Skip to main content
SpringerLink
Log in

Use of CPY* and Its Derivatives to Study Protein Quality Control in Various Cell Compartments

  • Protocol
  • First Online:
Ubiquitin Family Modifiers and the Proteasome

Part of the book series: Methods in Molecular Biology ((MIMB,volume 832))

Abstract

Mutated derivatives of carboxypeptidase yscY (CPY) are potent substrates to study protein quality control and protein degradation in different cell compartments in yeast. Depending on the subcellular compartment of interest, the design of the model substrate used has to be adapted. Here, we describe the derivatives of CPY* used in genetic screens based on a sensitive growth test in order to identify new components of the protein quality control systems in different degradation pathways (see Notes 1–3).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Wolf DH, Fink GR (1975) Proteinase C (carboxypeptidase Y) mutant of yeast. J Bacteriol 123:1150–1156.

    PubMed  CAS  Google Scholar 

  2. Heinemeyer W, Kleinschmidt JA, Saidowsky J et al (1991) Proteinase yscE, the yeast proteasome/multicatalytic-multifunctional proteinase: mutants unravel its function in stress induced proteolysis and uncover its necessity for cell survival. EMBO J 10:555–562.

    PubMed  CAS  Google Scholar 

  3. Wolf DH, Hilt W (2004) The proteasome: a proteolytic nanomachine of cell regulation and waste disposal. Biochim Biophys Acta 1695:19–31.

    Article  PubMed  CAS  Google Scholar 

  4. Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479.

    Article  PubMed  CAS  Google Scholar 

  5. Varshavsky A (1997) The ubiquitin system. Trends Biochem Sci 22:383–387.

    Article  PubMed  CAS  Google Scholar 

  6. Klausner RD, Sitia R (1990) Protein degradation in the endoplasmic reticulum. Cell 62:611–614.

    Article  PubMed  CAS  Google Scholar 

  7. Bonifacino JS, Lippincott-Schwartz J (1991) Degradation of proteins within the endoplasmic reticulum. Curr Opin Cell Biol 3:592–600.

    Article  PubMed  CAS  Google Scholar 

  8. Cheng SH, Gregory RJ, Marshall J et al (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63:827–834.

    Article  PubMed  CAS  Google Scholar 

  9. Jensen TJ, Loo MA, Pind S et al (1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83:129–135.

    Article  PubMed  CAS  Google Scholar 

  10. Ward CL, Omura S, Kopito RR (1995) Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83:121–127.

    Article  PubMed  CAS  Google Scholar 

  11. Sommer T, Jentsch S (1993) A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature 365:176–179.

    Article  PubMed  CAS  Google Scholar 

  12. Finger A, Knop M, Wolf DH (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.

    Article  PubMed  CAS  Google Scholar 

  13. Knop M, Finger A, Braun T et al (1996) Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. EMBO J 15:753–763.

    PubMed  CAS  Google Scholar 

  14. Hiller MM, Finger A, Schweiger M, Wolf DH (1996) ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science 273:1725–1728.

    Article  PubMed  CAS  Google Scholar 

  15. Linder P, Shore D, Hall MN. (2006) Landmark Papers in Yeast Biology, in Cold Spring Harbor Laboratory Press, pp 273–274.

    Google Scholar 

  16. Hampton RY, Gardner RG, 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.

    PubMed  CAS  Google Scholar 

  17. Bordallo J, Plemper RK, Finger A, Wolf DH (1998) Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins. Mol Biol Cell 9:209–222.

    PubMed  CAS  Google Scholar 

  18. Deak PM, Wolf DH (2001) Membrane topology and function of Der3/Hrd1p as a ubiquitin-protein ligase (E3) involved in endoplasmic reticulum degradation. J Biol Chem 276:10663–10669.

    Article  PubMed  CAS  Google Scholar 

  19. Knop M, Hauser N, Wolf DH (1996) N-Glycosylation affects endoplasmic reticulum degradation of a mutated derivative of carboxypeptidase yscY in yeast. Yeast 12:1229–1238.

    Article  PubMed  CAS  Google Scholar 

  20. Hitt R, Wolf DH (2004) Der1p, a protein required for degradation of malfolded soluble proteins of the endoplasmic reticulum: topology and Der1-like proteins. FEMS Yeast Res 4:721–729.

    Article  PubMed  CAS  Google Scholar 

  21. Jakob CA, Burda P, Roth J, 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.

    Article  PubMed  CAS  Google Scholar 

  22. Durr G, Strayle J, Plemper R et al (1998) The medial-Golgi ion pump Pmr1 supplies the yeast secretory pathway with Ca2+ and Mn2+ required for glycosylation, sorting, and endoplasmic reticulum-associated protein degradation. Mol Biol Cell 9:1149–1162.

    PubMed  CAS  Google Scholar 

  23. Clerc S, Hirsch C, Oggier DM et al (2009) Htm1 protein generates the N-glycan signal for glycoprotein degradation in the endoplasmic reticulum. J Cell Biol 184:159–172.

    Article  PubMed  CAS  Google Scholar 

  24. Quan EM, Kamiya Y, Kamiya D et al (2008) Defining the glycan destruction signal for endoplasmic reticulum-associated degradation. Mol Cell 32:870–877.

    Article  PubMed  CAS  Google Scholar 

  25. Jakob CA, Bodmer D, Spirig U et al (2001) Htm1p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast. EMBO Rep 2:423–430.

    PubMed  CAS  Google Scholar 

  26. Buschhorn BA, Kostova Z, Medicherla B, Wolf DH (2004) A genome-wide screen identifies Yos9p as essential for ER-associated degradation of glycoproteins. FEBS Lett 577:422–426.

    Article  PubMed  CAS  Google Scholar 

  27. Bhamidipati A, Denic V, Quan EM, Weissman JS (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.

    Article  PubMed  CAS  Google Scholar 

  28. Kim W, Spear ED, Ng DT (2005) Yos9p detects and targets misfolded glycoproteins for ER-associated degradation. Mol Cell 19:753–764.

    Article  PubMed  CAS  Google Scholar 

  29. Szathmary R, Bielmann R, Nita-Lazar M et al (2005) Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD. Mol Cell 19:765–775.

    Article  PubMed  CAS  Google Scholar 

  30. Kostova Z, Wolf DH (2005) Importance of carbohydrate positioning in the recognition of mutated CPY for ER-associated degradation. J Cell Sci 118:1485–1492.

    Article  PubMed  CAS  Google Scholar 

  31. Spear ED, Ng DT (2005) Single, context-specific glycans can target misfolded glycoproteins for ER-associated degradation. J Cell Biol 169:73–82.

    Article  PubMed  CAS  Google Scholar 

  32. Xie W, Kanehara K, Sayeed A, Ng DT (2009) Intrinsic conformational determinants signal protein misfolding to the Hrd1/Htm1 endoplasmic reticulum-associated degradation system. Mol Biol Cell 20:3317–3329.

    Article  PubMed  CAS  Google Scholar 

  33. Plemper RK, Bohmler S, Bordallo J et al (1997) Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation. Nature 388:891–895.

    Article  PubMed  CAS  Google Scholar 

  34. Brodsky JL, Werner ED, Dubas ME et al (1999) The requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct. J Biol Chem 274:3453–3460.

    Article  PubMed  CAS  Google Scholar 

  35. Nishikawa SI, Fewell SW, Kato Y et al (2001) Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation. J Cell Biol 153:1061–1070.

    Article  PubMed  CAS  Google Scholar 

  36. Plemper RK, Bordallo J, Deak PM et al (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 (Pt 22):4123–4134.

    PubMed  CAS  Google Scholar 

  37. Gardner RG, Swarbrick GM, Bays NW et al (2000) Endoplasmic reticulum degradation requires lumen to cytosol signaling. Trans-membrane control of Hrd1p by Hrd3p. J Cell Biol 151:69–82.

    Article  PubMed  CAS  Google Scholar 

  38. Gauss R, Sommer T, Jarosch E (2006) The Hrd1p ligase complex forms a linchpin between ER-lumenal substrate selection and Cdc48p recruitment. EMBO J 25:1827–1835.

    Article  PubMed  CAS  Google Scholar 

  39. Carvalho P, Goder V, Rapoport TA (2006) Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. Cell 126:361–373.

    Article  PubMed  CAS  Google Scholar 

  40. Denic V, Quan EM, Weissman JS (2006) A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation. Cell 126:349–359.

    Article  PubMed  CAS  Google Scholar 

  41. Horn SC, Hanna J, Hirsch C et al (2009) Usa1 functions as a scaffold of the HRD-ubiquitin ligase. Mol Cell 36:782–793.

    Article  PubMed  CAS  Google Scholar 

  42. Carvalho P, Stanley AM, Rapoport TA (2010) Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p. Cell 143:579–591.

    Article  PubMed  CAS  Google Scholar 

  43. Mehnert M, Sommer T, Jarosch E (2010) ERAD ubiquitin ligases: multifunctional tools for protein quality control and waste disposal in the endoplasmic reticulum. Bioessays 32:905–913.

    Article  PubMed  CAS  Google Scholar 

  44. Willer M, Forte GM, Stirling CJ (2008) Sec61p is required for ERAD-L: genetic dissection of the translocation and ERAD-L functions of Sec61P using novel derivatives of CPY. J Biol Chem 283:33883–33888.

    Article  PubMed  CAS  Google Scholar 

  45. Schafer A, Wolf DH (2009) Sec61p is part of the endoplasmic reticulum-associated degradation machinery. EMBO J 28:2874–2884.

    Article  PubMed  Google Scholar 

  46. Biederer T, Volkwein C, Sommer T (1997) Role of Cue1p in ubiquitination and degradation at the ER surface. Science 278:1806–1809.

    Article  PubMed  CAS  Google Scholar 

  47. Bordallo J, Wolf DH (1999) A RING-H2 finger motif is essential for the function of Der3/Hrd1 in endoplasmic reticulum associated protein degradation in the yeast Saccharomyces cerevisiae. FEBS Lett 448:244–248.

    Article  PubMed  CAS  Google Scholar 

  48. Jarosch E, Taxis C, Volkwein C et al (2002) Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Nat Cell Biol 4:134–139.

    Article  PubMed  CAS  Google Scholar 

  49. Ye Y, Meyer HH, Rapoport TA (2001) The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414:652–656.

    Article  PubMed  CAS  Google Scholar 

  50. Rabinovich E, Kerem A, Frohlich KU et al (2002) AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation. Mol Cell Biol 22:626–634.

    Article  PubMed  CAS  Google Scholar 

  51. Neuber O, Jarosch E, Volkwein C et al (2005) Ubx2 links the Cdc48 complex to ER-associated protein degradation. Nat Cell Biol 7:993–998.

    Article  PubMed  CAS  Google Scholar 

  52. Schuberth C, 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.

    Article  PubMed  CAS  Google Scholar 

  53. Alberts SM, Sonntag C, Schafer A, Wolf DH (2009) Ubx4 modulates cdc48 activity and influences degradation of misfolded proteins of the endoplasmic reticulum. J Biol Chem 284:16082–16089.

    Article  PubMed  CAS  Google Scholar 

  54. Medicherla B, Kostova Z, Schaefer A, Wolf DH (2004) A genomic screen identifies Dsk2p and Rad23p as essential components of ER-associated degradation. EMBO Rep 5:692–697.

    Article  PubMed  CAS  Google Scholar 

  55. Kohlmann S, Schafer A, Wolf DH (2008) Ubiquitin ligase Hul5 is required for fragment-specific substrate degradation in endoplasmic reticulum-associated degradation. J Biol Chem 283:16374–16383.

    Article  PubMed  CAS  Google Scholar 

  56. Taxis C, Hitt R, Park SH et al (2003) Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD. J Biol Chem 278:35903–35913.

    Article  PubMed  CAS  Google Scholar 

  57. Park SH, Bolender N, Eisele F et al (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.

    Article  PubMed  Google Scholar 

  58. Eisele F, Wolf DH (2008) Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1. FEBS Lett 582:4143–4146.

    Article  PubMed  CAS  Google Scholar 

  59. Heck JW, Cheung SK, Hampton RY (2010) Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1. Proc Natl Acad Sci U S A 107:1106–1111.

    Article  PubMed  CAS  Google Scholar 

  60. Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96.

    Article  PubMed  CAS  Google Scholar 

  61. Heilig JS, Elbing KL, Brent R (2001) Large-scale preparation of plasmid DNA. Curr Protoc Mol Biol Chapter 1:Unit1 7.

    Google Scholar 

Download references

Acknowledgments

We thank Frederik Eisele, Mario Scazzari, and Hisashi Hoshida for essential help and Hans Rudolph and Elena Martinez Benitez for critical reading. The work was supported by the EU network of excellence RUBICON, the Deutsche Forschungsgemeinschaft (Bonn, Germany), and the Fonds der Chemischen Industrie (Frankfurt, Germany).

Author information

Authors and Affiliations

  1. Institute of Biochemistry, University of Stuttgart, Stuttgart, Germany

    Alexandra Stolz & Dieter H. Wolf

Authors
  1. Alexandra Stolz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dieter H. Wolf
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dieter H. Wolf .

Editor information

Editors and Affiliations

  1. Institut für Genetik, Universität Köln, Zülpicher Str. 47, Köln, 50674, Germany

    R. Jürgen Dohmen

  2. Fak. Biologie, Universität Konstanz, Konstanz, 78457, Germany

    Martin Scheffner

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Stolz, A., Wolf, D.H. (2012). Use of CPY* and Its Derivatives to Study Protein Quality Control in Various Cell Compartments. In: Dohmen, R., Scheffner, M. (eds) Ubiquitin Family Modifiers and the Proteasome. Methods in Molecular Biology, vol 832. Humana Press. https://doi.org/10.1007/978-1-61779-474-2_35

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-474-2_35

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-473-5

  • Online ISBN: 978-1-61779-474-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation