Analysis of Protein Ubiquitination

Jeffrey D. Laney1, Mark Hochstrasser2

1 Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, 2 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.5
DOI:  10.1002/0471140864.ps1405s66
Online Posting Date:  November, 2011
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Abstract

Attachment of ubiquitin (Ub) to a protein requires a series of enzymes that recognize the substrate and promote Ub transfer. Several methods are described in this unit for determining if a protein has Ub‐transferring activity. They include immunoblotting of immunoprecipitated proteins, affinity purification using His‐tagged Ub, assaying for auto‐ubiquitination of E3, and assaying ubiquitination of a model substrate protein in vitro and in E. coli cells that express Ub‐ligation enzymes. These methods are suitable for a variety of eukaryotic cells, but techniques are specifically described for use with yeast and mammalian cells. Curr. Protoc. Protein Sci. 66:14.5.1‐14.5.13. © 2011 by John Wiley & Sons, Inc.

Keywords: ubiquitin; immunoprecipitation; ubiquitin‐protein ligase

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Immunoprecipitation of a Target Protein Followed by Anti‐Ub Immunoblotting
  • Basic Protocol 2: Affinity Purification of Ubiquitinated Proteins from Cells Expressing His6‐Ub
  • Basic Protocol 3: Assay for Auto‐Ubiquitination by E3 Ub‐Protein Ligases
  • Basic Protocol 4: Assay for Transfer of Ubiquitin to a Model Substrate Protein in Vitro
  • Basic Protocol 5: Reconstitution of the Transfer of Ubiquitin to a Substrate Protein in E. coli
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunoprecipitation of a Target Protein Followed by Anti‐Ub Immunoblotting

  Materials
  • Yeast or mammalian cells expressing the protein of interest and matching control cells
  • PBS ( appendix 2E)
  • RIPA buffer (see recipe) containing freshly dissolved 10 mM N‐ethylmaleimide (NEM) and protease inhibitors (see recipe), ice‐cold
  • H 2O, sterile
  • 100% ethanol containing freshly dissolved 50 mM NEM, ice‐cold
  • SDS buffer (see recipe)
  • Triton lysis buffer (see recipe) containing freshly dissolved 10 mM NEM and protease inhibitors, ice‐cold
  • Protein A‐agarose or protein G‐agarose beads
  • Triton lysis buffer (see recipe) supplemented with 0.05% (w/v) SDS
  • 2× SDS‐PAGE sample buffer (see recipe)
  • Blocking buffer—e.g., 5% (w/v) nonfat dry milk
  • Anti‐Ub antibodies (Covance)
  • Enhanced chemiluminescence (ECL), enhanced chemifluorescence (ECF), or quantum dot‐based detection (Invitrogen) reagents
  • 1.5‐ml microcentrifuge tubes
  • Centrifuge
  • 425‐ to 600‐µm acid‐washed glass beads (Sigma)
  • Vortexer
  • Speedvac Evaporator
  • Boiling water bath
  • End‐over‐end rotator
  • Micropipettor
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1), electroblotting (unit 10.7), and immunoblot detection (unit 10.10)

Basic Protocol 2: Affinity Purification of Ubiquitinated Proteins from Cells Expressing His6‐Ub

  Materials
  • Yeast or mammalian cells expressing the protein of interest and His 6‐tagged Ub (units 5.6 5.8) and matching control cells
  • PBS ( appendix 2E)
  • 6 M guanidine⋅HCl/100 mM sodium phosphate buffer, pH 8.0 ( appendix 2E), with and without 5 mM imidazole
  • Ni2+‐NTA‐agarose beads (e.g., Qiagen, Invitrogen, Clontech)
  • 6 M guanidine⋅HCl/100 mM sodium phosphate buffer, pH 5.8 ( appendix 2E)
  • Protein buffer (see recipe) with and without 10 and 200 mM imidazole
  • 10% (v/v) trichloroacetic acid (TCA)
  • 2× SDS‐PAGE sample buffer (see recipe)
  • Guanidine wash buffer (see recipe) with and without 20 mM imidazole
  • 1 M imidazole
  • Urea wash buffer, pH 8.0 (see recipe), containing 20 mM imidazole
  • Urea wash buffer, pH 6.0 (see recipe), containing 20 mM imidazole
  • 2× urea sample buffer (see recipe)
  • 2‐ and 1.5‐ml microcentrifuge tubes
  • Probe‐type sonicator with microtip
  • Centrifuge
  • End‐over‐end rotator
  • Boiling water bath
  • 425‐ to 600‐µm acid‐washed glass beads (Sigma)
  • Vortexer
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and immunoblot detection (unit 10.10)

Basic Protocol 3: Assay for Auto‐Ubiquitination by E3 Ub‐Protein Ligases

  Materials
  • 5× ubiquitination buffer (see recipe)
  • Purified GST‐fusion protein: glutathione‐S‐transferase (GST) fused with protein of interest (unit 6.6)
  • E1 Ub‐activating enzyme, E2 Ub‐conjugating enzyme, and Ub (purified from recombinant sources or commercially available from Calbiochem, Boston Biochem, or Sigma)
  • 2× SDS‐PAGE sample buffer (see recipe)
  • Anti‐Ub antibodies (Covance) or anti‐GST antibodies (Sigma)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and immunoblot detection (unit 10.10)

Basic Protocol 4: Assay for Transfer of Ubiquitin to a Model Substrate Protein in Vitro

  Materials
  • 5× ubiquitination buffer (see recipe)
  • Purified GST‐S peptide fusion protein: glutathione‐S‐transferase (GST), ribonuclease S peptide and the protein of interest (unit 6.6)
  • Ribonuclease S protein (Sigma)
  • E1 Ub‐activating enzyme, E2 Ub‐conjugating enzyme, and Ub (purified from recombinant sources or commercially available from Calbiochem, Boston Biochem, or Sigma)
  • 2× SDS‐PAGE sample buffer (see recipe)
  • Anti‐Ub antibodies (Covance)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and immunoblot detection (unit 10.10)

Basic Protocol 5: Reconstitution of the Transfer of Ubiquitin to a Substrate Protein in E. coli

  Materials
  • E. coli cells appropriate for protein expression from the T7 promoter
  • 1 M isopropyl‐β‐D‐1‐thiogalactopyranoside (IPTG)
  • BugBuster protein extraction reagent (Novagen) containing freshly dissolved protease inhibitors (see recipe)
  • Triton lysis buffer (see recipe) containing freshly dissolved protease inhibitors, ice‐cold
  • Ubiquitination cascade components [Ub, E1 (Uba1), E2 (appropriate Ubc), E3 (appropriate Ub‐protein ligase), and the substrate protein of interest] cloned behind the T7lac promoters in different Duet expression vectors (Novagen)
  • Protein A‐agarose or protein G‐agarose beads
  • 2× SDS‐PAGE sample buffer (see recipe)
  • Anti‐Ub antibodies (Covance)
  • Enhanced chemiluminescence (ECL) reagents for immunoblotting
  • 30°C incubator
  • Centrifuge
  • 1.5‐ml microcentrifuge tubes
  • End‐over‐end rotator
  • Micropipettor
  • Boiling water bath
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1), electroblotting (unit 10.7), and immunoblot detection (unit 10.10)
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Figures

Videos

Literature Cited

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   Ardley, H.C. and Robinson, P.A. 2005. E3 ubiquitin ligases. Essays Biochem. 41:15‐30.
   Bhat, K.P. and Greer, S.F. 2011. Proteolytic and non‐proteolytic roles of ubiquitin and the ubiquitin proteasome system in transcriptional regulation. Biochim. Biophys. Acta 1809:150‐155.
   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 243:1576‐1583.
   Ciechanover, A., Finley, D., and Varshavsky, A. 1984. Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell 37:57‐66.
   Clague, M.J. and Urbe, S. 2010. Ubiquitin: same molecule, different degradation pathways. Cell 143:682‐685.
   Deshaies, R.J. and Joazeiro, C.A. 2009. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78:399‐434.
   Finley, D., Sadis, S., Monia, B., Boucher, P., Ecker, D., Crooke, S., and Chau, V. 1994. Inhibition of proteolysis and cell cycle progression in a multiubiquitination‐deficient yeast mutant. Mol. Cell Biol. 14:5501‐5509.
   Ghaboosi, N. and Deshaies, R.J. 2007. A conditional yeast E1 mutant blocks the ubiquitin‐proteasome pathway and reveals a role for ubiquitin conjugates in targeting Rad23 to the proteasome. Mol. Biol. Cell 18:1953‐1963.
   Hershko, A. and Ciechanover, A. 1998. The ubiquitin system. Annu. Rev. Biochem. 67:425‐479.
   Hershko, A., Heller, H., Elias, S., and Ciechanover, A. 1983. Components of ubiquitin‐protein ligase system: resolution, affinity purification, and role in protein breakdown. J. Biol. Chem. 258:8206‐8214.
   Hislop, J.N. and von Zastrow, M. 2011. Role of ubiquitination in endocytic trafficking of G‐protein‐coupled receptors. Traffic 12:137‐148.
   Hodgins, R.R.W., Ellison, K.S., and Ellison, M.J. 1992. Expression of a ubiquitin derivative that conjugates to protein irreversibly produces phenotypes consistent with a ubiquitin deficiency. J. Biol. Chem. 267:8807‐8812.
   Ikeda, F. and Dikic, I. 2008. Atypical ubiquitin chains: New molecular signals. ‘Protein Modifications: Beyond the usual suspects' review series. EMBO Rep. 9:536‐542.
   Iwai, K. 2010. Functions of linear ubiquitin chains in the NF‐κB pathway: Linear polyubiquitin in NF‐κB signaling. Subcell. Biochem. 54:100‐106.
   Kaiser, P., Flick, K., Wittenberg, C., and Reed, S.I. 2000. Regulation of transcription by ubiquitination without proteolysis: Cdc34/SCF(Met30)‐mediated inactivation of the transcription factor Met4. Cell 102:303‐314.
   King, R.W., Peters, J.M., Tugendreich, S., Rolfe, M., Hieter, P., and Kirschner, M.W. 1995. A 20S complex containing CDC27 and CDC16 catalyzes the mitosis‐specific conjugation of ubiquitin to cyclin B. Cell 81:279‐288.
   Petroski, M.D. and Deshaies, R.J. 2005. Mechanism of lysine 48‐linked ubiquitin‐chain synthesis by the cullin‐RING ubiquitin‐ligase complex SCF‐Cdc34. Cell 123:1107‐1120.
   Pierce, N.W., Kleiger, G., Shan, S.O., and Deshaies, R.J. 2009. Detection of sequential polyubiquitylation on a millisecond timescale. Nature 462:615‐619.
   Scheffner, M., Huibregste, J.M., Vierstra, R.D., and Howley, P.M. 1993. The HPV E6 and E6‐AP complex functions as a ubiquitin‐protein ligase in the ubiquitination of p53. Cell 75:495‐505.
   Spencer, E., Jiang, J., and Chen, Z.J. 1999. Signal‐induced ubiquitination of IkappaBalpha by the F‐box protein Slimb/beta‐TrCP. Genes Dev. 13:284‐294.
   Swaminathan, S., Amerik, A.Y., and Hochstrasser, M. 1999. The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast. Mol. Biol. Cell 10:2583‐2594.
   Treier, M., Staszewski, L.M., and Bohmann, D. 1994. Ubiquitin‐dependent c‐jun degradation in vivo is mediated by the δ domain. Cell 78:787‐798.
   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.
   Verma, R., Chi, Y., and Deshaies, R.J. 1997. Cell‐free ubiquitination of cell cycle regulators in budding yeast extracts. Methods Enzymol. 283:366‐376.
   Xu, P., Duong, D.M., Seyfried, N.T., Cheng, D., Xie, Y., Robert, J., Rush, J., Hochstrasser, M., Finley, D., and Peng, J. 2009. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137:133‐145.
Key References
   Treier, M., Staszewski, L.M., and Bohmann, D. 1994. Ubiquitin‐dependent c‐Jun degradation in vivo is mediated by the δ domain. Cell 78:787‐798.
  Describes the affinity purification of ubiquitinated proteins from tissue culture cells using His6‐Ub and the mammalian expression vectors for the tagged forms of Ub.
   Laney, J.D. and Hochstrasser, M. 2002. Assaying protein ubiquitination in Saccharomyces cerevisiae. Methods Enzymol. 351:248‐257.
  Describes the yeast methods and the yeast expression vectors for mutant and tagged forms of Ub.
   Rosenbaum, J.C., Fredrickson, E.K., Oeser, M.L., Garrett‐Engele, C.M., Locke, M.N., Richardson, L.A., Nelson, Z.W., Hetrick, E.D., Milac, T.I., Gottschling, D.E., and Gardner, R.G. 2011. Disorder targets misorder in nuclear quality control degradation: A disordered ubiquitin ligase directly recognizes its misfolded substrates. Mol. Cell 41:93‐106.
  Describes the reconstitution of the ubiquitin transfer reaction in E. coli.
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