MISFOLDED WSC1P UNDERGO POST ER QUALITY CONTROL

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. A., Bentivoglio, C. M., and Barlowe, C. (2006). Erv26p directs pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles. Mol Biol Cell 17, 4780-4789. Busca, R., Martinez, M., Vilella, E., Pognonec, P., Deeb, S., Auwerx, J., Reina, M., and Vilaro, S. (1996). The mutation Gly142-->Glu in human lipoprotein lipase produces a missorted protein that is diverted to lysosomes. J Biol Chem 271, 2139-2146. Caldwell, S. R., Hill, K. J., and Cooper, A. A. (2001). Degradation of endoplasmic reticulum (ER) quality control substrates requires transport between the ER and Golgi. J Biol Chem 276, 23296-23303. Call, M. E., Pyrdol, J., Wiedmann, M., and Wucherpfennig, K. W. (2002). The organizing principle in the formation of the T cell receptor-CD3 complex. Cell 111, 967-979. Camirand, A., Heysen, A., Grondin, B., and Herscovics, A. (1991). Glycoprotein biosynthesis in Saccharomyces cerevisiae. Isolation and characterization of the gene encoding a specific processing alphamannosidase. J Biol Chem 266, 15120-15127. Carvalho, P., Goder, V., and Rapoport, T. A. (2006). Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. Cell 126, 361-373. Castillon, G. A., Watanabe, R., Taylor, M., Schwabe, T. M., and Riezman, H. (2009). Concentration of GPI-anchored proteins upon ER exit in yeast. Traffic 10, 186-200. Cereghino, J. L., Marcusson, E. G., and Emr, S. D. (1995). The cytoplasmic tail domain of the vacuolar protein sorting receptor Vps10p and a subset of VPS gene products regulate receptor stability, function, and localization. Mol Biol Cell 6, 1089-1102. Chang, A., and Fink, G. R. (1995). Targeting of the yeast plasma membrane [H+]ATPase: a novel gene AST1 prevents mislocalization of mutant ATPase to the vacuole. J Cell Biol 128, 39-49. Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K., and Varshavsky, A. (1989). A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243, 15761583. Chen, H. I., Einbond, A., Kwak, S. J., Linn, H., Koepf, E., Peterson, S., Kelly, J. W., and Sudol, M. (1997). Characterization of the WW domain of human yes-associated protein and its polyproline-containing ligands. J Biol Chem 272, 17070-17077. Chen, L., and Davis, N. G. (2002). Ubiquitin-independent entry into the yeast recycling pathway. Traffic 3, 110-123. Chen, X., Zhang, B., and Fischer, J. A. (2002). A specific protein substrate for a deubiquitinating enzyme: Liquid facets is the substrate of Fat facets. Genes Dev 16, 289-294. Cheng, S. H., Gregory, R. J., Marshall, J., Paul, S., Souza, D. W., White, G. A., O'Riordan, C. R., and Smith, A. E. (1990). Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63, 827-834. Cid, V. J., Duran, A., del Rey, F., Snyder, M. P., Nombela, C., and Sanchez, M. (1995). Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae. Microbiol Rev 59, 345-386. Clerc, S., Hirsch, C., Oggier, D. M., Deprez, P., Jakob, C., Sommer, T., and Aebi, M. (2009). Htm1 protein generates the N-glycan signal for glycoprotein degradation in the endoplasmic reticulum. J Cell Biol 184, 159-172. 153 Colley, N. J., Baker, E. K., Stamnes, M. A., and Zuker, C. S. (1991). The cyclophilin homolog ninaA is required in the secretory pathway. Cell 67, 255-263. Cooper, A. A., and Stevens, T. H. (1996). Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases. J Cell Biol 133, 529-541. Coughlan, C. M., Walker, J. L., Cochran, J. C., Wittrup, K. D., and Brodsky, J. L. (2004). Degradation of mutated bovine pancreatic trypsin inhibitor (BPTI) in the yeast vacuole suggests post-endoplasmic reticulum protein quality control. J Biol Chem. Dancourt, J., and Barlowe, C. (2009). Erv26p-dependent export of alkaline phosphatase from the ER requires lumenal domain recognition. Traffic 10, 1006-1018. Dancourt, J., and Barlowe, C. (2010). Protein sorting receptors in the early secretory pathway. Annu Rev Biochem 79, 777-802. de Melker, A. A., van der Horst, G., Calafat, J., Jansen, H., and Borst, J. (2001). c-Cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route. J Cell Sci 114, 2167-2178. Denic, V., Quan, E. M., and Weissman, J. S. (2006). A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation. Cell 126, 349-359. Denning, G. M., Anderson, M. P., Amara, J. F., Marshall, J., Smith, A. E., and Welsh, M. J. (1992). Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 358, 761-764. DeWitt, N. D., dos Santos, C. F., Allen, K. E., and Slayman, C. W. (1998). Phosphorylation region of the yeast plasma-membrane H+-ATPase. Role in protein folding and biogenesis. J Biol Chem 273, 2174421751. Di Guglielmo, G. M., Baass, P. C., Ou, W. J., Posner, B. I., and Bergeron, J. J. (1994). Compartmentalization of SHC, GRB2 and mSOS, and hyperphosphorylation of Raf-1 by EGF but not insulin in liver parenchyma. Embo J 13, 4269-4277. Dobson, C. M. (2002). Getting out of shape. Nature 418, 729-730. Dunn, R., and Hicke, L. (2001). Domains of the Rsp5 ubiquitin-protein ligase required for receptormediated and fluid-phase endocytosis. Mol Biol Cell 12, 421-435. Dupre, S., and Haguenauer-Tsapis, R. (2001). Deubiquitination step in the endocytic pathway of yeast plasma membrane proteins: crucial role of Doa4p ubiquitin isopeptidase. Mol Cell Biol 21, 4482-4494. Egner, R., and Kuchler, K. (1996). The yeast multidrug transporter Pdr5 of the plasma membrane is ubiquitinated prior to endocytosis and degradation in the vacuole. FEBS Lett 378, 177-181. Ellgaard, L., and Helenius, A. (2003). Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4, 181-191. Endrizzi, J. A., Breddam, K., and Remington, S. J. (1994). 2.8-Angstrom structure of yeast serine carboxypeptidase. Biochemistry 33, 11106-11120. Ettenberg, S. A., Magnifico, A., Cuello, M., Nau, M. M., Rubinstein, Y. R., Yarden, Y., Weissman, A. M., and Lipkowitz, S. (2001). Cbl-b-dependent coordinated degradation of the epidermal growth factor receptor 154 signaling complex. J Biol Chem 276, 27677-27684. Felder, S., Miller, K., Moehren, G., Ullrich, A., Schlessinger, J., and Hopkins, C. R. (1990). Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body. Cell 61, 623634. Fewell, S. W., Travers, K. J., Weissman, J. S., and Brodsky, J. L. (2001). The action of molecular chaperones in the early secretory pathway. Annu Rev Genet 35, 149-191. Finger, A., Knop, M., and Wolf, D. H. (1993). Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast. Eur J Biochem 218, 565-574. Fisk, H. A., and Yaffe, M. P. (1999). A role for ubiquitination in mitochondrial inheritance in Saccharomyces cerevisiae. J Cell Biol 145, 1199-1208. Fra, A. M., Fagioli, C., Finazzi, D., Sitia, R., and Alberini, C. M. (1993). Quality control of ER synthesized proteins: an exposed thiol group as a three-way switch mediating assembly, retention and degradation. Embo J 12, 4755-4761. Fujita, M., Yoko, O. T., and Jigami, Y. (2006). Inositol deacylation by Bst1p is required for the quality control of glycosylphosphatidylinositol-anchored proteins. Mol Biol Cell 17, 834-850. Fujita, Y., Krause, G., Scheffner, M., Zechner, D., Leddy, H. E., Behrens, J., Sommer, T., and Birchmeier, W. (2002). Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nat Cell Biol 4, 222-231. Futter, C. E., Pearse, A., Hewlett, L. J., and Hopkins, C. R. (1996). Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes. J Cell Biol 132, 1011-1023. Gajewska, B., Kaminska, J., Jesionowska, A., Martin, N. C., Hopper, A. K., and Zoladek, T. (2001). WW domains of Rsp5p define different functions: determination of roles in fluid phase and uracil permease endocytosis in Saccharomyces cerevisiae. Genetics 157, 91-101. Galan, J. M., and Haguenauer-Tsapis, R. (1997). Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein. Embo J 16, 5847-5854. Galan, J. M., Moreau, V., Andre, B., Volland, C., and Haguenauer-Tsapis, R. (1996). Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease. J Biol Chem 271, 10946-10952. Gardner, R. G., Swarbrick, G. M., Bays, N. W., Cronin, S. R., Wilhovsky, S., Seelig, L., Kim, C., and Hampton, R. Y. (2000). Endoplasmic reticulum degradation requires lumen to cytosol signaling. Transmembrane control of Hrd1p by Hrd3p. J Cell Biol 151, 69-82. Gauss, R., Jarosch, E., Sommer, T., and Hirsch, C. (2006). A complex of Yos9p and the HRD ligase integrates endoplasmic reticulum quality control into the degradation machinery. Nat Cell Biol 8, 849-854. Gelman, M. S., and Kopito, R. R. (2002). Rescuing protein conformation: prospects for pharmacological therapy in cystic fibrosis. J Clin Invest 110, 1591-1597. Gilchrist, A., Au, C. E., Hiding, J., Bell, A. W., Fernandez-Rodriguez, J., Lesimple, S., Nagaya, H., Roy, L., Gosline, S. J., Hallett, M., et al. (2006). Quantitative proteomics analysis of the secretory pathway. Cell 127, 1265-1281. 155 Gilstring, C. F., Melin-Larsson, M., and Ljungdahl, P. O. (1999). Shr3p mediates specific COPII coatomercargo interactions required for the packaging of amino acid permeases into ER-derived transport vesicles. Mol Biol Cell 10, 3549-3565. Giraudo, C. G., and Maccioni, H. J. (2003). Endoplasmic reticulum export of glycosyltransferases depends on interaction of a cytoplasmic dibasic motif with Sar1. Mol Biol Cell 14, 3753-3766. Gong, X., and Chang, A. (2001). A mutant plasma membrane ATPase, Pma1-10, is defective in stability at the yeast cell surface. Proc Natl Acad Sci U S A 98, 9104-9109. Gorden, P., Carpentier, J. L., Cohen, S., and Orci, L. (1978). Epidermal growth factor: morphological demonstration of binding, internalization, and lysosomal association in human fibroblasts. Proc Natl Acad Sci U S A 75, 5025-5029. Gray, J. V., Ogas, J. P., Kamada, Y., Stone, M., Levin, D. E., and Herskowitz, I. (1997). A role for the Pkc1 MAP kinase pathway of Saccharomyces cerevisiae in bud emergence and identification of a putative upstream regulator. Embo J 16, 4924-4937. Gruenberg, J., and Stenmark, H. (2004). The biogenesis of multivesicular endosomes. Nat Rev Mol Cell Biol 5, 317-323. Gurkan, C., Stagg, S. M., Lapointe, P., and Balch, W. E. (2006). The COPII cage: unifying principles of vesicle coat assembly. Nat Rev Mol Cell Biol 7, 727-738. Gurunathan, S., David, D., and Gerst, J. E. (2002). Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast. Embo J 21, 602-614. Haas, I. G., and Wabl, M. (1983). Immunoglobulin heavy chain binding protein. Nature 306, 387-389. Haglund, K., Sigismund, S., Polo, S., Szymkiewicz, I., Di Fiore, P. P., and Dikic, I. (2003). Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation. Nat Cell Biol 5, 461-466. Haigler, H. T., McKanna, J. A., and Cohen, S. (1979). Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431. J Cell Biol 81, 382-395. Halaban, R., Cheng, E., Zhang, Y., Moellmann, G., Hanlon, D., Michalak, M., Setaluri, V., and Hebert, D. N. (1997). Aberrant retention of tyrosinase in the endoplasmic reticulum mediates accelerated degradation of the enzyme and contributes to the dedifferentiated phenotype of amelanotic melanoma cells. Proc Natl Acad Sci U S A 94, 6210-6215. Hammond, C., and Helenius, A. (1994). Folding of VSV G protein: sequential interaction with BiP and calnexin. Science 266, 456-458. Harsay, E., and Schekman, R. (2002). A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway. J Cell Biol 156, 271-285. Harty, C., Strahl, S., and Romisch, K. (2001). O-mannosylation protects mutant alpha-factor precursor from endoplasmic reticulum-associated degradation. Mol Biol Cell 12, 1093-1101. Hebert, D. N., Bernasconi, R., and Molinari, M. (2010). ERAD substrates: which way out? Semin Cell Dev Biol 21, 526-532. Hegde, R. S., and Ploegh, H. L. (2010). Quality and quantity control at the endoplasmic reticulum. Curr Opin Cell Biol 22, 437-446. 156 Helenius, A., Marquardt, T., and Braakman, I. (1992). The endoplasmic reticulum as a protein-folding compartment. Trends Cell Biol 2, 227-231. Helliwell, S. B., Losko, S., and Kaiser, C. A. (2001). Components of a ubiquitin ligase complex specify polyubiquitination and intracellular trafficking of the general amino acid permease. J Cell Biol 153, 649662. Hershko, A., and Ciechanover, A. (1998). The ubiquitin system. Annu Rev Biochem 67, 425-479. Hicke, L. (2001). Protein regulation by monoubiquitin. Nat Rev Mol Cell Biol 2, 195-201. Hicke, L., and Dunn, R. (2003). Regulation of membrane protein transport by ubiquitin and ubiquitinbinding proteins. Annu Rev Cell Dev Biol 19, 141-172. Hill, K., and Cooper, A. A. (2000). Degradation of unassembled Vph1p reveals novel aspects of the yeast ER quality control system. Embo J 19, 550-561. Hirayama, H., Fujita, M., Yoko-o, T., and Jigami, Y. (2008). O-mannosylation is required for degradation of the endoplasmic reticulum-associated degradation substrate Gas1*p via the ubiquitin/proteasome pathway in Saccharomyces cerevisiae. J Biochem 143, 555-567. Hirsch, C., Gauss, R., Horn, S. C., Neuber, O., and Sommer, T. (2009). The ubiquitylation machinery of the endoplasmic reticulum. Nature 458, 453-460. Hofmann, K., and Bucher, P. (1995). The rsp5-domain is shared by proteins of diverse functions. FEBS Lett 358, 153-157. Hong, E., Davidson, A. R., and Kaiser, C. A. (1996). A pathway for targeting soluble misfolded proteins to the yeast vacuole. J Cell Biol 135, 623-633. Hoppe, T., Matuschewski, K., Rape, M., Schlenker, S., Ulrich, H. D., and Jentsch, S. (2000). Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing. Cell 102, 577-586. Hurley, J. H., and Emr, S. D. (2006). The ESCRT complexes: structure and mechanism of a membranetrafficking network. Annu Rev Biophys Biomol Struct 35, 277-298. Inadome, H., Noda, Y., Adachi, H., and Yoda, K. (2005). Immunoisolaton of the yeast Golgi subcompartments and characterization of a novel membrane protein, Svp26, discovered in the Sed5containing compartments. Mol Cell Biol 25, 7696-7710. Itin, C., Roche, A. C., Monsigny, M., and Hauri, H. P. (1996). ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins. Mol Biol Cell 7, 483-493. Jackson, D. D., and Stevens, T. H. (1997). VMA12 encodes a yeast endoplasmic reticulum protein required for vacuolar H+-ATPase assembly. J Biol Chem 272, 25928-25934. Jakob, C. A., Burda, P., Roth, J., and Aebi, M. (1998). Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure. J Cell Biol 142, 1223-1233. Jelinek-Kelly, S., and Herscovics, A. (1988). Glycoprotein biosynthesis in Saccharomyces cerevisiae. Purification of the alpha-mannosidase which removes one specific mannose residue from Man9GlcNAc. J Biol Chem 263, 14757-14763. 157 Jenness, D. D., Li, Y., Tipper, C., and Spatrick, P. (1997). Elimination of defective alpha-factor pheromone receptors. Mol Cell Biol 17, 6236-6245. Joazeiro, C. A., Wing, S. S., Huang, H., Leverson, J. D., Hunter, T., and Liu, Y. C. (1999). The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science 286, 309312. Jorgensen, M. U., Emr, S. D., and Winther, J. R. (1999). Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae. Eur J Biochem 260, 461-469. Kaether, C., Scheuermann, J., Fassler, M., Zilow, S., Shirotani, K., Valkova, C., Novak, B., Kacmar, S., Steiner, H., and Haass, C. (2007). Endoplasmic reticulum retention of the gamma-secretase complex component Pen2 by Rer1. EMBO Rep 8, 743-748. Kaganovich, D., Kopito, R., and Frydman, J. (2008). Misfolded proteins partition between two distinct quality control compartments. Nature 454, 1088-1095. Kappeler, F., Klopfenstein, D. R., Foguet, M., Paccaud, J. P., and Hauri, H. P. (1997). The recycling of ERGIC-53 in the early secretory pathway. ERGIC-53 carries a cytosolic endoplasmic reticulum-exit determinant interacting with COPII. J Biol Chem 272, 31801-31808. Katzmann, D. J., Babst, M., and Emr, S. D. (2001). Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106, 145-155. Katzmann, D. J., Odorizzi, G., and Emr, S. D. (2002). Receptor downregulation and multivesicular-body sorting. Nat Rev Mol Cell Biol 3, 893-905. Katzmann, D. J., Sarkar, S., Chu, T., Audhya, A., and Emr, S. D. (2004). Multivesicular body sorting: ubiquitin ligase Rsp5 is required for the modification and sorting of carboxypeptidase S. Mol Biol Cell 15, 468-480. Kawaguchi, S., Hsu, C. L., and Ng, D. T. (in press). Interplay of substrate retention and export signals in endoplasmic reticulum quality control. Plos One In press. Keleman, K., Rajagopalan, S., Cleppien, D., Teis, D., Paiha, K., Huber, L. A., Technau, G. M., and Dickson, B. J. (2002). Comm sorts robo to control axon guidance at the Drosophila midline. Cell 110, 415427. Kim, P. S., and Arvan, P. (1995). Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum. J Cell Biol 128, 29-38. Kim, W., Spear, E. D., and Ng, D. T. (2005). Yos9p detects and targets misfolded glycoproteins for ERassociated degradation. Mol Cell 19, 753-764. Kincaid, M. M., and Cooper, A. A. (2007). Misfolded proteins traffic from the endoplasmic reticulum (ER) due to ER export signals. Mol Biol Cell 18, 455-463. Kirisako, T., Baba, M., Ishihara, N., Miyazawa, K., Ohsumi, M., Yoshimori, T., Noda, T., and Ohsumi, Y. (1999). Formation process of autophagosome is traced with Apg8/Aut7p in yeast. J Cell Biol 147, 435-446. Kleijmeer, M., Ramm, G., Schuurhuis, D., Griffith, J., Rescigno, M., Ricciardi-Castagnoli, P., Rudensky, A. Y., Ossendorp, F., Melief, C. J., Stoorvogel, W., and Geuze, H. J. (2001). Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells. J Cell Biol 155, 53-63. 158 Klis, F. M. (1994). Review: cell wall assembly in yeast. Yeast 10, 851-869. Knittler, M. R., Dirks, S., and Haas, I. G. (1995). Molecular chaperones involved in protein degradation in the endoplasmic reticulum: quantitative interaction of the heat shock cognate protein BiP with partially folded immunoglobulin light chains that are degraded in the endoplasmic reticulum. Proc Natl Acad Sci U S A 92, 1764-1768. Kolling, R., and Hollenberg, C. P. (1994). The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants. Embo J 13, 3261-3271. Kopito, R. R. (1999). Biosynthesis and degradation of CFTR. Physiol Rev 79, S167-173. Kota, J., Gilstring, C. F., and Ljungdahl, P. O. (2007). Membrane chaperone Shr3 assists in folding amino acid permeases preventing precocious ERAD. J Cell Biol 176, 617-628. Kota, J., and Ljungdahl, P. O. (2005). Specialized membrane-localized chaperones prevent aggregation of polytopic proteins in the ER. J Cell Biol 168, 79-88. Kruse, K. B., Brodsky, J. L., and McCracken, A. A. (2006). Characterization of an ERAD gene as VPS30/ATG6 reveals two alternative and functionally distinct protein quality control pathways: one for soluble Z variant of human alpha-1 proteinase inhibitor (A1PiZ) and another for aggregates of A1PiZ. Mol Biol Cell 17, 203-212. Lara-Castro, C., Luo, N., Wallace, P., Klein, R. L., and Garvey, W. T. (2006). Adiponectin multimeric complexes and the metabolic syndrome trait cluster. Diabetes 55, 249-259. Lau, W. T., Howson, R. W., Malkus, P., Schekman, R., and O'Shea, E. K. (2000). Pho86p, an endoplasmic reticulum (ER) resident protein in Saccharomyces cerevisiae, is required for ER exit of the high-affinity phosphate transporter Pho84p. Proc Natl Acad Sci U S A 97, 1107-1112. Lee, M. C., Miller, E. A., Goldberg, J., Orci, L., and Schekman, R. (2004). Bi-directional protein transport between the ER and Golgi. Annu Rev Cell Dev Biol 20, 87-123. Levin, D. E. (2005). Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 69, 262-291. Levkowitz, G., Waterman, H., Ettenberg, S. A., Katz, M., Tsygankov, A. Y., Alroy, I., Lavi, S., Iwai, K., Reiss, Y., Ciechanover, A., et al. (1999). Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1. Mol Cell 4, 1029-1040. Levkowitz, G., Waterman, H., Zamir, E., Kam, Z., Oved, S., Langdon, W. Y., Beguinot, L., Geiger, B., and Yarden, Y. (1998). c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev 12, 3663-3674. Li, Y., Kane, T., Tipper, C., Spatrick, P., and Jenness, D. D. (1999). Yeast mutants affecting possible quality control of plasma membrane proteins. Mol Cell Biol 19, 3588-3599. Lilley, B. N., and Ploegh, H. L. (2004). A membrane protein required for dislocation of misfolded proteins from the ER. Nature 429, 834-840. Liu, Y., Sitaraman, S., and Chang, A. (2006). Multiple degradation pathways for misfolded mutants of the yeast plasma membrane ATPase, Pma1. J Biol Chem 281, 31457-31466. Ljungdahl, P. O., Gimeno, C. J., Styles, C. A., and Fink, G. R. (1992). SHR3: a novel component of the secretory pathway specifically required for localization of amino acid permeases in yeast. Cell 71, 463-478. 159 Lobert, V. H., Brech, A., Pedersen, N. M., Wesche, J., Oppelt, A., Malerod, L., and Stenmark, H. (2010). Ubiquitination of alpha beta integrin controls fibroblast migration through lysosomal degradation of fibronectin-integrin complexes. Dev Cell 19, 148-159. Lodder, A. L., Lee, T. K., and Ballester, R. (1999). Characterization of the Wsc1 protein, a putative receptor in the stress response of Saccharomyces cerevisiae. Genetics 152, 1487-1499. Lommel, M., Bagnat, M., and Strahl, S. (2004). Aberrant processing of the WSC family and Mid2p cell surface sensors results in cell death of Saccharomyces cerevisiae O-mannosylation mutants. Mol Cell Biol 24, 46-57. Lommel, M., and Strahl, S. (2009). Protein O-mannosylation: conserved from bacteria to humans. Glycobiology 19, 816-828. Losko, S., Kopp, F., Kranz, A., and Kolling, R. (2001). Uptake of the ATP-binding cassette (ABC) transporter Ste6 into the yeast vacuole is blocked in the doa4 Mutant. Mol Biol Cell 12, 1047-1059. Low, S. H., Tang, B. L., Wong, S. H., and Hong, W. (1995). Retardation of a surface protein chimera at the cis Golgi. Biochemistry 34, 5618-5626. Luo, W., and Chang, A. (1997). Novel genes involved in endosomal traffic in yeast revealed by suppression of a targeting-defective plasma membrane ATPase mutant. J Cell Biol 138, 731-746. Luo, W., and Chang, A. (2000). An endosome-to-plasma membrane pathway involved in trafficking of a mutant plasma membrane ATPase in yeast. Mol Biol Cell 11, 579-592. Lussier, M., Sdicu, A. M., Bussereau, F., Jacquet, M., and Bussey, H. (1997). The Ktr1p, Ktr3p, and Kre2p/Mnt1p mannosyltransferases participate in the elaboration of yeast O- and N-linked carbohydrate chains. J Biol Chem 272, 15527-15531. Ma, D., Zerangue, N., Lin, Y. F., Collins, A., Yu, M., Jan, Y. N., and Jan, L. Y. (2001). Role of ER export signals in controlling surface potassium channel numbers. Science 291, 316-319. Maldonado, A. M., de la Fuente, N., and Portillo, F. (1998). Characterization of an allele-nonspecific intragenic suppressor in the yeast plasma membrane H+-ATPase gene (Pma1). Genetics 150, 11-19. Malkus, P., Jiang, F., and Schekman, R. (2002). Concentrative sorting of secretory cargo proteins into COPII-coated vesicles. J Cell Biol 159, 915-921. Marcusson, E. G., Horazdovsky, B. F., Cereghino, J. L., Gharakhanian, E., and Emr, S. D. (1994). The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene. Cell 77, 579-586. Martinez-Menarguez, J. A., Geuze, H. J., Slot, J. W., and Klumperman, J. (1999). Vesicular tubular clusters between the ER and Golgi mediate concentration of soluble secretory proteins by exclusion from COPIcoated vesicles. Cell 98, 81-90. Massaad, M. J., Franzusoff, A., and Herscovics, A. (1999). The processing alpha1,2-mannosidase of Saccharomyces cerevisiae depends on Rer1p for its localization in the endoplasmic reticulum. Eur J Cell Biol 78, 435-440. Maxfield, F. R., and McGraw, T. E. (2004). Endocytic recycling. Nat Rev Mol Cell Biol 5, 121-132. McCracken, A. A., Karpichev, I. V., Ernaga, J. E., Werner, E. D., Dillin, A. G., and Courchesne, W. E. (1996). Yeast mutants deficient in ER-associated degradation of the Z variant of alpha-1-protease inhibitor. Genetics 144, 1355-1362. 160 Mellman, I. (1996). Endocytosis and molecular sorting. Annu Rev Cell Dev Biol 12, 575-625. Melnick, J., Dul, J. L., and Argon, Y. (1994). Sequential interaction of the chaperones BiP and GRP94 with immunoglobulin chains in the endoplasmic reticulum. Nature 370, 373-375. Molinari, M., Galli, C., Piccaluga, V., Pieren, M., and Paganetti, P. (2002). Sequential assistance of molecular chaperones and transient formation of covalent complexes during protein degradation from the ER. J Cell Biol 158, 247-257. Muchowski, P. J. (2002). Protein misfolding, amyloid formation, and neurodegeneration: a critical role for molecular chaperones? Neuron 35, 9-12. Mulholland, J., Konopka, J., Singer-Kruger, B., Zerial, M., and Botstein, D. (1999). Visualization of receptor-mediated endocytosis in yeast. Mol Biol Cell 10, 799-817. Muniz, M., Nuoffer, C., Hauri, H. P., and Riezman, H. (2000). The Emp24 complex recruits a specific cargo molecule into endoplasmic reticulum-derived vesicles. J Cell Biol 148, 925-930. Munro, S., and Pelham, H. R. B. (1987). A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899-907. Nakamoto, R. K., Verjovski-Almeida, S., Allen, K. E., Ambesi, A., Rao, R., and Slayman, C. W. (1998). Substitutions of aspartate 378 in the phosphorylation domain of the yeast PMA1 H+-ATPase disrupt protein folding and biogenesis. J Biol Chem 273, 7338-7344. Nakamura, N., Lyalin, D., and Panin, V. M. (2010). Protein O-mannosylation in animal development and physiology: from human disorders to Drosophila phenotypes. Semin Cell Dev Biol 21, 622-630. Nakano, A., Brada, D., and Schekman, R. (1988). A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast. J Cell Biol 107, 851-863. Ng, D. T., Brown, J. D., and Walter, P. (1996). Signal sequences specify the targeting route to the endoplasmic reticulum membrane. J Cell Biol 134, 269-278. Ng, D. T., Spear, E. D., and Walter, P. (2000). The unfolded protein response regulates multiple aspects of secretory and membrane protein biogenesis and endoplasmic reticulum quality control. J Cell Biol 150, 7788. Nichols, W. C., Seligsohn, U., Zivelin, A., Terry, V. H., Hertel, C. E., Wheatley, M. A., Moussalli, M. J., Hauri, H. P., Ciavarella, N., Kaufman, R. J., and Ginsburg, D. (1998). Mutations in the ER-Golgi intermediate compartment protein ERGIC-53 cause combined deficiency of coagulation factors V and VIII. Cell 93, 61-70. Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 1-6. Nishikawa, S., and Nakano, A. (1993). Identification of a gene required for membrane protein retention in the early secretory pathway. Proc Natl Acad Sci U S A 90, 8179-8183. Nishimura, N., and Balch, W. E. (1997). A di-acidic signal required for selective export from the endoplasmic reticulum. Science 277, 556-558. Nufer, O., Guldbrandsen, S., Degen, M., Kappeler, F., Paccaud, J. P., Tani, K., and Hauri, H. P. (2002). Role of cytoplasmic C-terminal amino acids of membrane proteins in ER export. J Cell Sci 115, 619-628. 161 Odorizzi, G., Babst, M., and Emr, S. D. (1998). Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body. Cell 95, 847-858. Okiyoneda, T., Barriere, H., Bagdany, M., Rabeh, W. M., Du, K., Hohfeld, J., Young, J. C., and Lukacs, G. L. (2010). Peripheral protein quality control removes unfolded CFTR from the plasma membrane. Science 329, 805-810. Orci, L., Ravazzola, M., Meda, P., Holcomb, C., Moore, H. P., Hicke, L., and Schekman, R. (1991). Mammalian Sec23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm. Proc Natl Acad Sci U S A 88, 8611-8615. Ostman, A., Thyberg, J., Westermark, B., and Heldin, C. H. (1992). PDGF-AA and PDGF-BB biosynthesis: proprotein processing in the Golgi complex and lysosomal degradation of PDGF-BB retained intracellularly. J Cell Biol 118, 509-519. Otte, S., and Barlowe, C. (2002). The Erv41p-Erv46p complex: multiple export signals are required in trans for COPII-dependent transport from the ER. Embo J 21, 6095-6104. Pagant, S., Kung, L., Dorrington, M., Lee, M. C., and Miller, E. A. (2007). Inhibiting endoplasmic reticulum (ER)-associated degradation of misfolded Yor1p does not permit ER export despite the presence of a diacidic sorting signal. Mol Biol Cell 18, 3398-3413. Paquet, M. E., Cohen-Doyle, M., Shore, G. C., and Williams, D. B. (2004). Bap29/31 influences the intracellular traffic of MHC class I molecules. J Immunol 172, 7548-7555. Park, S. H., Bolender, N., Eisele, F., Kostova, Z., Takeuchi, J., Coffino, P., and Wolf, D. H. (2007). The cytoplasmic Hsp70 chaperone machinery subjects misfolded and endoplasmic reticulum importincompetent proteins to degradation via the ubiquitin-proteasome system. Mol Biol Cell 18, 153-165. Petaja-Repo, U. E., Hogue, M., Laperriere, A., Bhalla, S., Walker, P., and Bouvier, M. (2001). Newly synthesized human delta opioid receptors retained in the endoplasmic reticulum are retrotranslocated to the cytosol, deglycosylated, ubiquitinated, and degraded by the proteasome. J Biol Chem 276, 4416-4423. Philip, B., and Levin, D. E. (2001). Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1. Mol Cell Biol 21, 271-280. Piao, H. L., Machado, I. M., and Payne, G. S. (2007). NPFXD-mediated endocytosis is required for polarity and function of a yeast cell wall stress sensor. Mol Biol Cell 18, 57-65. Piper, R. C., Bryant, N. J., and Stevens, T. H. (1997). The membrane protein alkaline phosphatase is delivered to the vacuole by a route that is distinct from the VPS-dependent pathway. J Cell Biol 138, 531545. Piper, R. C., Cooper, A. A., Yang, H., and Stevens, T. H. (1995). VPS27 controls vacuolar and endocytic traffic through a prevacuolar compartment in Saccharomyces cerevisiae. J Cell Biol 131, 603-617. Pizzirusso, M., and Chang, A. (2004). Ubiquitin-mediated targeting of a mutant plasma membrane ATPase, Pma1-7, to the endosomal/vacuolar system in yeast. Mol Biol Cell 15, 2401-2409. Ploegh, H. L. (2007). A lipid-based model for the creation of an escape hatch from the endoplasmic reticulum. Nature 448, 435-438. Powers, J., and Barlowe, C. (1998). Transport of axl2p depends on erv14p, an ER-vesicle protein related to the Drosophila cornichon gene product. J Cell Biol 142, 1209-1222. 162 Prasad, R., Kawaguchi, S., and Ng, D. T. (2010). A nucleus-based quality control mechanism for cytosolic proteins. Mol Biol Cell 21, 2117-2127. Qiang, L., Wang, H., and Farmer, S. R. (2007). Adiponectin secretion is regulated by SIRT1 and the endoplasmic reticulum oxidoreductase Ero1-L alpha. Mol Cell Biol 27, 4698-4707. Quan, E. M., Kamiya, Y., Kamiya, D., Denic, V., Weibezahn, J., Kato, K., and Weissman, J. S. (2008). Defining the glycan destruction signal for endoplasmic reticulum-associated degradation. Mol Cell 32, 870877. Raiborg, C., Malerod, L., Pedersen, N. M., and Stenmark, H. (2008). Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking. Exp Cell Res 314, 801-813. Raiborg, C., and Stenmark, H. (2009). The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature 458, 445-452. Rajavel, M., Philip, B., Buehrer, B. M., Errede, B., and Levin, D. E. (1999). Mid2 is a putative sensor for cell integrity signaling in Saccharomyces cerevisiae. Mol Cell Biol 19, 3969-3976. Ravid, T., Kreft, S. G., and Hochstrasser, M. (2006). Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways. Embo J 25, 533-543. Reggiori, F., Black, M. W., and Pelham, H. R. (2000). Polar transmembrane domains target proteins to the interior of the yeast vacuole. Mol Biol Cell 11, 3737-3749. Reggiori, F., and Pelham, H. R. (2001). Sorting of proteins into multivesicular bodies: ubiquitin-dependent and -independent targeting. Embo J 20, 5176-5186. Reggiori, F., and Pelham, H. R. (2002). A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies. Nat Cell Biol 4, 117-123. Roberts, M. J., Bentley, M. D., and Harris, J. M. (2002). Chemistry for peptide and protein PEGylation. Adv Drug Deliv Rev 54, 459-476. Rocca, A., Lamaze, C., Subtil, A., and Dautry-Varsat, A. (2001). Involvement of the ubiquitin/proteasome system in sorting of the interleukin receptor beta chain to late endocytic compartments. Mol Biol Cell 12, 1293-1301. Rotin, D., Staub, O., and Haguenauer-Tsapis, R. (2000). Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases. J Membr Biol 176, 1-17. Ryan, M., Graham, L. A., and Stevens, T. H. (2008). Voa1p functions in V-ATPase assembly in the yeast endoplasmic reticulum. Mol Biol Cell 19, 5131-5142. Saito, K., Chen, M., Bard, F., Chen, S., Zhou, H., Woodley, D., Polischuk, R., Schekman, R., and Malhotra, V. (2009). TANGO1 facilitates cargo loading at endoplasmic reticulum exit sites. Cell 136, 891-902. Saksena, S., Wahlman, J., Teis, D., Johnson, A. E., and Emr, S. D. (2009). Functional reconstitution of ESCRT-III assembly and disassembly. Cell 136, 97-109. Sambrook, J., Fritsch, E.M., and Maniatis, T. (1989). Molecular Cloning: a laboratory manual, 2nd edn.: Plainview, Cold Spring Habor Laboratory Press.). Sarkar, S., Perlstein, E. O., Imarisio, S., Pineau, S., Cordenier, A., Maglathlin, R. L., Webster, J. A., Lewis, T. A., O'Kane, C. J., Schreiber, S. L., and Rubinsztein, D. C. (2007). Small molecules enhance autophagy 163 and reduce toxicity in Huntington's disease models. Nat Chem Biol 3, 331-338. Sato, K., and Nakano, A. (2002). Emp47p and its close homolog Emp46p have a tyrosine-containing endoplasmic reticulum exit signal and function in glycoprotein secretion in Saccharomyces cerevisiae. Mol Biol Cell 13, 2518-2532. Sato, K., and Nakano, A. (2003). Oligomerization of a cargo receptor directs protein sorting into COPIIcoated transport vesicles. Mol Biol Cell 14, 3055-3063. Sato, K., Nishikawa, S., and Nakano, A. (1995). Membrane protein retrieval from the Golgi apparatus to the endoplasmic reticulum (ER): characterization of the RER1 gene product as a component involved in ER localization of Sec12p. Mol Biol Cell 6, 1459-1477. Sato, K., Sato, M., and Nakano, A. (2001). Rer1p, a retrieval receptor for endoplasmic reticulum membrane proteins, is dynamically localized to the Golgi apparatus by coatomer. J Cell Biol 152, 935-944. Sato, K., Sato, M., and Nakano, A. (2003). Rer1p, a retrieval receptor for ER membrane proteins, recognizes transmembrane domains in multiple modes. Mol Biol Cell 14, 3605-3616. Sato, M., Sato, K., and Nakano, A. (1996). Endoplasmic reticulum localization of Sec12p is achieved by two mechanisms: Rer1p-dependent retrieval that requires the transmembrane domain and Rer1pindependent retention that involves the cytoplasmic domain. J Cell Biol 134, 279-293. Sato, M., Sato, K., and Nakano, A. (2004). Endoplasmic reticulum quality control of unassembled iron transporter depends on Rer1p-mediated retrieval from the golgi. Mol Biol Cell 15, 1417-1424. Sawano, A., and Miyawaki, A. (2000). Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Res 28, E78. Scherer, P. E., Williams, S., Fogliano, M., Baldini, G., and Lodish, H. F. (1995). A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem 270, 26746-26749. Schimmoller, F., Singer-Kruger, B., Schroder, S., Kruger, U., Barlowe, C., and Riezman, H. (1995). The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi. Embo J 14, 1329-1339. Schubert, U., Anton, L. C., Gibbs, J., Norbury, C. C., Yewdell, J. W., and Bennink, J. R. (2000). Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 404, 770-774. Sevier, C. S., Weisz, O. A., Davis, M., and Machamer, C. E. (2000). Efficient export of the vesicular stomatitis virus G protein from the endoplasmic reticulum requires a signal in the cytoplasmic tail that includes both tyrosine-based and di-acidic motifs. Mol Biol Cell 11, 13-22. Sharma, M., Pampinella, F., Nemes, C., Benharouga, M., So, J., Du, K., Bache, K. G., Papsin, B., Zerangue, N., Stenmark, H., and Lukacs, G. L. (2004). Misfolding diverts CFTR from recycling to degradation: quality control at early endosomes. J Cell Biol 164, 923-933. Shenoy, S. K., McDonald, P. H., Kohout, T. A., and Lefkowitz, R. J. (2001). Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin. Science 294, 1307-1313. Sherwood, P. W., and Carlson, M. (1999). Efficient export of the glucose transporter Hxt1p from the endoplasmic reticulum requires Gsf2p. Proc Natl Acad Sci U S A 96, 7415-7420. Shih, S. C., Katzmann, D. J., Schnell, J. D., Sutanto, M., Emr, S. D., and Hicke, L. (2002). Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis. Nat Cell Biol 4, 389- 164 393. Shtiegman, K., and Yarden, Y. (2003). The role of ubiquitylation in signaling by growth factors: implications to cancer. Semin Cancer Biol 13, 29-40. Sikorski, R. S., and Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19-27. Sitia, R., and Cattaneo, A. (1995). in The antibodies (eds Zanetti, M., and Capra, J.D.): Harwood Academic, Luxembourg). Sitia, R., Neuberger, M., Alberini, C., Bet, P., Fra, A., Valetti, C., Williams, G., and Milstein, C. (1990). Developmental regulation of IgM secretion: the role of the carboxy-terminal cysteine. Cell 60, 781-790. Spasic, D., Raemaekers, T., Dillen, K., Declerck, I., Baert, V., Serneels, L., Fullekrug, J., and Annaert, W. (2007). Rer1p competes with APH-1 for binding to nicastrin and regulates gamma-secretase complex assembly in the early secretory pathway. J Cell Biol 176, 629-640. Spear, E. D., and Ng, D. T. (2003). Stress tolerance of misfolded carboxypeptidase Y requires maintenance of protein trafficking and degradative pathways. Mol Biol Cell 14, 2756-2767. Spear, E. D., and Ng, D. T. (2005). Single, context-specific glycans can target misfolded glycoproteins for ER-associated degradation. J Cell Biol 169, 73-82. Spormann, D. O., Heim, J., and Wolf, D. H. (1992). Biogenesis of the yeast vacuole (lysosome). The precursor forms of the soluble hydrolase carboxypeptidase yscS are associated with the vacuolar membrane. J Biol Chem 267, 8021-8029. Springael, J. Y., and Andre, B. (1998). Nitrogen-regulated ubiquitination of the Gap1 permease of Saccharomyces cerevisiae. Mol Biol Cell 9, 1253-1263. Stang, E., Johannessen, L. E., Knardal, S. L., and Madshus, I. H. (2000). Polyubiquitination of the epidermal growth factor receptor occurs at the plasma membrane upon ligand-induced activation. J Biol Chem 275, 13940-13947. Staub, O., Gautschi, I., Ishikawa, T., Breitschopf, K., Ciechanover, A., Schild, L., and Rotin, D. (1997). Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination. Embo J 16, 6325-6336. Stevens, T., Esmon, B., and Schekman, R. (1982). Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell 30, 439-448. Stevens, T. L., Blum, J. H., Foy, S. P., Matsuuchi, L., and DeFranco, A. L. (1994). A mutation of the mu transmembrane that disrupts endoplasmic reticulum retention. Effects on association with accessory proteins and signal transduction. J Immunol 152, 4397-4406. Strous, G. J., van Kerkhof, P., Govers, R., Ciechanover, A., and Schwartz, A. L. (1996). The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor. Embo J 15, 3806-3812. Swanson, R., Locher, M., and Hochstrasser, M. (2001). A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation. Genes Dev 15, 2660-2674. Szathmary, R., Bielmann, R., Nita-Lazar, M., Burda, P., and Jakob, C. A. (2005). Yos9 protein is essential 165 for degradation of misfolded glycoproteins and may function as lectin in ERAD. Mol Cell 19, 765-775. Tan, P. K., Howard, J. P., and Payne, G. S. (1996). The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae. J Cell Biol 135, 1789-1800. Teis, D., Saksena, S., and Emr, S. D. (2009). SnapShot: the ESCRT machinery. Cell 137, 182-182 e181. Terrell, J., Shih, S., Dunn, R., and Hicke, L. (1998). A function for monoubiquitination in the internalization of a G protein-coupled receptor. Mol Cell 1, 193-202. Thrower, J. S., Hoffman, L., Rechsteiner, M., and Pickart, C. M. (2000). Recognition of the polyubiquitin proteolytic signal. Embo J 19, 94-102. Tonelli, J., Li, W., Kishore, P., Pajvani, U. B., Kwon, E., Weaver, C., Scherer, P. E., and Hawkins, M. (2004). Mechanisms of early insulin-sensitizing effects of thiazolidinediones in type diabetes. Diabetes 53, 1621-1629. Trilla, J. A., Duran, A., and Roncero, C. (1999). Chs7p, a new protein involved in the control of protein export from the endoplasmic reticulum that is specifically engaged in the regulation of chitin synthesis in Saccharomyces cerevisiae. J Cell Biol 145, 1153-1163. Trombetta, E. S., and Parodi, A. J. (2003). Quality control and protein folding in the secretory pathway. Annu Rev Cell Dev Biol 19, 649-676. Tsai, B., Rodighiero, C., Lencer, W. I., and Rapoport, T. A. (2001). Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin. Cell 104, 937-948. van Munster, E. B., van Vliet, L. J., and Aten, J. A. (1997). Reconstruction of optical pathlength distributions from images obtained by a wide-field differential interference contrast microscope. J Microsc 188, 149-157. VanSlyke, J. K., Deschenes, S. M., and Musil, L. S. (2000). Intracellular transport, assembly, and degradation of wild-type and disease-linked mutant gap junction proteins. Mol Biol Cell 11, 1933-1946. Vashist, S., Kim, W., Belden, W. J., Spear, E. D., Barlowe, C., and Ng, D. T. (2001). Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding. J Cell Biol 155, 355-368. Vashist, S., and Ng, D. T. (2004). Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control. J Cell Biol 165, 41-52. Vay, H. A., Philip, B., and Levin, D. E. (2004). Mutational analysis of the cytoplasmic domain of the Wsc1 cell wall stress sensor. Microbiology 150, 3281-3288. Vembar, S. S., and Brodsky, J. L. (2008). One step at a time: endoplasmic reticulum-associated degradation. Nat Rev Mol Cell Biol 9, 944-957. Verna, J., Lodder, A., Lee, K., Vagts, A., and Ballester, R. (1997). A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 94, 13804-13809. Vida, T. A., and Emr, S. D. (1995). A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128, 779-792. Votsmeier, C., and Gallwitz, D. (2001). An acidic sequence of a putative yeast Golgi membrane protein 166 binds COPII and facilitates ER export. Embo J 20, 6742-6750. Wang, G., McCaffery, J. M., Wendland, B., Dupre, S., Haguenauer-Tsapis, R., and Huibregtse, J. M. (2001). Localization of the Rsp5p ubiquitin-protein ligase at multiple sites within the endocytic pathway. Mol Cell Biol 21, 3564-3575. Wang, G., Yang, J., and Huibregtse, J. M. (1999). Functional domains of the Rsp5 ubiquitin-protein ligase. Mol Cell Biol 19, 342-352. Wang, Q., and Chang, A. (1999). Eps1, a novel PDI-related protein involved in ER quality control in yeast. Embo J 18, 5972-5982. Wang, S., and Ng, D. T. (2010). Evasion of endoplasmic reticulum surveillance makes Wsc1p an obligate substrate of Golgi quality control. Mol Biol Cell 21, 1153-1165. Wang, Z. V., Schraw, T. D., Kim, J. Y., Khan, T., Rajala, M. W., Follenzi, A., and Scherer, P. E. (2007). Secretion of the adipocyte-specific secretory protein adiponectin critically depends on thiol-mediated protein retention. Mol Cell Biol 27, 3716-3731. Watanabe, R., and Riezman, H. (2004). Differential ER exit in yeast and mammalian cells. Curr Opin Cell Biol 16, 350-355. Weissman, A. M. (2001). Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol 2, 169-178. Wieland, F. T., Gleason, M. L., Serafini, T. A., and Rothman, J. E. (1987). The rate of bulk flow from the endoplasmic reticulum to the cell surface. Cell 50, 289-300. Wileman, T., Carson, G. R., Concino, M., Ahmed, A., and Terhorst, C. (1990). The gamma and epsilon subunits of the CD3 complex inhibit pre-Golgi degradation of newly synthesized T cell antigen receptors. J Cell Biol 110, 973-986. Wiseman, R. L., Powers, E. T., Buxbaum, J. N., Kelly, J. W., and Balch, W. E. (2007). An adaptable standard for protein export from the endoplasmic reticulum. Cell 131, 809-821. Wolins, N., Bosshart, H., Kuster, H., and Bonifacino, J. S. (1997). Aggregation as a determinant of protein fate in post-Golgi compartments: role of the luminal domain of furin in lysosomal targeting. J Cell Biol 139, 1735-1745. Wollert, T., and Hurley, J. H. (2010). Molecular mechanism of multivesicular body biogenesis by ESCRT complexes. Nature 464, 864-869. Wollert, T., Wunder, C., Lippincott-Schwartz, J., and Hurley, J. H. (2009). Membrane scission by the ESCRT-III complex. Nature 458, 172-177. Xie, W., Kanehara, K., and Ng, D. T. (2009). Intrinsic conformational determinants signal protein misfolding to the Hrd1/Htm1 ERAD system. Mol Biol Cell In Press. Xie, W., and Ng, D. T. (2010). ERAD substrate recognition in budding yeast. Semin Cell Dev Biol 21, 533539. Ye, Y., Shibata, Y., Yun, C., Ron, D., and Rapoport, T. A. (2004). A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature 429, 841-847. Yokouchi, M., Kondo, T., Houghton, A., Bartkiewicz, M., Horne, W. C., Zhang, H., Yoshimura, A., and Baron, R. (1999). Ligand-induced ubiquitination of the epidermal growth factor receptor involves the 167 interaction of the c-Cbl RING finger and UbcH7. J Biol Chem 274, 31707-31712. Zhang, B., Chang, A., Kjeldsen, T. B., and Arvan, P. (2001). Intracellular retention of newly synthesized insulin in yeast is caused by endoproteolytic processing in the Golgi complex. 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