTRODUCTION...........................................................................................................................12 Cells are defined by their organization.......................................................................................12 Organization of the secretory pathway.......................................................................................13 Exploring cells with a centrifuge................................................................................................14 Organellar proteomics ................................................................................................................15 LITERATURE REVIEW................................................................................................................17 Overview of cellular proteins involved in cargo processing ......................................................17 Post-transcriptional regulation of cargo messenger RNA: P-bodies and the ER........................18 Cargo synthesis and translocation (entry) into the endoplasmic reticulum ................................19 Removal of the signal sequence .................................................................................................21 The folding environment of the endoplasmic reticulum.............................................................23 Folding energetics in the endoplasmic reticulum .......................................................................24 Chaperones of the endoplasmic reticulum..................................................................................26 The glycoprotein recognition system.....................................................................................27 Disulfide bond formation and oxidative protein folding........................................................30 Hydrophobic domain recognition ..........................................................................................33 N-terminal proline recognition...............................................................................................35 Cytosolic chaperones .............................................................................................................36 ER-associated degradation (ERAD)...........................................................................................37 Selection of terminally misfolded/destabilized proteins for degradation...............................37 Retrotranslocation into the cytoplasm....................................................................................39 Proteasomal degradation........................................................................................................40 Non-proteasomal degradation................................................................................................41 Secretion of cargo out of the ER: engaging the COPII coated vesicle .......................................42 ER retention signals....................................................................................................................43 Protein synthesis and folding generates a secretory “load”........................................................44 The unfolded protein response relieves secretory load...............................................................44 The mechanism of sensing protein folding in the ER.................................................................46 Increasing the capacity of protein folding through signal transduction......................................47 Signalling downstream of Ire1...............................................................................................47 Signalling downstream of PERK ...........................................................................................48 Signalling downstream of ATF-6 ..........................................................................................49 Interpretation of ER stress by the different transducers.........................................................49 Limiting protein entry into the ER during times of protein overload .........................................50 Translational inhibition of secretory proteins ........................................................................50 Co-translocational degradation ..............................................................................................52 Transcriptional repression of secretory proteins ....................................................................54 ER stress-induced cell death.......................................................................................................54