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.

  • Letter
  • Published:

Hexameric assembly of the proteasomal ATPases is templated through their C termini

Nature volume 459, pages 866–870 (2009)Cite this article

Abstract

Substrates of the proteasome are recognized and unfolded by the regulatory particle, and then translocated into the core particle (CP) to be degraded1. A hetero-hexameric ATPase ring, containing subunits Rpt1-6, is situated within the base subassembly of the regulatory particle1. The ATPase ring sits atop the CP, with the Rpt carboxy termini inserted into pockets in the CP2,3,4,5,6. Here we identify a previously unknown function of the Rpt proteins in proteasome biogenesis through deleting the C-terminal residue from each Rpt in the yeast Saccharomyces cerevisiae. Our results indicate that assembly of the hexameric ATPase ring is templated on the CP. We have also identified an apparent intermediate in base assembly, BP1, which contains Rpn1, three Rpts and Hsm3, a chaperone for base assembly. The Rpt proteins with the strongest assembly phenotypes, Rpt4 and Rpt6, were absent from BP1. We propose that Rpt4 and Rpt6 form a nucleating complex to initiate base assembly, and that this complex is subsequently joined by BP1 to complete the Rpt ring. Our studies show that assembly of the proteasome base is a rapid yet highly orchestrated process.

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: rpt4 -Δ1 and rpt6 -Δ1 mutants are proteasome hypomorphs with defective proteasome assembly.
Figure 2: Identification of a base assembly intermediate.
Figure 3: Pulse-chase analysis of the BP1 base assembly intermediate.
Figure 4: Interactions between Rpt C termini and the CP control RP-chaperone release.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Forster, A., Masters, E. I., Whitby, F. G., Robinson, H. & Hill, C. P. The 1.9 Å structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions. Mol. Cell 18, 589–599 (2005)

    Article  Google Scholar 

  3. Rabl, J. et al. Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases. Mol. Cell 30, 360–368 (2008)

    Article  CAS  Google Scholar 

  4. Smith, D. M. et al. Docking of the proteasomal ATPases’ carboxyl termini in the 20S proteasome’s alpha ring opens the gate for substrate entry. Mol. Cell 27, 731–744 (2007)

    Article  CAS  Google Scholar 

  5. Whitby, F. G. et al. Structural basis for the activation of 20S proteasomes by 11S regulators. Nature 408, 115–120 (2000)

    Article  ADS  CAS  Google Scholar 

  6. Gillette, T. G., Kumar, B., Thompson, D., Slaughter, C. A. & Demartino, G. N. Differential roles of the C-termini of AAA subunits of PA700 (19S regulator) in asymmetric assembly and activation of the 26s proteasome. J. Biol. Chem. 283, 31813–31822 (2008)

    Article  CAS  Google Scholar 

  7. Borissenko, L. & Groll, M. 20S proteasome and its inhibitors: crystallographic knowledge for drug development. Chem. Rev. 107, 687–717 (2007)

    Article  CAS  Google Scholar 

  8. Mannhaupt, G., Schnall, R., Karpov, V., Vetter, I. & Feldmann, H. Rpn4p acts as a transcription factor by binding to PACE, a nonamer box found upstream of 26S proteasomal and other genes in yeast. FEBS Lett. 450, 27–34 (1999)

    Article  CAS  Google Scholar 

  9. Xie, Y. & Varshavsky, A. RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: a negative feedback circuit. Proc. Natl Acad. Sci. USA 98, 3056–3061 (2001)

    Article  ADS  CAS  Google Scholar 

  10. Isono, E. et al. The assembly pathway of the 19S regulatory particle of the yeast 26S proteasome. Mol. Biol. Cell 18, 569–580 (2007)

    Article  CAS  Google Scholar 

  11. Kusmierczyk, A. R., Kunjappu, M. J., Funakoshi, M. & Hochstrasser, M. A multimeric assembly factor controls the formation of alternative 20S proteasomes. Nature Struct. Mol. Biol. 15, 237–244 (2008)

    Article  CAS  Google Scholar 

  12. Le Tallec, B., Barrault, M. B., Guerois, R., Carre, T. & Peyroche, A. Hsm3/S5b participates in the assembly pathway of the 19S regulatory particle of the proteasome. Mol. Cell 33, 389–399 (2009)

    Article  CAS  Google Scholar 

  13. Saeki, Y., Toh-e, A. & Yokosawa, H. Rapid isolation and characterization of the yeast proteasome regulatory complex. Biochem. Biophys. Res. Commun. 273, 509–515 (2000)

    Article  CAS  Google Scholar 

  14. Leggett, D. S., Glickman, M. H. & Finley, D. Purification of proteasomes, proteasome subcomplexes, and proteasome-associated proteins from budding yeast. Methods Mol. Biol. 301, 57–70 (2005)

    CAS  PubMed  Google Scholar 

  15. 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)

    Article  CAS  Google Scholar 

  16. Roelofs, J. et al. Chaperone-mediated pathway of proteasome regulatory particle assembly. Nature 10.1038/nature08063 (this issue)

  17. Nakamura, Y. et al. Structural basis for the recognition between the regulatory particles Nas6 and Rpt3 of the yeast 26S proteasome. Biochem. Biophys. Res. Commun. 359, 503–509 (2007)

    Article  CAS  Google Scholar 

  18. Dawson, S. et al. Gankyrin is an ankyrin-repeat oncoprotein that interacts with CDK4 kinase and the S6 ATPase of the 26 S proteasome. J. Biol. Chem. 277, 10893–10902 (2002)

    Article  CAS  Google Scholar 

  19. Kress, W., Mutschler, H. & Weber-Ban, E. Assembly pathway of an AAA+ protein: tracking ClpA and ClpAP complex formation in real time. Biochemistry 46, 6183–6193 (2007)

    Article  CAS  Google Scholar 

  20. Richmond, C., Gorbea, C. & Rechsteiner, M. Specific interactions between ATPase subunits of the 26 S protease. J. Biol. Chem. 272, 13403–13411 (1997)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Gorbea, C., Taillandier, D. & Rechsteiner, M. Mapping subunit contacts in the regulatory complex of the 26S proteasome. S2 and S5b form a tetramer with ATPase subunits S4 and S7. J. Biol. Chem. 275, 875–882 (2000)

    Article  CAS  Google Scholar 

  23. Rosenzweig, R., Osmulski, P. A., Gaczynska, M. & Glickman, M. H. The central unit within the 19S regulatory particle of the proteasome. Nature Struct. Mol. Biol. 15, 573–580 (2008)

    Article  CAS  Google Scholar 

  24. Guthrie, C. & Fink, G. R. Guide to Yeast Genetics and Molecular Biology. (Academic Press, 1991)

    Google Scholar 

  25. 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, 3425–3439 (2000)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Li, X., Kusmierczyk, A. R., Wong, P., Emili, A. & Hochstrasser, M. Beta-subunit appendages promote 20S proteasome assembly by overcoming an Ump1-dependent checkpoint. EMBO J. 26, 2339–2349 (2007)

    Article  CAS  Google Scholar 

  28. Kleijnen, M. F. et al. Stability of the proteasome can be regulated allosterically through engagement of its proteolytic active sites. Nature Struct. Mol. Biol. 14, 1180–1188 (2007)

    Article  CAS  Google Scholar 

  29. Hanna, J. et al. Deubiquitinating enzyme Ubp6 functions noncatalytically to delay proteasomal degradation. Cell 127, 99–111 (2006)

    Article  CAS  Google Scholar 

  30. Peng, J. & Gygi, S. P. Proteomics: the move to mixtures. J. Mass Spectrom. 36, 1083–1091 (2001)

    Article  ADS  CAS  Google Scholar 

  31. Eng, J. K., McCormack, A. L. & Yates, J. R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. D. Orth for discussion and technical assistance, and C. Hill for discussions. We also thank J. W. Hanna, S. Elsasser and all members of Finley laboratory for comments on the manuscript. The work was supported by grants from US National Institutes of Health (NIH) to D.F. (GM043601) and S.P.G. (GM67945), an EMBO long-term fellowship to J.R. and an NIH NRSA postdoctoral fellowship (5F32GM75737-2) to S.P.

Author Contributions S.P. conducted all experiments. J.R. and D.F. contributed to designing experiments. W.K. performed mass spectrometry. S.P. and D.F. wrote the paper. All authors provided comments.

Author information

Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA,

    Soyeon Park, Jeroen Roelofs, Woong Kim, Jessica Robert, Steven P. Gygi & Daniel Finley

  2. Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Forchheimer Building, Room 305, Bronx, New York 10461, USA,

    Marion Schmidt

Authors
  1. Soyeon Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeroen Roelofs
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Woong Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jessica Robert
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marion Schmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Steven P. Gygi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Daniel Finley
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel Finley.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-5 with Legends, Supplementary Tables 1-2 and Supplementary References. (PDF 2487 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Park, S., Roelofs, J., Kim, W. et al. Hexameric assembly of the proteasomal ATPases is templated through their C termini. Nature 459, 866–870 (2009). https://doi.org/10.1038/nature08065

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08065

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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