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A cryptic protease couples deubiquitination and degradation by the proteasome

Abstract

The 26S proteasome is responsible for most intracellular proteolysis in eukaryotes1,2. Efficient substrate recognition relies on conjugation of substrates with multiple ubiquitin molecules and recognition of the polyubiquitin moiety by the 19S regulatory complex—a multisubunit assembly that is bound to either end of the cylindrical 20S proteasome core. Only unfolded proteins can pass through narrow axial channels into the central proteolytic chamber of the 20S core, so the attached polyubiquitin chain must be released to allow full translocation of the substrate polypeptide. Whereas unfolding is rate-limiting for the degradation of some substrates and appears to involve chaperone-like activities associated with the proteasome3,4,5, the importance and mechanism of degradation-associated deubiquitination has remained unclear. Here we report that the POH1 (also known as Rpn11 in yeast) subunit of the 19S complex is responsible for substrate deubiquitination during proteasomal degradation. The inability to remove ubiquitin can be rate-limiting for degradation in vitro and is lethal to yeast. Unlike all other known deubiquitinating enzymes (DUBs) that are cysteine proteases6,7, POH1 appears to be a Zn2+-dependent protease.

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Figure 1: Proteasomal degradation of UbOM is targeted by means of the unfolded ovomucoid moiety.
Figure 2: A ubiquitin-aldehyde (Ubal)-insensitive DUB couples release of ubiquitin with substrate degradation (western blots).
Figure 3: Alignment of the MPN domain of MPN family proteins.
Figure 4: Evidence that Rpn11/POH1 is a Zn2+-dependent DUB and characterization of rpn11 mutants.

References

  1. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998)

    CAS  Google Scholar 

  2. Zwickl, P., Baumeister, W. & Steven, A. Dis-assembly lines: the proteasome and related ATPase-assisted proteases. Curr. Opin. Struct. Biol. 10, 242–250 (2000)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  5. Liu, C. et al. Conformational remodeling of proteasomal substrates by PA700, the 19S regulatory complex of the 26S proteasome. J. Biol. Chem. 277, 26815–26820 (2002)

    CAS  Article  Google Scholar 

  6. D'Andrea, A. & Pellman, D. Deubiquitinating enzymes: a new class of biological regulators. Crit. Rev. Biochem. Mol. Biol. 33, 337–352 (1998)

    CAS  Article  Google Scholar 

  7. Amerik, A. Y., Li, S. J. & Hochstrasser, M. Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae. Biol. Chem. 381, 981–992 (2000)

    CAS  Article  Google Scholar 

  8. DeKoster, G. T. & Robertson, A. D. Thermodynamics of unfolding for Kazal-type serine protease inhibitors: entropic stabilization of ovomucoid first domain by glycosylation. Biochemistry 36, 2323–2331 (1997)

    CAS  Article  Google Scholar 

  9. Johnson, E. S., Ma, P. C., Ota, I. M. & Varshavsky, A. A proteolytic pathway that recognizes ubiquitin as a degradation signal. J. Biol. Chem. 270, 17442–17456 (1995)

    CAS  Article  Google Scholar 

  10. Hershko, A. & Rose, I. A. Ubiquitin-aldehyde: a general inhibitor of ubiquitin-recycling processes. Proc. Natl Acad. Sci. USA 84, 1829–1833 (1987)

    ADS  CAS  Article  Google Scholar 

  11. 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)

    ADS  CAS  Article  Google Scholar 

  12. Wintrode, P. L., Makhatadze, G. I. & Privalov, P. L. Thermodynamics of ubiquitin unfolding. Proteins 18, 246–253 (1994)

    CAS  Article  Google Scholar 

  13. Borodovsky, A. et al. A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J. 20, 5187–5196 (2001)

    CAS  Article  Google Scholar 

  14. 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)

    ADS  CAS  Article  Google Scholar 

  15. Rubin, D. M., Glickman, M. H., Larsen, C. N., Dhruvakumar, S. & Finley, D. Active site mutants in the six regulatory particle ATPases reveal multiple roles for ATP in the proteasome. EMBO J. 17, 4909–4919 (1998)

    CAS  Article  Google Scholar 

  16. Leggett, D. S. et al. Multiple associated proteins regulate proteasome structure and function. Mol. Cell (in the press)

  17. Papa, F. R. & Hochstrasser, M. The yeast DOA4 gene encodes a deubiquitinating enzyme related to a product of the human tre-2 oncogene. Nature 366, 313–319 (1993)

    ADS  CAS  Article  Google Scholar 

  18. Papa, F. R., Amerik, A. Y. & Hochstrasser, M. Interaction of the Doa4 deubiquitinating enzyme with the yeast 26S proteasome. Mol. Biol. Cell 10, 741–756 (1999)

    CAS  Article  Google Scholar 

  19. Glickman, M. H. et al. A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell 94, 615–623 (1998)

    CAS  Article  Google Scholar 

  20. Lyapina, S. et al. Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome. Science 292, 1382–1385 (2001)

    ADS  CAS  Article  Google Scholar 

  21. Hofmann, K. & Bucher, P. The PCI domain: a common theme in three multiprotein complexes. Trends Biochem. Sci. 23, 204–205 (1998)

    CAS  Article  Google Scholar 

  22. Glickman, M. H., Rubin, D. M., Fried, V. A. & Finley, D. The regulatory particle of the Saccharomyces cerevisiae proteasome. Mol. Cell Biol. 18, 3149–3162 (1998)

    CAS  Article  Google Scholar 

  23. Alberts, I. L., Nadassy, K. & Wodak, S. J. Analysis of zinc binding sites in protein crystal structures. Protein Sci. 7, 1700–1716 (1998)

    CAS  Article  Google Scholar 

  24. Eytan, E., Armon, T., Heller, H., Beck, S. & Hershko, A. Ubiquitin C-terminal hydrolase activity associated with the 26 S protease complex. J. Biol. Chem. 268, 4668–4674 (1993)

    CAS  PubMed  Google Scholar 

  25. Wilkinson, K. D. Regulation of ubiquitin-dependent processes by deubiquitinating enzymes. FASEB J. 11, 1245–1256 (1997)

    CAS  Article  Google Scholar 

  26. Ponting, C. P., Aravind, L., Schultz, J., Bork, P. & Koonin, E. V. Eukaryotic signalling domain homologues in archaea and bacteria. Ancient ancestry and horizontal gene transfer. J. Mol. Biol. 289, 729–745 (1999)

    CAS  Article  Google Scholar 

  27. Thick, J., Mak, Y. F., Metcalfe, J., Beatty, D. & Taylor, A. M. A gene on chromosome Xq28 associated with T-cell prolymphocytic leukemia in two patients with ataxia telangiectasia. Leukemia 8, 564–573 (1994)

    CAS  PubMed  Google Scholar 

  28. Beers, E. P. & Callis, J. Utility of polyhistidine-tagged ubiquitin in the purification of ubiquitin-protein conjugates and as an affinity ligand for the purification of ubiquitin-specific hydrolases. J. Biol. Chem. 268, 21645–21649 (1993)

    CAS  PubMed  Google Scholar 

  29. Hoffman, L., Pratt, G. & Rechsteiner, M. Multiple forms of the 20S multicatalytic and the 26S ubiquitin/ATP- dependent proteases from rabbit reticulocyte lysate. J. Biol. Chem. 267, 22362–22368 (1992)

    CAS  PubMed  Google Scholar 

  30. Piotrowski, J. et al. Inhibition of the 26S proteasome by polyubiquitin chains synthesized to have defined lengths. J. Biol. Chem. 272, 23712–23721 (1997)

    CAS  Article  Google Scholar 

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Acknowledgements

We thank D. Finley and D. Leggett for yeast strains, advice on proteasome purification, and a sample of yeast proteasomes; G. DeMartino for bovine PA700; A. Robertson for ovomucoid protein; C. Pickart for the Ub(V76)DHFR plasmid; K. Wilkinson, H. Ploegh and A. Borodovsky for UbVS, recombinant Ubp6 protein and anti-human Ubp6; C. Pickart, A. Lam and K. Wilkinson for discussions; and L. Weisman and C. Pickart for comments on the manuscript. This work was supported by a grant from the NIH and a UI Biocatalysis and Bioprocessing Fellowship to T.Y.

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Correspondence to Robert E. Cohen.

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Yao, T., Cohen, R. A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419, 403–407 (2002). https://doi.org/10.1038/nature01071

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