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. Together all our data reveal a new function of the nucleus as a compartment central to the quality control of cytosolic proteins. viii LIST OF TABLES Table 2.1 List of yeast strains used in this study 33 Table 2.2 List of plasmids used in this study 38 Table 2.3 List of oligonucleotide primers used in this study 39 ix REFERENCES 1. Arndt V., Rogon C., and Höhfeld J. (2007). To be, or not to be-molecular chaperones in protein degradation. Cell. Mol. Life Sci. 64, 2525–2541. 2. Arndt V., Dick N., Tawo R., Dreiseidler M., Wenzel D., Hesse M., Fürst D. O., Saftig P., Saint R., Fleischmann B. K. (2010). Chaperone-assisted selective autophagy is essential for muscle maintenance. Curr. Biol. 20, 143–148. 3. Arnold I., and Langer T. (2002). Membrane protein degradation by AAA proteases in mitochondria. Biochim. Biophys. Acta. 1592, 89–96. 4. Ballinger C. A.,Connell P.,Wu Y.,Hu Z.,Thompson L. J.,Yin L. Y.,and Patterson C. (1999). Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions. Mol. Cell Biol. 19, 4535–4545. 5. Bartel B., Wunning I., and Varshavsky A. (1990). The recognition component of the N-end rule pathway. EMBO J. 9, 3179–3189. 6. Becker J., Walter W., Yan W., and Craig E. A. (1996). Functional interaction of cytosolic hsp70 and a DnaJ-related protein, Ydj1p, in protein translocation in vivo. Mol. Cell Biol. 16, 4378–4386. 7. Bhamidipati A., Denic V., Quan E. M., and Weissman J. S. (2005). Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen. Mol. Cell 19, 741–751. 8. Blachly-Dyson E., and Stevens T. H. (1987). Yeast carboxypeptidase Y can be translocated and glycosylated without its amino-terminal signal sequence. J. Cell Biol. 104, 1183–1191. 9. Blachly-Dyson E., and Stevens T. H. (1987). Yeast carboxypeptidase Y can be translocated and glycosylated without its amino-terminal signal sequence. J. Cell Biol. 104, 1183–1191. 10. Borkovich K. A., Farrelly F. W., Finkelstein D. B., Taulien J., and Lindquist S. (1989). Hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures. Mol. Cell Biol. 9, 3919–3930. 11. Braun S., Matuschewski K., Rape M., Thoms S., and Jentsch S. (2002) Role of the ubiquitin-selective CDC48(UFD1/NPL4) chaperone (segregase) in ERAD of OLE1 and other substrates. EMBO J. 21, 615–621. 12. Brehmer D., Rudiger S., Gassler C. S., Klostermeier D., Packschies L., Reinstein J., Mayer M. P., and Bukau B. (2001). Tuning of chaperone activity of Hsp70 proteins by modulation of nucleotide exchange. Nat. Struct. Biol. 8, 427–432. 13. Buchberger A., Bukau B., and Sommer T. (2010). Protein Quality Control in the Cytosol and the Endoplasmic Reticulum: Brothers in Arms. Molecular Cell 40, 238-252. 14. Bukau B., Horwich A. L. (1998). The Hsp70 and Hsp60 chaperone machines, Cell 92, 135 351–366. 15. Bukau B., Weissman J., and Horwich A. (2006). Molecular chaperones and protein quality control. Cell 125, 443–451. 16. Cajo G. C., Horne B. E., Kelley W. L., Schwager F., Georgopoulos C., and Genevaux P. (2006). The role of the DIF motif of the DnaJ (Hsp40) co-chaperone in the regulation of the DnaK (Hsp70) chaperone cycle. J. Biol. Chem. 281, 12436–12444. 17. Caplan A. J., Cyr D. M., and Douglas M. G. (1992). YDJ1p facilitates polypeptide translocation across different intracellular membranes by a conserved mechanism. Cell 71, 1143–1155. 18. Caplan A. J., and Douglas M. G. (1991). Characterization of YDJ1: a yeast homologue of the bacterial dnaJ protein. J. Cell Biol. 114, 609–621. 19. Carra S., Seguin S.J., Lambert H., and Landry J. (2008). HspB8 chaperone activity toward poly(Q)-containing proteins depends on its association with Bag3, a stimulator of macroautophagy. J. Biol. Chem. 283, 1437–1444. 20. 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. 21. Chadli A., Bouhouche I., Sullivan W., Stensgard B., McMahon N., Catelli M. G., and Toft D. O. (2000). Dimerization and N-terminal domain proximity underlie the function of the molecular chaperone heat shock protein 90. Proc. Natl. Acad. Sci. 97, 12524–12529. 22. Chalfie M., Tu Y., Euskirchen G., Ward W. W., and Prasher D. C. (1994). Green fluorescent protein as a marker for gene expression. Science 263, 802-805. 23. Cheetham M. E., and Caplan A. J. (1998). Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function. Cell Stress Chaperones 3, 28–36. 24. Chiti F., and Dobson C. M., (2006). Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333–366. 25. Chughtai Z. S., Rassadi R., Matusiewicz N., and Stochaj U. (2001). Starvation promotes nuclear accumulation of the hsp70 Ssa4p in yeast cells. J. Biol. Chem. 276, 20261–20266. 26. Ciechanover A., and Brundin P. (2003). The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron 40, 427–446. 27. 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. 28. Connell P., Ballinger C. A., Jiang J., Wu Y., Thompson L. J., Höhfeld J., and Patterson C. (2001). The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins. Nat. Cell Biol. 3, 93–96. 29. Craig E., Ziegelhoffer T., Nelson J., Laloraya S., and Halladay J. (1995). Complex multigene family of functionally distinct Hsp70s of yeast. Cold Spring Harb. Symp. 136 Quant. Biol. 60, 441–449. 30. Cuervo A.M., Stefanis L., Fredenburg R., Lansbury P.T., and Sulzer D. (2004) Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science 305, 1292–1295. 31. Cullen B. R. (2003). Nuclear mRNA export: insights from virology. Trends Biochem. Sci. 28, 419–424. 32. Cyr D. M. (1995). Cooperation of the molecular chaperone Ydj1 with specific Hsp70 homologs to suppress protein aggregation. FEBS Lett. 359, 129–132. 33. Demand J., Luders J., and Hohfeld J. (1998). The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors. Mol. Cell Biol. 18, 2023–2028. 34. Deng M., and Hochstrasser M. (2006). Spatially regulated ubiquitin ligation by an ER/nuclear membrane ligase. Nature 443, 827–831. 35. 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. 36. Deshaies R. J., Koch B. D., Werner-Washburne M., Craig E. A., and Schekman R. (1988). A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature 332, 800–805. 37. Deveraux Q., Ustrell V., Pickart C., and Rechsteiner M. A. (1994). 26S protease subunit that binds ubiquitin conjugates. J. Biol. Chem. 269, 7059–7061. 38. Diamant S., Ben-Zvi A. P., Bukau B., and Goloubinoff P. (2000). Size-dependent disaggregation of stable protein aggregates by the DnaK chaperone machinery. J. Biol. Chem. 275, 21107–21113. 39. Dice J. F. (2007). Chaperone-mediated autophagy. Autophagy 3, 295–299. 40. Dobson C. M. (2004). Principles of protein folding, misfolding and aggregation. Semin Cell Dev Biol. 15, 3–16. 41. Dragovic Z., Broadley S. A., Shomura Y., Bracher A., and Hartl F. U. (2006). Molecular chaperones of the Hsp110 family act as nucleotide exchange factors of Hsp70s. Embo J. 25, 2519–2528. 42. Dreveny I., Kondo H., Uchiyama K., Shaw A., Zhang X., and Freemont P. S. (2004). Structural basis of the interaction between the AAA ATPase p97/VCP and its adaptor protein p47. EMBO J. 23, 1030–1039. 43. Eisele F., and Wolf D. H. (2008). Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1. FEBS Lett. 582, 4143–4146. 44. Ellgaard L., and Helenius A. (2003). Quality control in the endoplasmic reticulum. Nat. Rev. Mol. Cell Biol. 4, 181–191. 45. Espelin C. W., Simons K. T., Harrison S. C., and Sorger P.K. (2003). Binding of the essential Saccharomyces cerevisiae kinetochore protein Ndc10p to CDEII. Mol Biol 137 Cell. 14, 557–568. 46. Esser C., Alberti S., and Hohfeld J. (2004). Cooperation of molecular chaperones with the ubiquitin/proteasome system. Biochim. Biophys. Acta 1695, 171–188. 47. Fan C. Y., Lee S., Ren H. Y., and Cyr D. M. (2004). Exchangeable chaperone modules contribute to specification of type I and type II Hsp40 cellular function. Mol. Biol. Cell 15, 761–773. 48. 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. 49. Flaherty K. M., Deluca-Flaherty C., and Mckay D. B. (1990). Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature 346, 623–628. 50. Fleming J. A., Lightcap E. S., Sadis S., Thoroddsen V., Bulawa C. E., and Blackman R. K. (2002). Complementary whole-genome technologies reveal the cellular response to proteasome inhibition by PS-341. Proc. Natl. Acad. Sci. USA 99, 1461–1466. 51. Flynn G. C., Chappell T. G., and Rothman J. E. (1989). Peptide binding and release by proteins implicated as catalysts of protein assembly. Science 245, 385–390. 52. Flynn G. C., Pohl J., Flocco M. T., Rothman J. E. (1991). Peptide-binding specificity of the molecular chaperone BiP. Nature 353, 726–30. 53. Frydman J. (2001). Folding of newly translated proteins in vivo: the role of molecular chaperones, Annu. Rev. Biochem. 70, 603–647. 54. Gamerdinger M., Hajieva P., Kaya A. M., Wolfrum U., Hartl F. U., and Behl C. (2009). Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3. EMBO J. 28, 889–901. 55. Gardner R. G., Nelson Z. W., and Gottschling D. E. (2005). Degradation-mediated protein quality control in the nucleus. Cell 120, 803–815. 56. 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. 57. Garza R. M., Sato B. K., and Hampton R. Y. (2009). In vitro analysis of Hrd1pmediated retrotranslocation of its multispanning membrane substrate 3-hydroxy- 3-methylglutaryl (HMG)-CoA reductase. J. Biol. Chem. 284, 14710–14722. 58. Gassler C. S., Wiederkehr T., Brehmer D., Bukau B., and Mayer M. P. (2001). Bag-1M accelerates nucleotide release for human Hsc70 and Hsp70 and can act concentration-dependent as positive and negative cofactor. J. Biol. Chem. 276, 32538–32544. 59. Gauss R., Sommer T., and Jarosch E. (2006). The Hrd1p ligase complex forms a linchpin between ER-lumenal substrate selection and Cdc48p recruitment. Embo J. 138 25, 1827–1835. 60. Geoffrey S. W. Blake M. S., Joel B., and Thomas C. T. (1999). Rapid protein-folding assay using green fluorescent protein. Nature Biotechnology 17, 691–695. 61. Glickman M. H., Rubin D. M., Coux O., Wefes I., Pfeifer G., Cjeka Z., Baumeister W., Fried V. A., and Finley D. (1998). A subcomplex of the proteasome regulatory particle required for ubiquitin–conjugate degradation and related to the COP9-signalosome and eIF3. Cell 94, 615–623. 62. Glover J. R., and Lindquist S. (1998). Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94, 73–82. 63. Goeckeler J. L., Petruso A. P., Aguirre J., Clement C. C., Chiosis G., and Brodsky J. L. (2008). The yeast Hsp110, Sse1p, exhibits high-affinity peptide binding. FEBS Lett. 582, 2393–2396. 64. Goloubinoff P., and De Los Rios P. (2007). The mechanism of Hsp70 chaperones: (entropic) pulling the models together. Trends Biochem. Sci. 32, 372–380. 65. Grenert J. P., Johnson B. D., and Toft D. O. (1999). The importance of ATP binding and hydrolysis by hsp90 in formation and function of protein heterocomplexes. J. Biol. Chem. 274, 17525–17533. 66. Guthrie C., and Fink G. R. (1991). Guide to yeast genetics and molecular biology, in: J.N. Abelson, M.I. Simon (Eds.), Methods in Enzymology, vol. 194, Academic Press, San Diego, p. 933. 67. Hampton R. Y., and Garza R. M. (2009). Protein Quality Control as a Strategy for Cellular Regulation: Lessons from Ubiquitin-Mediated Regulation of the Sterol Pathway. Chem. Rev. 109, 1561–1574. 68. Hampton R. Y. (2002). ER-associated degradation in protein quality control and cellular regulation. Curr. Opin. Cell Biol. 14, 476–482. 69. Hampton R. Y., Gardner R. G., and Rine J. (1996). Role of 26S proteasome and HRD genes in the degradation of 3- hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. Mol. Biol. Cell 7, 2029–2044. 70. Han W., and Christen P. (2003). Interdomain communication in the molecular chaperone DnaK. Biochem. J. 369, 627–634. 71. Hara T., Nakamura K., Matsui M., Yamamoto A., Nakahara Y., Suzuki-Migishima R., Yokoyama M., Mishima K., Saito I., Okano H., and Mizushima N. (2006). Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 441, 885–889. 72. Harris S. F., Shiau A. K., and Agard D. A. (2004). The crystal structure of the carboxy-terminal dimerization domain of htpG, the Escherichia coli Hsp90, reveals a potential substrate binding site. Structure 12, 1087–97. 73. Hartl F. U., and Hayer-Hartl M. (2002). Molecular chaperones in the cytosol: from nascent chain to folded protein, Science 295, 1852–1858. 139 74. Hartl F. U. and Hayer-Hartl M. (2009). Converging concepts of protein folding in vitro and in vivo. Nat. Struct. Mol. Biol. 16, 574–581. 75. Hartmann-Petersen R., Seeger M., and Gordon C. (2003). Transferring substrates to the 26S proteasome. Trends Biochem. Sci. 28, 26–31. 76. Heck J. W., Cheung S. K., and Hampton R. Y. (2010). Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1. Proc. Natl. Acad. Sci. USA 107, 1106–1111. 77. Helenius A., and Aebi M. (2004). Roles of N-linked glycans in the endoplasmic reticulum. Annu. Rev. Biochem. 73, 1019–1049. 78. Hennessy F., Cheetham M. E., Dirr H. W., and Blatch G. L. (2000). Analysis of the levels of conservation of the J domain among the various types of DnaJ-like proteins. Cell Stress Chaperones 5, 347–358. 79. Hennessy F., Nicoll W. S., Zimmermann R., Cheetham M. E., and Blatch G. L. (2005). Not all domains are created equal: implications for the specificity of Hsp40-Hsp70 interactions. Protein Sci. 14, 1697–709. 80. Herscovics A. (2001). Structure and function of Class I alpha 1,2-mannosidases involved in glycoprotein synthesis and endoplasmic reticulum quality control. Biochimie 83, 757–762. 81. Hershko A., and Ciechanover A. (1998). The ubiquitin system. Annu Rev Biochem 67, 425–479. 82. Hettema E. H., Ruigrok C. C., Koerkamp M. G., van den Berg M., Tabak H. F., Distel B., and Braakman I. (1998). The cytosolic DnaJ-like protein djp1p is involved specifically in peroxisomal protein import. J. Cell Biol. 142, 421–434. 83. Hiller M. M , Finger A., Schweiger M., and Wolf D. H. (1996). ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science 273, 1725–1728. 84. Hirsch C., Gauss R., Horn S. C., Neuber O., and Sommer T. (2009). The ubiquitylation machinery of the endoplasmic reticulum. Nature 458, 453–460. 85. Horton L. E., James P., Craig E. A., and Hensold J. O. (2001). The yeast hsp70 homologue Ssa is required for translation and interacts with Sis1 and Pab1 on translating ribosomes. J. Biol. Chem. 276, 14426–14433. 86. Horton P., Park K. J., Obayashi T., Fujita N., Harada H., Adams-Collier C. J., and Nakai K. (2007). WoLF PSORT: protein localization predictor. Nucleic Acids Res 35, W585–587. 87. Hoseki J., Ushioda R., and Nagata K. (2010). Mechanism and components of endoplasmic reticulum associated Degradation. J. Biochem. 147, 19–25. 88. Huyer G., Piluek W. F., Fansler Z., Kreft S. G., Hochstrasser M., Brodsky J. L., and Michaelis S. (2004). Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein. J. Biol. Chem. 279, 38369–38378. 140 89. Ingolia T. D., Slater M. R., and Craig E. A. (1982). Saccharomyces cerevisiae contains a complex multigene family related to the major heat shock-inducible gene of Drosophila. Mol. Cell. Biol. 2, 1388–1398. 90. Ismail N., and Ng D. T. (2006). Have you HRD? Understanding ERAD Is DOAble!. Cell 126, 237–239. 91. Iwata A., Christianson J. C., Bucci M., Ellerby L. M., Nukina N., Forno L. S., and Kopito R. (2005). Increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation. Proc. Natl. Acad. Sci. USA 102, 13135–13140. 92. Jakob C. A., Bodmer D., Spirig U., Battig P., Marcil A. (2001). Htm1p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast. EMBO Rep. 2, 423–430. 93. Jarosch E., Taxis C., Volkwein C., Bordallo J., Finley D., Wolf D. H., and Sommer T. (2002). Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Nat. Cell Biol. 4, 134–139. 94. Johnston, J. A., Ward, C. L., and Kopito, R. R. (1998). Aggresomes: a cellular response to misfolded proteins. J. Cell Biol. 143, 1883–1898. 95. Jordan R., and McMacken R. (1995). Modulation of the ATPase activity of the molecular chaperone DnaK by peptides and the DnaJ and GrpE heat shock proteins. J. Biol. Chem. 270, 4563–4569. 96. Kabani M., Kelley S. S., Morrow M. W., Montgomery D. L., Sivendran R. (2003). Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP. Mol. Biol. Cell 14, 3437–3448. 97. Kabani M., Beckerich J. M., and Brodsky J. L. (2002). Nucleotide exchange factor for the yeast Hsp70 molecular chaperone Ssa1p. Mol. Cell Biol. 22, 4677–4689. 98. Kabani M., McLellan C., Raynes D. A., Guerriero V., and Brodsky J. L. (2002). HspBP1, a homologue of the yeast Fes1 and Sls1 proteins, is an Hsc70 nucleotide exchange factor. FEBS Lett. 531, 339–342. 99. Kabani M., Kelley S. S., Morrow M. W., Montgomery D. L., Sivendran R., Rose M. D., Gierasch L. M., and Brodsky J. L. (2003). Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP. Mol. Biol. Cell 14, 3437– 3448. 100. Kaganovich D., Kopito R., and Frydman J. (2008). Misfolded proteins partition between two distinct quality control compartments. Nature 454, 1088–1095. 101. Karzai A. W., and McMacken R. (1996). A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein. J. Biol. Chem. 271, 11236–11246 102. Kawaguchi Y., Kovacs J. J., McLaurin A., Vance J. M., Ito A., and Yao T. P. (2003). The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell 115, 727–738. 103. Kerscher O., Felberbaum R., and Hochstrasser M. (2006). Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell. Dev. Biol. 22, 159–180. 141 104. Kettern N., Dreiseidler M., Tawo R. and Höhfeld J. (2010). Chaperone-assisted degradation: multiple paths to destruction. J. Biol. Chem. 391, 481–489. 105. Kiffin R., Christian C., Knecht E., and Cuervo A.M. (2004). Activation of chaperone-mediated autophagy during oxidative stress. Mol. Biol. Cell. 15, 4829–4840. 106. Kim W., Spear E. D., and Ng D. T. (2005). Yos9p detects and targets misfolded glycoproteins for ER-associated degradation. Mol. Cell 19, 753–764. 107. Kirkin V., Lamark T., Sou Y. S., Bjorkoy G., Nunn J. L., Bruun J. A., Shvets E., McEwan D. G., Clausen T. H., Wild P., Bilusic I., Theurillat J. P., Overvatn A., Ishii T., Elazar Z., Komatsu M., Dikic I., and Johansen T. (2009). A role for NBR1 in autophagosomal degradation of ubiquitinated substrates. Mol. Cell 33, 505–516. 108. Klionsky D. J., Banta L. M., and Emr S. D. (1988). Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information. Mol. Cell. Biol. 8, 2105–2116. 109. Knop M., Finger A., Braun T., Hellmuth K., and Wolf D. H. (1996). Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. EMBO J. 15, 753–763. 110. Komatsu M., Waguri S., Chiba T., Murata S., Iwata J., Tanida I., Ueno T., Koike M., Uchiyama Y., Kominami E., and Tanaka K. (2006). Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 441, 880–884. 111. Komatsu M., Waguri S., Koike M., Sou Y. S., Ueno T., Hara T., Mizushima N., Iwata J., Ezaki J., Murata S., Hamazaki J., Nishito Y., Iemura S., Natsume T., Yanagawa T., Uwayama J., Warabi E., Yoshida H., Ishii T., Kobayashi A., Yamamoto M., Yue Z., Uchiyama Y., Kominami E., and Tanaka K. (2007). Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 131, 1149–1163. 112. Kopito R. R. (2000). Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol. 10, 524–530. 113. Kruse K. B., Brodsky J. L., and McCracken A. A. (2006). Autophagy: an ER protein quality control process. Autophagy 2, 135–137. 114. Kruse K. B., Brodsky J. L., and McCracken A. A. (2006). Characterization of an ERAD ene 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. 115. Kryndushkin D. S., Smirnov V. N., Ter-Avanesyan M. D., and Kushnirov V. V. (2002). Increased expression of Hsp40 chaperones, transcriptional factors, and ribosomal protein Rpp0 can cure yeast prions. J. Biol. Chem. 277, 23702–23708 116. Lam Y. A., Lawson T. G., Velayutham M., Zweier J. L., and Pickart, C. M. (2002). A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal. Nature 416, 763–767. 117. Langer T., Lu C., Echols H., Flanagan J., Hayer M. K., and Hartl F. U. (1992). 142 Successive action of DnaK, DnaJ and GroEL along chaperone-mediated protein folding. Nature 356, 683–689. the pathway of 118. Laporte D., Salin B., Daignan-Fornier B., and Sagot I. (2008). Reversible cytoplasmic localization of the proteasome in quiescent yeast cells. J. Cell Biol. 181, 737–745. 119. Laufen T., Mayer M. P., Beisel C., Klostermeier D., Mogk A., Reinstein J., Bukau B. (1999). Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones. Proc Natl Acad Sci USA 96, 5452–5457. 120. Lee C., Schwartz M. P., Prakash S., Iwakura M., and Matouschek A. (2001). ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. Mol. Cell 7, 627–637. 121. Lewis M. J., and Pelham H. R. (2009). Inefficient quality control of thermosensitive proteins on the plasma membrane. PLoS One 4, e5038. 122. Lian H. Y., Zhang H., Zhang Z. R., Loovers H. M., Jones G. W., Rowling P. J., Itzhaki L. S., Zhou J. M., and Perrett S. (2007). Hsp40 interacts directly with the native state of the yeast prion protein Ure2 and inhibits formation of amyloid-like fibrils. J. Biol. Chem. 282, 11931–11940. 123. Liberek K., Marszalek J., Ang D., Georgopoulos C., Zylicz M. (1991). Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc. Natl. Acad. Sci. USA 88, 2874–2878. 124. Lipford J. R., Smith G. T., Chi Y., and Deshaies R. J. (2005). A putative stimulatory role for activator turnover in gene expression. Nature 438, 113–116. 125. Loayza D., Tam A., Schmidt W.K., and Michaelis S. (1998). Ste6p mutants defective in exit from the endoplasmic reticulum (ER) reveal aspects of an ER quality control pathway in Saccharomyces cerevisiae. Mol. Biol. Cell. 9, 2767–2784. 126. Lu Z., and Cyr D. M. (1998). Protein folding activity of Hsp70 is modified differentially by the hsp40 cochaperones Sis1 and Ydj1. J. Biol. Chem. 273, 27824–27830. 127. Lupas A., Koster A. J., and Baumeister W. (1993). Structural features of 26S and 20S proteasomes. Enzyme Protein 47, 252–273. 128. Macara I. G. (2001). Transport into and out of the nucleus. Microbiol. Mol. Biol. Rev. 65, 570–594. 129. Maruya M., Sameshima M., Nemoto T., and Yahara I. (1999). Monomer arrangement in HSP90 dimer as determined by decoration with N and C-terminal region specific antibodies. J. Mol. Biol. 285, 903–907. 130. Mayer M. P., and Bukau B. (2005). Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol. Life Sci. 62, 670–684. 131. McCarty J. S., Buchberger A., Reinstein J., and Bukau B. (1995). The role of ATP in the functional cycle of the DnaK chaperone system. J. Mol. Biol. 249, 126–37. 132. McClellan A. J., Scott M. D., and Frydman J. (2005). Folding and quality control of 143 the HL tumor suppressor proceed through distinct chaperone pathways. Cell 121, 739–748. 133. McClellan A. J., Tam A., Kaganovich D., and Frydman J. (2005). Protein quality control: chaperones culling corrupt conformations. Nat. Cell. Biol. 7, 736–741. 134. McDonough H., and Patterson C. (2003). CHIP: a link between, the chaperone and proteasome systems. Cell Stress Chaperones 8, 303–308. 135. Metzger M. B., and Michaelis S. (2009). Analysis of quality control substrates in distinct cellular compartments reveals a unique role for Rpn4p in tolerating misfolded membrane proteins. Mol. Biol. Cell. 20, 1006–1019. 136. Metzger M. B., Maurer M. J., Dancy B. M., and Michaelis S. (2008). Degradation of a cytosolic protein requires endoplasmic reticulum-associated degradation machinery. J. Biol. Chem. 283, 32302–32316. 137. Meyer P., Prodromou C., Hu B., Vaughan C., and Roe S. M. (2003). Structural and functional analysis of the middle segment of hsp90: implications for ATP hydrolysis and client protein and cochaperone interactions. Mol. Cell 11, 647–658. 138. Mizushima N., Levine B., Cuervo A. M., and Klionsky D. J. (2008). Autophagy fights disease through cellular self-digestion. Nature 451, 1069–1075. 139. Molinari M., Calanca V., Galli C., Lucca P., Paganetti P. (2003). Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle. Science 299, 1397–400. 140. Morimoto R. I. (2008). Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging. Genes Dev. 22, 1427–1438. 141. Murata S., Minami Y., Minami M., Chiba T., and Tanaka K. (2001). CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein. EMBO Rep. 2, 1133–1138. 142. Nakatogawa H., Suzuki K., Kamada Y., and Ohsumi Y. (2009). Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat. Rev. Mol. Cell Biol. 10, 458–467. 143. Nakatsukasa K., and Brodsky J. L. (2008). The recognition and retrotranslocation of misfolded proteins from the endoplasmic reticulum. Traffic 9, 861–870. 144. Neuber O., Jarosch E., Volkwein C., Walter J., and Sommer T. (2005) Ubx2 links the Cdc48 complex to ERassociated protein degradation. Nat. Cell Biol. 7, 993–998. 145. Ng D.T. (2005). Screening for mutants defective in secretory protein maturation and ER quality control. Methods 35, 366-372. 146. Nillegoda N. B., Theodoraki M. A., Mandal A. K., Mayo K. J., Ren H. Y., Sultana R., Wu K., Johnson J., Cyr D. M., and Caplan A. J. (2010). Ubr1 and Ubr2 function in a quality control pathway for degradation of unfolded cytosolic proteins. Mol. Biol. Cell 21, 2102–2116 147. Nishikawa S, Brodsky JL, Nakatsukasa K. (2005). Roles of molecular chaperones in endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD). 144 J. Biochem. (Tokyo) 137, 551– 555. 148. Nishikawa S. I., Fewell S. W., Kato Y., Brodsky J. L., and Endo T. (2001). Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation. J. Cell Biol. 153, 1061–1070. 149. Nuoffer C., Jeno P., Conzelmann A., and Riezman H. (1991). Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiae GAS1 protein to the plasma membrane. Mol. Cell. Biol. 11, 27–37. 150. Obermann W. M., Sondermann H., Russo A. A., Pavletich N. P., and Hartl F. U. (1998). In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis. J. Cell Biol. 143, 901–910. 151. Ormo M., Cubitt A. B., Kallio K., Gross L. A., Tsien R. Y., and Remington S. J. (1996). Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395. 152. Panaretou B., Prodromou C., Roe S. M., O’Brien R., Ladbury J. E., and Piper P. W. (1998). ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo. EMBO J. 17, 4829–36. 153. 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 import-incompetent proteins to degradation via the ubiquitin-proteasome system. Mol. Biol. Cell 18, 153–165. 154. Pickart C. M. (2001). Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533. 155. Pickart C. M., and Cohen R. E. (2004). Proteasomes and their kin: proteases in the machine age. Nat. Rev. Mol. Cell Biol. 5, 177–187. 156. Plemper R. K., Böhmler S., Bordallo J., Sommer T., and Wolf D. H. (1997). Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation. Nature 388, 891–895. 157. Plemper R. K., Bordallo J., Deak P. M., Taxis C., Hitt R., and Wolf D. H. (1999). Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation. J. Cell Sci. 112, 4123–4134. 158. Polier S., Dragovic Z., Hartl F. U., and Bracher A. (2008). Structural basis for the cooperation of Hsp70 and Hsp110 chaperones in protein folding. Cell 133, 1068–1079. 159. Powers E. T., Morimoto R. I., Dillin A., Kelly J. W., and Balch W. E. (2009). Biological and chemical approaches to diseases of proteostasis deficiency. Annu. Rev. Biochem. 78, 959–991. 160. Prasad R., Kawaguchi S., and Ng D. T. (2010). A nucleus-based quality control mechanism for cytosolic proteins. Mol. Biol. Cell 21, 2117–2127. 161. Prip-Buus C., Westerman B., Schmitt M., Langer T., Neupert W. and Schwarz E. (1996). Role of the mitochondrial DnaJ homologue, Mdj1p, in the prevention of 145 heat-induced protein aggregation. FEBS Lett. 380, 142–146. 162. Prodromou C., Panaretou B., Chohan S., Siligardi G., O’Brien R., and Ladbury J. E. (2000). The ATPase cycle of Hsp90 drives a molecular ‘clamp’ via transient dimerization of the N-terminal domains. EMBO J. 19, 4383–92. 163. Prodromou C., Roe S. M., O’Brien R., Ladbury J. E., Piper P. W., and Pearl L. H. (1997). Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell 90, 65–75. 164. Qiu X. B., Shao Y. M., Miao S., and Wang L. (2006). The diversity of the DnaJ/Hsp40 family, the crucial partners for Hsp70 chaperones. Cell Mol. Life. Sci. 63, 2560–2570. 165. 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, 870–877. 166. 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. 167. Raviol H., Sadlish H., Rodriguez F., Mayer M. P., and Bukau B. (2006). Chaperone network in the yeast cytosol: Hsp110 is revealed as an Hsp70 nucleotide exchange factor. EMBO J. 25, 2510–2518. 168. Rechsteiner M., and Hill C. P. (2005). Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors. Trends Cell Biol. 15, 27–33. 169. Reits E. A. J., Benham A. M., Plougastel B., Neefjes J., and Trowsdale J. (1997). Dynamics of proteasome distribution in living cells. EMBO J. 16, 6087–6094. 170. Richly H., Rape M., Braun S., Rumpf S., Hoege C., and Jentsch S. (2005). A series of ubiquitin binding factors connects CDC48/p97 to substrate multiubiquitylation and proteasomal targeting. Cell 120, 73–84. 171. Rock K. L., Gramm C., Rothstein L., Clark K., Stein R., Dick L., Hwang D., and Goldberg A. L. (1994). Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78, 761–771. 172. Romisch K. (2005). Endoplasmic Reticulum-Associated Degradtion. Annu. Rev. Cell Dev. Biol. 21, 435–56. 173. Rosser M. F., Washburn E., Muchowski P. J., Patterson C., and Cyr D. M. (2007). Chaperone functions of the E3 ubiquitin ligase CHIP. J. Biol. Chem. 282, 22267–22277. 174. Rubinsztein D. C. (2006). The roles of intracellular protein-degradation pathways in neurodegeneration. Nature 443, 780–786. 175. Rüdiger S., Germeroth L., Schneider-Mergener J., and Bukau B. (1997). Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 16, 1501–1507. 146 176. Russell R., Wali Karzai A., Mehl A. F., and McMacken R. (1999). DnaJ dramatically stimulates ATP hydrolysis by DnaK: insight into targeting of Hsp70 proteins to polypeptide substrates. Biochemistry 38, 4165–4176. 177. Russell, S. J., Steger, K. A., and Johnston, S. A. (1999). Subcellular localization, stoichiometry, and protein levels of 26 S proteasome subunits in yeast. J. Biol. Chem. 274, 21943–21952. 178. Sambrook, J., Fritsch, E. M., and Maniatis, T. (1989). Molecular Cloning: a laboratory manual, 2nd edn (Plainview, Cold Spring Harbor Laboratory Press). 179. Schuberth C., and Buchberger A. (2005). Membrane-bound Ubx2 recruits Cdc48 to ubiquitin ligases and their substrates to ensure efficient ER-associated protein degradation. Nat. Cell Biol. 7, 999–1006. 180. Schwimmer C., and Masison D. C. (2002). Antagonistic interactions between yeast [PSI+] and [URE3] prions and curing of [URE3] by Hsp70 protein chaperone Ssa1p but not by Ssa2p. Mol. Cell Biol. 22, 3590–3598. 181. Shaner L., Wegele H., Buchner J., and Morano K. A. (2005). The yeast Hsp110 Sse1 functionally interacts with the Hsp70 chaperones Ssa and Ssb. J. Biol. Chem. 280, 41262–41269. 182. Sharma D., and Masison D. C. (2009). Hsp70 Structure, Function, Regulation and Influence on Yeast Prions. Protein Pept. Lett. 16, 571–581. 183. Shi Y., Mosser D. D., and Morimoto R. I. (1998). Molecular chaperones as HSF1-specific transcriptional repressors. Genes Dev. 12, 654–666. 184. Sommer T., and Jentsch S. (1993). A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature 365, 176–179. 185. Shomura Y., Dragovic Z., Chang H. C., Tzvetkov N., Young J. C., Brodsky J. L., Guerriero V., Hartl F. U., and Bracher A. (2005). Regulation of Hsp70 function by HspBP1: structural analysis reveals an alternate mechanism for Hsp70 nucleotide exchange. Mol. Cell 17, 367–379. 186. Shulga N., Roberts P., Gu Z., Spitz L., Tabb M. M., Nomura M., and Goldfarb D. S. (1996). In vivo nuclear transport kinetics in Saccharomyces cerevisiae: a role for heat shock protein 70 during targeting and translocation. J. Cell Biol. 135, 329–339. 187. Skowyra D., Georgopoulos C., and Zylicz M. (1990). The E. coli dnaK gene product, the hsp70 homolog, can reactivate heat-inactivated RNA polymerase in an ATP hydrolysisdependent manner. Cell 62, 939–944. 188. 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. 189. Stade K., Ford C. S., Guthrie C., and Weis K. (1997). Exportin (Crm1p) is an essential nuclear export factor. Cell 90, 1041–1050. 190. Stankiewicz M., Nikolay R., Rybin V., and Mayer M. P. (2010). CHIP participates in protein triage decisions by preferentially ubiquitinating Hsp70-bound substrates. FEBS J. 277, 3353–3367. 147 191. Stebbins C. E., Russo A. A., Schneider C., Rosen N., Hartl F. U., and Pavletich N. P. (1997). Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell 89, 239– 250. 192. Stirling C. J., and Hewitt E. W. (1992). The Saccharomyces cerevisiae SEC65 gene encodes a component of the yeast signal recognition particle with homology to human SRP19. Nature 356, 534–537. 193. Swanson R., Locher M., and Hochstrasser M. (2001). A conserved ubiquitin ligase of the nuclear. Genes Dev. 20, 2660-2674 194. Szabo A., Langer T., Schroder H., Flanagan J., and Bukau B. (1994). The ATP hydrolysisdependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE. Proc. Natl. Acad. Sci. USA 91, 10345–10349. 195. Szathmary R., Bielmann R., Nita-Lazar M., Burda P., and Jakob C. A. (2005). Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD. Mol Cell 19, 765–775. 196. Takayama S., Bimston D. N., Matsuzawa S., Freeman B. C., Aime-Sempe C., Xie Z. (1997). BAG-1 modulates the chaperone activity of Hsp70/Hsc70. EMBO J. 16, 4887–4896. 197. Talcott B., and Moore M. S. (1999). Getting across the nuclear pore complex. Trends Cell Biol. 9, 312–318. 198. Taniuchi H., and Anfinsen C. B. (1969). An experimental approach to the study of the folding of staphylococcal nuclease. J. Biol. Chem. 244, 3864–3875. 199. Terada K., and Mori M. (2000). Human DnaJ homologs dj2 and dj3, and bag-1 are positive cochaperones of hsc70. J. Biol. Chem. 275, 24728–24734. 200. Teresa M. B., Christine M. W., and Jeffrey L. B. (2007). The Activities and Function of Molecular Chaperones in the Endoplasmic Reticulum. Semin Cell Dev. Biol. 18, 751–761. 201. Theyssen H., Schuster H. P., Packschies L., Bukau B., and Reinstein J. (1996). The second step of ATP binding to DnaK induces peptide release. J. Mol. Biol. 263, 657–70. 202. Tian G., Xiang S., Noiva R., Lennarz W. J., and Schindelin H. (2006). The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites. Cell 124, 61–73. 203. 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. 204. Tsien R. Y. (1998). The green fluorescent protein. Annu. Rev. Biochem. 67, 509-544. 205. Tu B. P., Weissman J. S. (2004). Oxidative protein folding in eukaryotes: mechanisms and consequences. J. Cell Biol. 164, 341–346. 206. Ungewickell E., Ungewickell H. Holstein S. E., Lindner R., and Prasad K. (1995). 148 Role of auxilin in uncoating clathrin-coated vesicles. Nature 378, 632–635. 207. van der Straten A., Rommel C., Dickson B., and Hafen E. (1997). The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila. EMBO J. 16, 1961–1969. 208. 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. 209. 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. 210. 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. 211. Verma R., and Deshaies R. (2000). A proteasome howdunit: the case of the missing signal. Cell 101, 341–344. 212. Vogel J. P., Misra L. M., and Rose M. D. (1990). Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast. J. Cell Biol. 110, 1885–1895. 213. Walsh P., Bursac D., Law Y. C., Cyr D., and Lithgow T. (2004). The J-protein family: modulating protein assembly, disassembly and translocation. EMBO Rep. 5, 567–571. 214. Wang T. F., Chang J. H., and Wang C. (1993). Identification of the peptide binding domain of hsc70. 18-Kilodalton fragment located immediately after ATPase domain is sufficient for high affinity binding. J. Biol. Chem. 268, 26049–26051. 215. Wegele H., Haslbeck M., Reinstein J., and Buchner J. (2003). Sti1 is a novel activator of the Ssa proteins. J. Biol. Chem. 278, 25970–25976. 216. Weissman A. M. (2001). Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell. Biol. 2, 169–178. 217. Werner-Washburne M., Stone D. E., and Craig E. A. (1987). Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevisiae. Mol. Cell Biol. 7, 2568–2577. 218. Wu Y., Swulius M. T., Moremen K. W., Sifers R. N. (2003). Elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation. Proc. Natl. Acad. Sci. USA 100, 8229–8234. 219. Xie W., and Ng D. T. (2010). ERAD substrate recognition in budding yeast. Semin Cell Dev. Biol. 21, 533–539. 220. Xie Y., and Varshavsky A. (2000). Physical association of ubiquitin ligases and the 26S proteasome. Proc. Natl. Acad. Sci. USA 97, 2497–502 221. Ye Y., Meyer H. H., and Rapoport T. A. (2001). The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414, 652–656. 222. Ye Y., Shibata Y., Yun C., Ron D., and Rapoport T. A. (2004) A membrane protein complex mediates retrotranslocation from the ER lumen into the cytosol. Nature 429, 149 841–847. 223. Yoon H. J., and Carbon J. (1995). Genetic and biochemical interactions between an essential kinetochore protein, Cbf2p/Ndc10p, and the CDC34 ubiquitin-conjugating enzyme. Mol. Cell. Biol. 15, 4835–4842. 224. Youker R. T., Walsh P., Beilharz T., Lithgow T., and Brodsky J. L. (2004). Distinct roles for the Hsp40 and Hsp90 molecular chaperones during cystic fibrosis transmembrane conductance regulator degradation in yeast. Mol. Biol. Cell 15, 4787–4797. 225. Young J. C., Agashe V. R., Siegers K., and Hartl F. U. (2004). Pathways of chaperone-mediated protein folding in the cytosol. Nat. Rev. Mol. Cell. Biol. 5, 781–791. 226. Zaher, H. S., and Green, R. (2009). Fidelity at the molecular level: lessons from protein synthesis. Cell 136, 746–762. 227. Zemp I., and Kutay U. (2007). Nuclear export and cytoplasmic maturation of ribosomal subunits. FEBS Lett. 581, 2783–2793. 228. Zhang Y., Nijbroek G., Sullivan M. L., McCracken A. A., Watkins S. C., Michaelis S., and Brodsky J. L. (2001). Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast. Mol. Biol. Cell 12, 1303–1314. 229. Zhao R., and Houry W. A. (2005). Hsp90: a chaperone for protein folding and gene regulation. Biochem. Cell Biol. 83, 703–710. 230. Zhu X., Zhao X., Burkholder W. F., Gragerov A., Ogata C. M., Gottesman M. E., and Hendrickson W. A. (1996). Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272, 1606–1614. 231. Zou J., Guo Y., Guettouche T., Smith D. F., and Voellmy R. (1998). Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell 94, 471–480. 150 [...]... 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