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:

An unstructured initiation site is required for efficient proteasome-mediated degradation

Nature Structural & Molecular Biology volume 11, pages 830–837 (2004)Cite this article

Abstract

The proteasome is the main ATP-dependent protease in eukaryotic cells and controls the concentration of many regulatory proteins in the cytosol and nucleus. Proteins are targeted to the proteasome by the covalent attachment of polyubiquitin chains. The ubiquitin modification serves as the proteasome recognition element but by itself is not sufficient for efficient degradation of folded proteins. We report that proteolysis of tightly folded proteins is accelerated greatly when an unstructured region is attached to the substrate. The unstructured region serves as the initiation site for degradation and is hydrolyzed first, after which the rest of the protein is digested sequentially. These results identify the initiation site as a novel component of the targeting signal, which is required to engage the proteasome unfolding machinery efficiently. The proteasome degrades a substrate by first binding to its ubiquitin modification and then initiating unfolding at an unstructured region.

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: Sequential degradation of two-domain proteins by the proteasome in two directions.
Figure 2: Proteasome degradation is accelerated by an unstructured region in the substrate protein.
Figure 3: Proteasome degradation initiates at an unstructured region in the substrate protein.
Figure 4: Tightly folded substrates must contain an unstructured region for efficient proteasome degradation.
Figure 5: Characterization of degradation initiation sites.
Figure 6: Schematic representation of the proposed proteasome degradation cycle.

Similar content being viewed by others

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. 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 

  3. Groll, M. et al. Structure of 20S proteasome from yeast at 2.4 Å resolution. Nature 386, 463–471 (1997).

    Article  CAS  Google Scholar 

  4. Groll, M. et al. A gated channel into the proteasome core particle. Nat. Struct. Biol. 7, 1062–1067 (2000).

    Article  CAS  Google Scholar 

  5. Wenzel, T. & Baumeister, W. Conformational constraints in protein degradation by the 20S proteasome. Nat. Struct. Biol. 2, 199–204 (1995).

    Article  CAS  Google Scholar 

  6. Johnston, J.A., Johnson, E.S., Waller, P.R.H. & 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 

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

    CAS  PubMed  Google Scholar 

  8. 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 

  9. Braun, B.C. et al. The base of the proteasome regulatory particle exhibits chaperone-like activity. Nat. Cell Biol. 1, 221–226 (1999).

    Article  CAS  Google Scholar 

  10. 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 

  11. Kenniston, J.A., Baker, T.A., Fernandez, J.M. & Sauer, R.T. Linkage between ATP consumption and mechanical unfolding during the protein processing reactions of an AAA+ degradation machine. Cell 114, 511–520 (2003).

    Article  CAS  Google Scholar 

  12. Breitschopf, K., Bengal, E., Ziv, T., Admon, A. & Ciechanover, A. A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein. EMBO J. 17, 5964–5973 (1998).

    Article  CAS  Google Scholar 

  13. Pickart, C.M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001).

    Article  CAS  Google Scholar 

  14. 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 

  15. Verma, R. et al. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298, 611–615 (2002).

    Article  CAS  Google Scholar 

  16. Petroski, M.D. & Deshaies, R.J. Context of multiubiquitin chain attachment influences the rate of Sic1 degradation. Mol. Cell 11, 1435–1444 (2003).

    Article  CAS  Google Scholar 

  17. Hoskins, J.R., Yanagihara, K., Mizuuchi, K. & Wickner, S. ClpAP and ClpXP degrade proteins with tags located in the interior of the primary sequence. Proc. Natl. Acad. Sci. USA 99, 11037–11042 (2002).

    Article  CAS  Google Scholar 

  18. Kihara, A., Akiyama, Y. & Ito, K. Dislocation of membrane proteins in FtsH-mediated proteolysis. EMBO J. 18, 2970–2981 (1999).

    Article  CAS  Google Scholar 

  19. Reid, B.G., Fenton, W.A., Horwich, A.L. & Weber-Ban, E.U. ClpA mediates directional translocation of the substrate proteins into the ClpP protease. Proc. Natl. Acad. Sci. USA 98, 3768–3772 (2001).

    Article  CAS  Google Scholar 

  20. Herman, C., Prakash, S., Lu, C.Z., Matouschek, A. & Gross, C.A. Lack of a robust unfoldase activity confers a unique level of substrate specificity to the universal AAA protease FtsH. Mol. Cell 11, 659–669 (2003).

    Article  CAS  Google Scholar 

  21. 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 

  22. Orian, A. et al. Structural motifs involved in ubiquitin-mediated processing of the NF-κB precursor p105: roles of the glycine-rich region and a downstream ubiquitination domain. Mol. Cell Biol. 19, 3664–3673 (1999).

    Article  CAS  Google Scholar 

  23. Orlowski, M. & Wilk, S. Ubiquitin-independent proteolytic functions of the proteasome. Arch. Biochem. Biophys. 415, 1–5 (2003).

    Article  CAS  Google Scholar 

  24. 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 

  25. Uversky, V.N. What does it mean to be natively unfolded? Eur. J. Biochem. 269, 2–12 (2002).

    Article  CAS  Google Scholar 

  26. Viitanen, P.V., Donaldson, G.K., Lorimer, G.H., Lubben, T.H. & Gatenby, A.A. Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase. Biochemistry 30, 9716–9723 (1991).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. 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 

  29. Liu, C.W., Corboy, M.J., DeMartino, G.N. & Thomas, P.J. Endoproteolytic activity of the proteasome. Science 299, 408–411 (2003).

    Article  CAS  Google Scholar 

  30. Peng, J. et al. A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 21, 921–926 (2003).

    Article  CAS  Google Scholar 

  31. Scherer, D.C., Brockman, J.A., Chen, Z., Maniatis, T. & Ballard, D.W. Signal-induced degradation of IkBa requires site-specific ubiquitination. Proc. Natl. Acad. Sci. USA 92, 11259–11263 (1995).

    Article  CAS  Google Scholar 

  32. Glotzer, M., Murray, A.W. & Kirschner, M.W. Cyclin is degraded by the ubiquitin pathway. Nature 349, 132–138 (1991).

    Article  CAS  Google Scholar 

  33. Rodriguez, M.S., Desterro, J.M., Lain, S., Lane, D.P. & Hay, R.T. Multiple C-terminal lysine residues target p53 for ubiquitin-proteasome–mediated degradation. Mol. Cell Biol. 20, 8458–8467 (2000).

    Article  CAS  Google Scholar 

  34. Hoppe, T. et al. Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing. Cell 102, 577–586 (2000).

    Article  CAS  Google Scholar 

  35. Delagoutte, E. & von Hippel, P.H. Helicase mechanisms and the coupling of helicases within macromolecular machines. Part I: Structures and properties of isolated helicases. Q. Rev. Biophys. 35, 431–478 (2002).

    Article  CAS  Google Scholar 

  36. Tsu, C.A., Kossen, K. & Uhlenbeck, O.C. The Escherichia coli DEAD protein DbpA recognizes a small RNA hairpin in 23S rRNA. RNA 7, 702–709 (2001).

    Article  CAS  Google Scholar 

  37. Levchenko, I., Seidel, M., Sauer, R.T. & Baker, T.A. A specificity-enhancing factor for the ClpXP degradation machine. Science 289, 2354–2356 (2000).

    Article  CAS  Google Scholar 

  38. Neher, S.B., Sauer, R.T. & Baker, T.A. Distinct peptide signals in the UmuD and UmuD′ subunits of UmuD/D′ mediate tethering and substrate processing by the ClpXP protease. Proc. Natl. Acad. Sci. USA 100, 13219–13224 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  40. Alberti, S. et al. Ubiquitylation of BAG-1 suggests a novel regulatory mechanism during the sorting of chaperone substrates to the proteasome. J. Biol. Chem. 277, 45920–45927 (2002).

    Article  CAS  Google Scholar 

  41. Rao, H. & Sastry, A. Recognition of specific ubiquitin conjugates is important for the proteolytic functions of the ubiquitin-associated domain proteins Dsk2 and Rad23. J. Biol. Chem. 277, 11691–11695 (2002).

    Article  CAS  Google Scholar 

  42. Raasi, S. & Pickart, C.M. Rad23 ubiquitin-associated domains (UBA) inhibit 26S proteasome-catalyzed proteolysis by sequestering lysine 48–linked polyubiquitin chains. J. Biol. Chem. 278, 8951–8959 (2003).

    Article  CAS  Google Scholar 

  43. Kleijnen, M.F., Alarcon, R.M. & Howley, P.M. The ubiquitin-associated domain of hPLIC-2 interacts with the proteasome. Mol. Biol. Cell 14, 3868–3875 (2003).

    Article  CAS  Google Scholar 

  44. Bateman, A. et al. The Pfam protein families database. Nucleic Acids Res. 32 (Database issue), D138–D141 (2004).

    Article  CAS  Google Scholar 

  45. Dai, R.M., Chen, E., Longo, D.L., Gorbea, C.M. & Li, C.C. Involvement of valosin-containing protein, an ATPase co-purified with IκBα and 26 S proteasome, in ubiquitin-proteasome–mediated degradation of IκBα. J. Biol. Chem. 273, 3562–3573 (1998).

    Article  CAS  Google Scholar 

  46. Lam, Y.A., Xu, W., DeMartino, G.N. & Cohen, R.E. Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature 385, 737–740 (1997).

    Article  CAS  Google Scholar 

  47. Leggett, D.S. et al. Multiple associated proteins regulate proteasome structure and function. Mol. Cell 10, 495–507 (2002).

    Article  CAS  Google Scholar 

  48. Hartley, R.W. A two state conformational transition of the extracellular ribonuclease of Bacillus amyloliquefaciens (barnase) induced by sodium dodecyl sulfate. Biochemistry 14, 2367–2370 (1975).

    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. Iwakura, M., Nakamura, T., Yamane, C. & Maki, K. Systematic circular permutation of an entire protein reveals essential folding elements. Nat. Struct. Biol. 7, 580–585 (2000).

    Article  CAS  Google Scholar 

  51. Varshavsky, A. The N-end rule. Cell 69, 725–735 (1992).

    Article  CAS  Google Scholar 

  52. Matouschek, A. et al. Active unfolding of precursor proteins during mitochondrial protein import. EMBO J. 16, 6727–6736 (1997).

    Article  CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Pickart, L. Hicke, J. Widom, C. Holmberg and O. Uhlenbeck for advice and helpful comments on the manuscript. We also thank P. Bellare, J. Schnell, N. Jaffe and A. Wilcox for carefully reading and correcting the manuscript. M. Fisher (University of Kansas Medical Center) provided purified GroEL and advice. We acknowledge the use of the instruments at the Keck Biophysics Facility of the Robert H. Lurie Comprehensive Cancer Center at Northwestern University. The work was supported by US National Institutes of Health grant R01GM63004, by a Scholar Award to A.M. from the Leukemia and Lymphoma Society and by a Gramm Travel Fellowship Award to S.P. from the Robert H. Lurie Comprehensive Cancer Center at Northwestern University.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, 60208, Illinois, USA

    Sumit Prakash, Lin Tian, Kevin S Ratliff, Rebecca E Lehotzky & Andreas Matouschek

  2. Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, 60611, Illinois, USA

    Sumit Prakash, Lin Tian, Kevin S Ratliff, Rebecca E Lehotzky & Andreas Matouschek

Authors
  1. Sumit Prakash
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lin Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kevin S Ratliff
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rebecca E Lehotzky
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andreas Matouschek
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andreas Matouschek.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Prakash, S., Tian, L., Ratliff, K. et al. An unstructured initiation site is required for efficient proteasome-mediated degradation. Nat Struct Mol Biol 11, 830–837 (2004). https://doi.org/10.1038/nsmb814

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb814

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