Skip to main content
SpringerLink
Log in
Book cover

Proteostasis pp 117–142Cite as

Studying Protein Ubiquitylation in Yeast

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1449))

Abstract

Ubiquitylation is a reversible posttranslational modification that is critical for most, if not all, cellular processes and essential for viability. Ubiquitin conjugates to substrate proteins either as a single moiety (monoubiquitylation) or as polymers composed of ubiquitin molecules linked to each other with various topologies and structures (polyubiquitylation). This contributes to an elaborate ubiquitin code that is decrypted by specific ubiquitin-binding proteins. Indeed, these different types of ubiquitylation have different functional outcomes, notably affecting the stability of the substrate, its interactions, its activity, or its subcellular localization. In this chapter, we describe protocols to determine whether a protein is ubiquitylated, to identify the site that is ubiquitylated, and provide direction to study the topology of the ubiquitin modification, in the yeast Saccharomyces cerevisiae.

This is a preview of subscription content, 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   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Komander D, Rape M (2012) The ubiquitin code. Annu Rev Biochem 81:203–229. doi:10.1146/annurev-biochem-060310-170328

    Article  CAS  PubMed  Google Scholar 

  2. Husnjak K, Dikic I (2012) Ubiquitin-binding proteins: decoders of ubiquitin-mediated cellular functions. Annu Rev Biochem 81:291–322. doi:10.1146/annurev-biochem-051810-094654

    Article  CAS  PubMed  Google Scholar 

  3. Kulathu Y, Komander D (2012) Atypical ubiquitylation – the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat Rev Mol Cell Biol 13:508. doi:10.1038/nrm3394

    Article  CAS  PubMed  Google Scholar 

  4. Ulrich HD, Jentsch S (2000) Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J 19:3388–3397. doi:10.1093/emboj/19.13.3388

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Spence J, Sadis S, Haas AL, Finley D (1995) A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol Cell Biol 15:1265–1273

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hofmann RM, Pickart CM (1999) Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96:645–653

    Article  CAS  PubMed  Google Scholar 

  7. Signaling to NF-kappaB: regulation by ubiquitination (2010) 2:a003350. http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=20300215&retmode=ref&cmd=prlinks

  8. Versatile roles of k63-linked ubiquitin chains in trafficking (2014) 3:1027–1088. http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=25396681&retmode=ref&cmd=prlinks.

  9. Galan JM, Haguenauer-Tsapis R (1997) Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein. EMBO J 16:5847–5854. doi:10.1093/emboj/16.19.5847

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Xu P, Duong DM, Seyfried NT, Cheng D, Xie Y et al (2009) Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137:133–145. doi:10.1016/j.cell.2009.01.041

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ziv I, Matiuhin Y, Kirkpatrick DS, Erpapazoglou Z, Léon S et al (2011) A perturbed ubiquitin landscape distinguishes between ubiquitin in trafficking and in proteolysis. Mol Cell Proteomics 10:M111.009753. doi:10.1074/mcp.M111.009753

    Article  PubMed  PubMed Central  Google Scholar 

  12. Wagner SA, Beli P, Weinert BT, Nielsen ML, Cox J et al (2011) A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol Cell Proteomics 10:M111.013284. doi:10.1074/mcp.M111.013284

    Article  PubMed  PubMed Central  Google Scholar 

  13. Kim W, Bennett EJ, Huttlin EL, Guo A, Li J et al (2011) Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell 44:325–340. doi:10.1016/j.molcel.2011.08.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease (2011) 286:10457–10465. http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=21278249&retmode=ref&cmd=prlinks

  15. Clague MJ, Coulson JM, Urbé S (2012) Cellular functions of the DUBs. J Cell Sci 125:277–286. doi:10.1242/jcs.090985

    Article  CAS  PubMed  Google Scholar 

  16. Dohmen RJ, Stappen R, McGrath JP, Forrová H, Kolarov J et al (1995) An essential yeast gene encoding a homolog of ubiquitin-activating enzyme. J Biol Chem 270:18099–18109

    Article  CAS  PubMed  Google Scholar 

  17. Gwizdek C, Iglesias N, Rodriguez MS, Ossareh-Nazari B, Hobeika M et al (2006) Ubiquitin-associated domain of Mex67 synchronizes recruitment of the mRNA export machinery with transcription. Proc Natl Acad Sci U S A 103:16376–16381. doi:10.1073/pnas.0607941103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Histidine-tagged ubiquitin substitutes for wild-type ubiquitin in Saccharomyces cerevisiae and facilitates isolation and identification of in vivo substrates of the ubiquitin pathway (2000) 282:54–64. http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=10860499&retmode=ref&cmd=prlinks.

  19. Peng J, Schwartz D, Elias JE, Thoreen CC, Cheng D et al (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21:921–926. doi:10.1038/nbt849

    Article  CAS  PubMed  Google Scholar 

  20. Mayor T, Deshaies RJ (2005) Two-step affinity purification of multiubiquitylated proteins from Saccharomyces cerevisiae. Meth Enzymol 399:385–392. doi:10.1016/S0076-6879(05)99026-5

    Article  CAS  PubMed  Google Scholar 

  21. Guerrero C, Tagwerker C, Kaiser P, Huang L (2006) An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26S proteasome-interacting network. Mol Cell Proteomics 5:366–378. doi:10.1074/mcp.M500303-MCP200

    Article  CAS  PubMed  Google Scholar 

  22. Tagwerker C, Flick K, Cui M, Guerrero C, Dou Y et al (2006) A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking. Mol Cell Proteomics 5:737–748. doi:10.1074/mcp.M500368-MCP200

    Article  CAS  PubMed  Google Scholar 

  23. Becuwe M, Vieira N, Lara D, Gomes-Rezende J, Soares-Cunha C et al (2012) A molecular switch on an arrestin-like protein relays glucose signaling to transporter endocytosis. J Cell Biol 196:247–259. doi:10.1083/jcb.201109113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Laney JD, Hochstrasser M (2002) Assaying protein ubiquitination in Saccharomyces cerevisiae. Meth Enzymol 351:248–257

    Article  CAS  PubMed  Google Scholar 

  25. Kaiser P, Tagwerker C (2005) Is this protein ubiquitinated? Meth Enzymol 399:243–248. doi:10.1016/S0076-6879(05)99016-2

    Article  CAS  PubMed  Google Scholar 

  26. Kragt A, Voorn-Brouwer T, van den Berg M, Distel B (2005) The Saccharomyces cerevisiae peroxisomal import receptor Pex5p is monoubiquitinated in wild type cells. J Biol Chem 280:7867–7874. doi:10.1074/jbc.M413553200

    Article  CAS  PubMed  Google Scholar 

  27. Léon S, Zhang L, McDonald WH, Yates J, Cregg JM et al (2006) Dynamics of the peroxisomal import cycle of PpPex20p: ubiquitin-dependent localization and regulation. J Cell Biol 172:67–78. doi:10.1083/jcb.200508096

    Article  PubMed  PubMed Central  Google Scholar 

  28. Stawiecka-Mirota M, Pokrzywa W, Morvan J, Zoladek T, Haguenauer-Tsapis R et al (2007) Targeting of Sna3p to the endosomal pathway depends on its interaction with Rsp5p and multivesicular body sorting on its ubiquitylation. Traffic 8:1280–1296. doi:10.1111/j.1600-0854.2007.00610.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA et al (2005) Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev 19:2816–2826. doi:10.1101/gad.1362105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Danielsen JMR, Sylvestersen KB, Bekker-Jensen S, Szklarczyk D, Poulsen JW et al (2011) Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level. Mol Cell Proteomics 10:M110.003590. doi:10.1074/mcp.M110.003590

    Article  PubMed  Google Scholar 

  31. Newton K, Matsumoto ML, Ferrando RE, Wickliffe KE, Rape M et al (2012) Using linkage-specific monoclonal antibodies to analyze cellular ubiquitylation. Methods Mol Biol 832:185–196. doi:10.1007/978-1-61779-474-2_13

    Article  CAS  PubMed  Google Scholar 

  32. Aillet F, Lopitz-Otsoa F, Hjerpe R, Torres-Ramos M, Lang V et al (2012) Isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. Methods Mol Biol 832:173–183. doi:10.1007/978-1-61779-474-2_12

    Article  CAS  PubMed  Google Scholar 

  33. Hjerpe R, Aillet F, Lopitz-Otsoa F, Lang V, England P et al (2009) Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep 10:1250–1258. doi:10.1038/embor.2009.192

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sims JJ, Scavone F, Cooper EM, Kane LA, Youle RJ et al (2012) Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling. Nat Meth 9:303–309. doi:10.1038/nmeth.1888

    Article  CAS  Google Scholar 

  35. Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant (1994) 14:5501–5509. http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=8035826&retmode=ref&cmd=prlinks

  36. Selective inhibitors of the proteasome-dependent and vacuolar pathways of protein degradation in Saccharomyces cerevisiae (1996) 271:27280–27284. http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=8910302&retmode=ref&cmd=prlinks

  37. Combined chemical and genetic approach to inhibit proteolysis by the proteasome (2010) 27:965–974. http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=20625982&retmode=ref&cmd=prlinks

  38. Liu C, Apodaca J, Davis LE, Rao H (2007) Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells. Biotechniques 42:158–160

    Article  CAS  PubMed  Google Scholar 

  39. Léon S, Erpapazoglou Z, Haguenauer-Tsapis R (2008) Ear1p and Ssh4p are new adaptors of the ubiquitin ligase rsp5p for cargo ubiquitylation and sorting at multivesicular bodies. Mol Biol Cell 19:2379–2388. doi:10.1091/mbc.E08-01-0068

    Article  PubMed  PubMed Central  Google Scholar 

  40. Erpapazoglou Z, Dhaoui M, Pantazopoulou M, Giordano F, Mari M et al (2012) A dual role for K63-linked ubiquitin chains in multivesicular body biogenesis and cargo sorting. Mol Biol Cell 23:2170–2183. doi:10.1091/mbc.E11-10-0891

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Dan Finley (Harvard Medical School, Boston, MA, USA) for providing us with the SUB strains. This work was funded by the CNRS, the Fondation ARC pour la Recherche sur le Cancer (SFI20101201844 and SFI20121205762) and the Ligue contre le Cancer – Comité de Paris (RS13/75-45 and RS14/75-120) to SL. The authors would like to acknowledge networking support by the Proteostasis COST Action (BM1307).

Author information

Authors and Affiliations

  1. Institut Jacques Monod, UMR 7592 CNRS/Univ. Paris Diderot, Sorbonne Paris Cité, Paris, France

    Junie Hovsepian, Michel Becuwe & Sébastien Léon

  2. Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA, 02115, USA

    Michel Becuwe

  3. Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia

    Oded Kleifeld

  4. Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

    Michael H. Glickman

  5. Membrane Trafficking, Ubiquitin and Signaling Lab, Institut Jacques Monod, UMR7592 CNRS/Université Paris-Diderot, 15 Rue Hélène Brion, Bât. Buffon, 75205, Paris Cedex 13, France

    Sébastien Léon

Authors
  1. Junie Hovsepian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michel Becuwe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Oded Kleifeld
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael H. Glickman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sébastien Léon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sébastien Léon .

Editor information

Editors and Affiliations

  1. Computational and Experimental Biology Group Department of Health Promotion and Chronic Diseases, National Health Institute Dr. Ricardo Jorge, INSA, I.P., Lisboa, Portugal

    Rune Matthiesen

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Hovsepian, J., Becuwe, M., Kleifeld, O., Glickman, M.H., Léon, S. (2016). Studying Protein Ubiquitylation in Yeast. In: Matthiesen, R. (eds) Proteostasis. Methods in Molecular Biology, vol 1449. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3756-1_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3756-1_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3754-7

  • Online ISBN: 978-1-4939-3756-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation