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:

Defining the geometry of the two-component proteasome degron

Nature Chemical Biology volume 7, pages 161–167 (2011)Cite this article

Subjects

Abstract

The eukaryotic 26S proteasome controls cellular processes by degrading specific regulatory proteins. Most proteins are targeted for degradation by a signal or degron that consists of two parts: a proteasome-binding tag, typically covalently attached polyubiquitin chains, and an unstructured region that serves as the initiation region for proteasomal proteolysis. Here we have characterized how the arrangement of the two degron parts in a protein affects degradation. We found that a substrate is degraded efficiently only when its initiation region is of a certain minimal length and is appropriately separated in space from the proteasome-binding tag. Regions that are located too close or too far from the proteasome-binding tag cannot access the proteasome and induce degradation. These spacing requirements are different for a polyubiquitin chain and a ubiquitin-like domain. Thus, the arrangement and location of the proteasome initiation region affect a protein's fate and are important in selecting proteins for proteasome-mediated 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-mediated degradation depends on initiation region length.
Figure 2: Separating the proteasome-binding tag and initiation region in degradation substrates.
Figure 3: Immunoglobulin domain I27 insertions increase the distance between proteasome-binding tag and the initiation region.
Figure 4: Proteasome-mediated degradation depends on the spacing between proteasome-binding tag and initiation region.
Figure 5: Spacing requirements for the two degron parts as initiation region length varies.
Figure 6: Schematic representation of the length and spacing requirement for the initiation region.

Similar content being viewed by others

References

  1. Pickart, C.M. Back to the future with ubiquitin. Cell 116, 181–190 (2004).

    Article  CAS  Google Scholar 

  2. 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 

  3. Takeuchi, J., Chen, H. & Coffino, P. Proteasome substrate degradation requires association plus extended peptide. EMBO J. 26, 123–131 (2007).

    Article  CAS  Google Scholar 

  4. Schrader, E.K., Harstad, K.G. & Matouschek, A. Targeting proteins for degradation. Nat. Chem. Biol. 5, 815–822 (2009).

    Article  CAS  Google Scholar 

  5. 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 

  6. 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  Google Scholar 

  7. 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 

  8. Husnjak, K. et al. Proteasome subunit Rpn13 is a novel ubiquitin receptor. Nature 453, 481–488 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Finley, D. Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513 (2009).

    Article  CAS  Google Scholar 

  11. Hiyama, H. et al. Interaction of hHR23 with S5a. The ubiquitin-like domain of hHR23 mediates interaction with S5a subunit of 26 S proteasome. J. Biol. Chem. 274, 28019–28025 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Saeki, Y., Sone, T., Toh-e, A. & Yokosawa, H. Identification of ubiquitin-like protein-binding subunits of the 26S proteasome. Biochem. Biophys. Res. Commun. 296, 813–819 (2002).

    Article  CAS  Google Scholar 

  14. Prakash, S., Inobe, T., Hatch, A.J. & Matouschek, A. Substrate selection by the proteasome during degradation of protein complexes. Nat. Chem. Biol. 5, 29–36 (2009).

    Article  CAS  Google Scholar 

  15. 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 

  16. 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 

  17. 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 

  18. 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 

  19. 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 

  20. Komander, D. et al. Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains. EMBO Rep. 10, 466–473 (2009).

    Article  CAS  Google Scholar 

  21. Saeki, Y. et al. Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome. EMBO J. 28, 359–371 (2009).

    Article  CAS  Google Scholar 

  22. Watkins, J.F., Sung, P., Prakash, L. & Prakash, S. The Saccharomyces cerevisiae DNA repair gene RAD23 encodes a nuclear protein containing a ubiquitin-like domain required for biological function. Mol. Cell. Biol. 13, 7757–7765 (1993).

    Article  CAS  Google Scholar 

  23. Schauber, C. et al. Rad23 links DNA repair to the ubiquitin/proteasome pathway. Nature 391, 715–718 (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. Verhoef, L.G. et al. Minimal length requirement for proteasomal degradation of ubiquitin-dependent substrates. FASEB J. 23, 123–133 (2009).

    Article  CAS  Google Scholar 

  26. Politou, A.S., Gautel, M., Pfuhl, M., Labeit, S. & Pastore, A. Immunoglobulin-type domains of titin: same fold, different stability? Biochemistry 33, 4730–4737 (1994).

    Article  CAS  Google Scholar 

  27. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J.M. & Gaub, H.E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–1112 (1997).

    Article  CAS  Google Scholar 

  28. Improta, S., Politou, A.S. & Pastore, A. Immunoglobulin-like modules from titin I-band: extensible components of muscle elasticity. Structure 4, 323–337 (1996).

    Article  CAS  Google Scholar 

  29. von Castelmur, E. et al. A regular pattern of Ig super-motifs defines segmental flexibility as the elastic mechanism of the titin chain. Proc. Natl. Acad. Sci. USA 105, 1186–1191 (2008).

    Article  CAS  Google Scholar 

  30. Yang, T.T., Cheng, L. & Kain, S.R. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res. 24, 4592–4593 (1996).

    Article  CAS  Google Scholar 

  31. Adams, S.R. et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063–6076 (2002).

    Article  CAS  Google Scholar 

  32. Beskow, A. et al. A conserved unfoldase activity for the p97 AAA-ATPase in proteasomal degradation. J. Mol. Biol. 394, 732–746 (2009).

    Article  CAS  Google Scholar 

  33. 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 

  34. Larsen, C.N. & Finley, D. Protein translocation channels in the proteasome and other proteases. Cell 91, 431–434 (1997).

    Article  CAS  Google Scholar 

  35. Baumeister, W., Walz, J., Zühl, F. & Seemüller, E. The proteasome: paradigm of a self-compartmentalizing protease. Cell 92, 367–380 (1998).

    Article  CAS  Google Scholar 

  36. Keiler, K.C., Waller, P.R. & Sauer, R.T. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271, 990–993 (1996).

    Article  CAS  Google Scholar 

  37. Gottesman, S., Roche, E., Zhou, Y. & Sauer, R.T. The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. Genes Dev. 12, 1338–1347 (1998).

    Article  CAS  Google Scholar 

  38. Flynn, J.M., Neher, S.B., Kim, Y.I., Sauer, R.T. & Baker, T.A. Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals. Mol. Cell 11, 671–683 (2003).

    Article  CAS  Google Scholar 

  39. Gonzalez, M., Frank, E.G., Levine, A.S. & Woodgate, R. Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: in vitro degradation and identification of residues required for proteolysis. Genes Dev. 12, 3889–3899 (1998).

    Article  CAS  Google Scholar 

  40. Yamada-Inagawa, T., Okuno, T., Karata, K., Yamanaka, K. & Ogura, T. Conserved pore residues in the AAA protease FtsH are important for proteolysis and its coupling to ATP hydrolysis. J. Biol. Chem. 278, 50182–50187 (2003).

    Article  CAS  Google Scholar 

  41. Hinnerwisch, J., Fenton, W.A., Furtak, K.J., Farr, G.W. & Horwich, A.L. Loops in the central channel of ClpA chaperone mediate protein binding, unfolding, and translocation. Cell 121, 1029–1041 (2005).

    Article  CAS  Google Scholar 

  42. Martin, A., Baker, T.A. & Sauer, R.T. Diverse pore loops of the AAA+ ClpX machine mediate unassisted and adaptor-dependent recognition of ssrA-tagged substrates. Mol. Cell 29, 441–450 (2008).

    Article  CAS  Google Scholar 

  43. Zhang, F. et al. Mechanism of substrate unfolding and translocation by the regulatory particle of the proteasome from Methanocaldococcus jannaschii. Mol. Cell 34, 485–496 (2009).

    Article  CAS  Google Scholar 

  44. Djuranovic, S. et al. Structure and activity of the N-terminal substrate recognition domains in proteasomal ATPases. Mol. Cell 34, 580–590 (2009).

    Article  CAS  Google Scholar 

  45. Miller, W.G. & Goebel, C.V. Dimensions of protein random coils. Biochemistry 7, 3925–3935 (1968).

    Article  CAS  Google Scholar 

  46. 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 

  47. Levchenko, I., Grant, R.A., Flynn, J.M., Sauer, R.T. & Baker, T.A. Versatile modes of peptide recognition by the AAA+ adaptor protein SspB. Nat. Struct. Mol. Biol. 12, 520–525 (2005).

    Article  CAS  Google Scholar 

  48. McGinness, K.E., Bolon, D.N., Kaganovich, M., Baker, T.A. & Sauer, R.T. Altered tethering of the SspB adaptor to the ClpXP protease causes changes in substrate delivery. J. Biol. Chem. 282, 11465–11473 (2007).

    Article  CAS  Google Scholar 

  49. Rood, J.I., Laird, A.J. & Williams, J.W. Cloning of the Escherichia coli K-12 dihydrofolate reductase gene following mu-mediated transposition. Gene 8, 255–265 (1980).

    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 S. Elsasser (Harvard University Medical School), D. Finley (Harvard University Medical School), B.S. Glick (University of Chicago) and Y. Saeki (Tokyo Metropolitan Institute of Medical Science) for providing plasmids and yeast strains and members of the Matouschek lab for advice and comments. The authors greatly appreciate and gratefully acknowledge N. Nukina (RIKEN) for his support and the use of his laboratory equipment. We also thank G. Leigh for editing the manuscript. The work was supported by grants R01GM063004 and U54CA143869 from the US National Institutes of Health, by Ministry of Education, Culture, Sports, Science and Technology of Japan Grant-in-Aid 22770137 (T.I.) and by the Robert H. Lurie Comprehensive Cancer Center at Northwestern University. T.I. also gratefully acknowledges a Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad and the RIKEN Special Postdoctoral Researchers Program.

Author information

Authors and Affiliations

  1. Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA

    Tomonao Inobe, Susan Fishbain, Sumit Prakash & Andreas Matouschek

  2. Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois, USA

    Tomonao Inobe, Susan Fishbain, Sumit Prakash & Andreas Matouschek

  3. Laboratory for Structural Neuropathology, RIKEN Brain Science Institute, Wako, Saitama, Japan

    Tomonao Inobe

Authors
  1. Tomonao Inobe
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Susan Fishbain
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sumit Prakash
    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

T.I., S.F., S.P. and A.M. designed and interpreted the experiments and wrote the manuscript. T.I., S.F. and S.P. performed the experiments.

Corresponding author

Correspondence to Andreas Matouschek.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Methods (PDF 1163 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Inobe, T., Fishbain, S., Prakash, S. et al. Defining the geometry of the two-component proteasome degron. Nat Chem Biol 7, 161–167 (2011). https://doi.org/10.1038/nchembio.521

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.521

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