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A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal


The 26S proteasome is the chief site of regulatory protein turnover in eukaryotic cells1. It comprises one 20S catalytic complex (composed of four stacked rings of seven members) and two axially positioned 19S regulatory complexes (each containing about 18 subunits) that control substrate access to the catalytic chamber2. In most cases, targeting to the 26S proteasome depends on tagging of the substrate with a specific type of polyubiquitin chain3,4,5,6. Recognition of this signal is followed by substrate unfolding and translocation, which are presumably catalysed by one or more of six distinct AAA ATPases located in the base—a ring-like 19S subdomain that abuts the axial pore of the 20S complex and exhibits chaperone activity in vitro7,8,9. Despite the importance of polyubiquitin chain recognition in proteasome function, the site of this signal's interaction with the 19S complex has not been identified previously. Here we use crosslinking to a reactive polyubiquitin chain to show that a specific ATPase subunit, S6′ (also known as Rpt5), contacts the bound chain. The interaction of this signal with 26S proteasomes is modulated by ATP hydrolysis. Our results suggest that productive recognition of the proteolytic signal, as well as proteasome assembly and substrate unfolding, are ATP-dependent events.

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Figure 1: Crosslinking strategy.
Figure 2: Specific crosslinking of Ub4 to proteasomes.
Figure 3: The 50K crosslinked protein is S6′.
Figure 4: ATP hydrolysis modulates the polyubiquitin chain–proteasome interaction (EPR).


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

    CAS  Article  Google Scholar 

  2. Voges, D., Zwickl, P. & Baumeister, W. The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem. 68, 1015–1068 (1999).

    CAS  Article  Google Scholar 

  3. Chau, V. et al. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243, 1576–1583 (1989).

    ADS  CAS  Article  Google Scholar 

  4. Finley, D. et al. Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant. Mol. Cell. Biol. 14, 5501–5509 (1994).

    CAS  Article  Google Scholar 

  5. Pickart, C .M. Ubiquitin in chains. Trends Biochem. Sci. 25, 544–548 (2000).

    CAS  Article  Google Scholar 

  6. 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 

  7. 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 

  8. 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 

  9. Strickland, E., Hakala, K., Thomas, P. J. & DeMartino, G. N. Recognition of misfolded proteins by PA700, the regulatory subcomplex of the 26 S proteasome. J. Biol. Chem. 275, 5565–5572 (2000).

    CAS  Article  Google Scholar 

  10. Beal, R., Deveraux, Q., Xia, G., Rechsteiner, M. & Pickart, C. Surface hydrophobic residues of multiubiquitin chains essential for proteolytic targeting. Proc. Natl Acad. Sci. USA 93, 861–866 (1996).

    ADS  CAS  Article  Google Scholar 

  11. Vijay-Kumar, S., Bugg, C. E. & Cook, W. J. Structure of ubiquitin refined at 1.8 Å resolution. J. Mol. Biol. 194, 531–544 (1987).

    CAS  Article  Google Scholar 

  12. Cook, W. J., Jeffrey, L. C., Kasperek, E. & Pickart, C. M. Structure of tetraubiquitin shows how multiubiquitin chains can be formed. J. Mol. Biol. 236, 601–609 (1994).

    CAS  Article  Google Scholar 

  13. 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).

    CAS  Article  Google Scholar 

  14. 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 

  15. Fu, H. et al. Multiubiquitin chain binding and protein degradation are mediated by distinct domains within the 26 S proteasome subunit Mcb1. J. Biol. Chem. 273, 1970–1981 (1998).

    CAS  Article  Google Scholar 

  16. van Nocker, S. et al. The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover. Mol. Cell. Biol. 16, 6020–6028 (1996).

    CAS  Article  Google Scholar 

  17. Hartmann-Petersen, R., Tanaka, K. & Hendil, K. B. Quarternary structure of the ATPase complex of human 26S proteasomes determined by chemical cross-linking. Arch. Biochem. Biophys. 386, 89–94 (2001).

    CAS  Article  Google Scholar 

  18. Davy, A. et al. A protein–protein interaction map of the Caenorhabditis elegans 26S proteasome. EMBO Rep. 2, 821–828 (2001).

    CAS  Article  Google Scholar 

  19. DeMartino, G. N. et al. Identification, purification, and characterization of a PA700-dependent activator of the proteasome. J. Biol. Chem. 271, 3112–3118 (1996).

    CAS  Article  Google Scholar 

  20. Hoffman, L. & Rechsteiner, M. Nucleotidase activities of the 26S proteasome and its regulatory complex. J. Biol. Chem. 271, 32538–32545 (1997).

    Article  Google Scholar 

  21. Verma, R. et al. Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol. Biol. Cell 11, 3425–3429 (2000).

    CAS  Article  Google Scholar 

  22. Qin, P. Z., Butcher, S. E., Feigon, J. & Hubbell, W. L. Quantitative analysis of the isolated GAAA tetraloop/receptor interaction in solution: a site-directed spin labeling study. Biochemistry 40, 6929–6936 (2001).

    CAS  Article  Google Scholar 

  23. Dai, R. M. & Li, C.-C. H. Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation. Nature Cell Biol. 3, 740–744 (2001).

    CAS  Article  Google Scholar 

  24. Rubin, C. 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 

  25. Kohler, A. et al. The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release. Mol. Cell 7, 1143–1152 (2001).

    ADS  CAS  Article  Google Scholar 

  26. 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 

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

    CAS  Article  Google Scholar 

  28. Sankarapandi, S. & Zweier, J. L. Bicarbonate is required for the peroxidase function of Cu,Zn-superoxide dismutase at physiological pH. J. Biol. Chem. 274, 1226–1232 (1999).

    CAS  Article  Google Scholar 

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We thank G. DeMartino for a gift of modulator complex; A. Varshavksy for the GST–Rpt5 construct; A. Mildvan for discussions; and M. Hochstrasser and B. Cohen for comments on the manuscript. This work was supported by a grant from the National Institutes of Health. Y.A.L. is a senior fellow of the Leukemia and Lymphoma Society.

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Correspondence to Cecile M. Pickart.

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Lam, Y., Lawson, T., Velayutham, M. et al. A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal. Nature 416, 763–767 (2002).

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