Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Substrate selection by the proteasome during degradation of protein complexes

Nature Chemical Biology volume 5, pages 29–36 (2009)Cite this article

Abstract

The proteasome controls the turnover of many cellular proteins. Two structural features are typically required for proteins to be degraded: covalently attached ubiquitin polypeptides that allow binding to the proteasome and an unstructured region in the targeted protein that initiates proteolysis. Here, we have tested the degradation of model proteins to further explore how the proteasome selects its substrates. Using purified yeast proteasome and mammalian proteasome in cell lysate, we have demonstrated that the two structural features can act in trans when separated onto different proteins in a multisubunit complex. In such complexes, the location of the unstructured initiation site and its chemical properties determine which subunit is degraded. Thus, our findings reveal the molecular basis of subunit specificity in the degradation of protein complexes. In addition, our data provide a plausible explanation for how adaptor proteins can bind to otherwise stable proteins and target them for degradation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Proteasome substrates and N-degron–mediated degradation.
Figure 2: The proteasome degrades an unubiquitinated subunit in a heterodimeric complex.
Figure 3: trans initiation by the proteasome.
Figure 4: Substrate selection in reticulocyte lysate.
Figure 5: Substrate selection by the purified proteasome.
Figure 6: Subunit-specific degradation by the proteasome.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Glickman, M.H. & Ciechanover, A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, 373–428 (2002).

    Article  CAS  Google Scholar 

  2. Hochstrasser, M. Lingering mysteries of ubiquitin-chain assembly. Cell 124, 27–34 (2006).

    Article  Google Scholar 

  3. Deveraux, Q., Ustrell, V., Pickart, C. & Rechsteiner, M. A 26 S protease subunit that binds ubiquitin conjugates. J. Biol. Chem. 269, 7059–7061 (1994).

    CAS  PubMed  Google Scholar 

  4. Lam, Y.A., Lawson, T.G., Velayutham, M., Zweier, J.L. & Pickart, C.M. A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal. Nature 416, 763–767 (2002).

    Article  CAS  Google Scholar 

  5. Lee, C., Schwartz, M.P., Prakash, S., Iwakura, M. & Matouschek, A. ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. Mol. Cell 7, 627–637 (2001).

    Article  CAS  Google Scholar 

  6. Johnson, E.S., Gonda, D.K. & Varshavsky, A. Cis-trans recognition and subunit-specific degradation of short-lived proteins. Nature 346, 287–291 (1990).

    Article  CAS  Google Scholar 

  7. Hochstrasser, M. & Varshavsky, A. In vivo degradation of a transcriptional regulator: the yeast alpha 2 repressor. Cell 61, 697–708 (1990).

    Article  CAS  Google Scholar 

  8. Nishiyama, A. et al. A nonproteolytic function of the proteasome is required for the dissociation of Cdc2 and cyclin B at the end of M phase. Genes Dev. 14, 2344–2357 (2000).

    Article  CAS  Google Scholar 

  9. Verma, R., McDonald, H., Yates, J.R. & Deshaies, R.J. Selective degradation of ubiquitinated Sic1 by purified 26S proteasome yields active S phase cyclin-Cdk. Mol. Cell 8, 439–448 (2001).

    Article  CAS  Google Scholar 

  10. Elsasser, S. et al. Proteasome subunit Rpn1 binds ubiquitin-like protein domains. Nat. Cell Biol. 4, 725–730 (2002).

    Article  CAS  Google Scholar 

  11. Goebl, M.G., Goetsch, L. & Byers, B. The Ubc3 (Cdc34) ubiquitin-conjugating enzyme is ubiquitinated and phosphorylated in vivo. Mol. Cell. Biol. 14, 3022–3029 (1994).

    Article  CAS  Google Scholar 

  12. Heessen, S., Masucci, M.G. & Dantuma, N.P. The Uba2 domain functions as an intrinsic stabilization signal that protects Rad23 from proteasomal degradation. Mol. Cell 18, 225–235 (2005).

    Article  CAS  Google Scholar 

  13. Schauber, C. et al. Rad23 links DNA repair to the ubiquitin/proteasome pathway. Nature 391, 715–718 (1998).

    Article  CAS  Google Scholar 

  14. Prakash, S., Tian, L., Ratliff, K.S., Lehotzky, R.E. & Matouschek, A. An unstructured initiation site is required for efficient proteasome-mediated degradation. Nat. Struct. Mol. Biol. 11, 830–837 (2004).

    Article  CAS  Google Scholar 

  15. Hartley, R.W. Directed mutagenesis and barnase-barstar recognition. Biochemistry 32, 5978–5984 (1993).

    Article  CAS  Google Scholar 

  16. Schreiber, G. & Fersht, A.R. Interaction of barnase with its polypeptide inhibitor barstar studied by protein engineering. Biochemistry 32, 5145–5150 (1993).

    Article  CAS  Google Scholar 

  17. Bachmair, A., Finley, D. & Varshavsky, A. In vivo half-life of a protein is a function of its amino-terminal residue. Science 234, 179–186 (1986).

    Article  CAS  Google Scholar 

  18. Bachmair, A. & Varshavsky, A. The degradation signal in a short-lived protein. Cell 56, 1019–1032 (1989).

    Article  CAS  Google Scholar 

  19. Gonda, D.K. et al. Universality and structure of the N-end rule. J. Biol. Chem. 264, 16700–16712 (1989).

    CAS  PubMed  Google Scholar 

  20. Ciechanover, A. & Ben-Saadon, R. N-terminal ubiquitination: more protein substrates join in. Trends Cell Biol. 14, 103–106 (2004).

    Article  CAS  Google Scholar 

  21. Stack, J.H., Whitney, M., Rodems, S.M. & Pollok, B.A. A ubiquitin-based tagging system for controlled modulation of protein stability. Nat. Biotechnol. 18, 1298–1302 (2000).

    Article  CAS  Google Scholar 

  22. Thrower, J.S., Hoffman, L., Rechsteiner, M. & Pickart, C.M. Recognition of the polyubiquitin proteolytic signal. EMBO J. 19, 94–102 (2000).

    Article  CAS  Google Scholar 

  23. Beal, R.E., Toscano-Cantaffa, D., Young, P., Rechsteiner, M. & Pickart, C.M. The hydrophobic effect contributes to polyubiquitin chain recognition. Biochemistry 37, 2925–2934 (1998).

    Article  CAS  Google Scholar 

  24. Johnston, J.A., Johnson, E.S., Waller, P.R. & Varshavsky, A. Methotrexate inhibits proteolysis of dihydrofolate reductase by the N-end rule pathway. J. Biol. Chem. 270, 8172–8178 (1995).

    Article  CAS  Google Scholar 

  25. Tian, L., Holmgren, R.A. & Matouschek, A. A conserved processing mechanism regulates the activity of transcription factors cubitus interruptus and NF-κB. Nat. Struct. Mol. Biol. 12, 1045–1053 (2005).

    Article  CAS  Google Scholar 

  26. Gonzalez, S.L., Stremlau, M., He, X., Basile, J.R. & Münger, K. Degradation of the retinoblastoma tumor suppressor by the human papillomavirus type 16 E7 oncoprotein is important for functional inactivation and is separable from proteasomal degradation of E7. J. Virol. 75, 7583–7591 (2001).

    Article  CAS  Google Scholar 

  27. Berezutskaya, E. & Bagchi, S. The human papillomavirus E7 oncoprotein functionally interacts with the S4 subunit of the 26S proteasome. J. Biol. Chem. 272, 30135–30140 (1997).

    Article  CAS  Google Scholar 

  28. Whitby, F.G. & Hill, C.P. A versatile platform for inactivation and destruction. Structure 15, 137–138 (2007).

    Article  CAS  Google Scholar 

  29. Dang, Y., Siew, L.M. & Zheng, Y.H. APOBEC3g is degraded by the proteasomal pathway in a Vif-dependent manner without being polyubiquitylated. J. Biol. Chem. 283, 13124–13131 (2008).

    Article  CAS  Google Scholar 

  30. Chen, L. & Madura, K. Rad23 promotes the targeting of proteolytic substrates to the proteasome. Mol. Cell. Biol. 22, 4902–4913 (2002).

    Article  CAS  Google Scholar 

  31. Elsasser, S. & Finley, D. Delivery of ubiquitinated substrates to protein-unfolding machines. Nat. Cell Biol. 7, 742–749 (2005).

    Article  CAS  Google Scholar 

  32. McClellan, A.J., Tam, S., Kaganovich, D. & Frydman, J. Protein quality control: chaperones culling corrupt conformations. Nat. Cell Biol. 7, 736–741 (2005).

    Article  CAS  Google Scholar 

  33. Xie, Y. & Varshavsky, A. Physical association of ubiquitin ligases and the 26S proteasome. Proc. Natl. Acad. Sci. USA 97, 2497–2502 (2000).

    Article  CAS  Google Scholar 

  34. Verma, R., Oania, R., Graumann, J. & Deshaies, R.J. Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system. Cell 118, 99–110 (2004).

    Article  CAS  Google Scholar 

  35. Jeffrey, P.D. et al. Mechanism of Cdk activation revealed by the structure of a Cyclin A-Cdk2 complex. Nature 376, 313–320 (1995).

    Article  CAS  Google Scholar 

  36. King, R.W., Glotzer, M. & Kirschner, M.W. Mutagenic analysis of the destruction signal of mitotic cyclins and structural characterization of ubiquitinated intermediates. Mol. Biol. Cell 7, 1343–1357 (1996).

    Article  CAS  Google Scholar 

  37. Klotzbücher, A., Stewart, E., Harrison, D. & Hunt, T. The 'destruction box' of cyclin A allows B-type cyclins to be ubiquitinated, but not efficiently destroyed. EMBO J. 15, 3053–3064 (1996).

    Article  Google Scholar 

  38. Verhoef, L.G. et al. Minimal length requirement for proteasomal degradation of ubiquitin-dependent substrates. FASEB J. published online, doi:10.1096/fj.08-115055 (16 September 2008).

  39. Zhang, M. & Coffino, P. Repeat sequence of Epstein-Barr virus-encoded nuclear antigen 1 protein interrupts proteasome substrate processing. J. Biol. Chem. 279, 8635–8641 (2004).

    Article  CAS  Google Scholar 

  40. Bienkiewicz, E.A., Adkins, J.N. & Lumb, K.J. Functional consequences of preorganized helical structure in the intrinsically disordered cell-cycle inhibitor p27(Kip1). Biochemistry 41, 752–759 (2002).

    Article  CAS  Google Scholar 

  41. Lee, C., Prakash, S. & Matouschek, A. Concurrent translocation of multiple polypeptide chains through the proteasomal degradation channel. J. Biol. Chem. 277, 34760–34765 (2002).

    Article  CAS  Google Scholar 

  42. Sharon, M. et al. 20S proteasomes have the potential to keep substrates in store for continual degradation. J. Biol. Chem. 281, 9569–9575 (2006).

    Article  CAS  Google Scholar 

  43. Stankunas, K. & Crabtree, G.R. Exploiting protein destruction for constructive use. Proc. Natl. Acad. Sci. USA 104, 11511–11512 (2007).

    Article  CAS  Google Scholar 

  44. Banaszynski, L.A., Chen, L.C., Maynard-Smith, L.A., Ooi, A.G. & Wandless, T.J. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126, 995–1004 (2006).

    Article  CAS  Google Scholar 

  45. Stankunas, K. et al. Conditional protein alleles using knockin mice and a chemical inducer of dimerization. Mol. Cell 12, 1615–1624 (2003).

    Article  CAS  Google Scholar 

  46. Pratt, M.R., Schwartz, E.C. & Muir, T.W. Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt. Proc. Natl. Acad. Sci. USA 104, 11209–11214 (2007).

    Article  CAS  Google Scholar 

  47. Matsuzawa, S., Cuddy, M., Fukushima, T. & Reed, J.C. Method for targeting protein destruction by using a ubiquitin-independent, proteasome-mediated degradation pathway. Proc. Natl. Acad. Sci. USA 102, 14982–14987 (2005).

    Article  CAS  Google Scholar 

  48. Janse, D.M., Crosas, B., Finley, D. & Church, G.M. Localization to the proteasome is sufficient for degradation. J. Biol. Chem. 279, 21415–21420 (2004).

    Article  CAS  Google Scholar 

  49. Sakamoto, K.M. et al. Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. USA 98, 8554–8559 (2001).

    Article  CAS  Google Scholar 

  50. Saeki, Y., Isono, E. & Toh-E, A. Preparation of ubiquitinated substrates by the PY motif-insertion method for monitoring 26S proteasome activity. Methods Enzymol. 399, 215–227 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R.W. Carthew, A.C. Rosenzweig, J. Widom and members of the Matouschek lab (Northwestern University), as well as R.J. Deshaies (Caltech), for advice and comments, and we thank G. Leigh for editing the manuscript. The work was supported by grant R01GM64003 from the US National Institutes of Health, by the Leukemia and Lymphoma Society and by the Robert H. Lurie Comprehensive Cancer Center at Northwestern University. T.I. gratefully acknowledges a Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2205 Tech Drive, Evanston, 60208, Illinois, USA

    Sumit Prakash, Tomonao Inobe, Ace Joseph Hatch & Andreas Matouschek

  2. Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 303 East Superior Street L3-125, Chicago, 60611, Illinois, USA

    Sumit Prakash, Tomonao Inobe, Ace Joseph Hatch & Andreas Matouschek

Authors
  1. Sumit Prakash
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tomonao Inobe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ace Joseph Hatch
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andreas Matouschek
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.P., T.I. and A.M. designed the experiments and wrote the manuscript. S.P. and T.I. performed most of the experiments. A.J.H. assisted S.P. in performing some of the degradation experiments, in purifying proteins and in constructing many of the genes.

Corresponding author

Correspondence to Andreas Matouschek.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, and Supplementary Methods (PDF 960 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Prakash, S., Inobe, T., Hatch, A. et al. Substrate selection by the proteasome during degradation of protein complexes. Nat Chem Biol 5, 29–36 (2009). https://doi.org/10.1038/nchembio.130

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.130

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing