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Chaperone-mediated pathway of proteasome regulatory particle assembly


The proteasome is a protease that controls diverse processes in eukaryotic cells. Its regulatory particle (RP) initiates the degradation of ubiquitin–protein conjugates by unfolding the substrate and translocating it into the proteasome core particle (CP) to be degraded1. The RP has 19 subunits, and their pathway of assembly is not understood. Here we show that in the yeast Saccharomyces cerevisiae three proteins are found associated with RP but not with the RP–CP holoenzyme: Nas6, Rpn14 and Hsm3. Mutations in the corresponding genes confer proteasome loss-of-function phenotypes, despite their virtual absence from the holoenzyme. These effects result from deficient RP assembly. Thus, Nas6, Rpn14 and Hsm3 are RP chaperones. The RP contains six ATPases–the Rpt proteins–and each RP chaperone binds to the carboxy-terminal domain of a specific Rpt. We show in an accompanying study2 that RP assembly is templated through the Rpt C termini, apparently by their insertion into binding pockets in the CP. Thus, RP chaperones may regulate proteasome assembly by directly restricting the accessibility of Rpt C termini to the CP. In addition, competition between the RP chaperones and the CP for Rpt engagement may explain the release of RP chaperones as proteasomes mature.

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Figure 1: Nas6, Hsm3 and Rpn14 bind to free RP.
Figure 2: Phenotypic analysis of nas6 Δ, hsm3 Δ and rpn14 Δ mutants.
Figure 3: RP chaperones bind to the C-domains of Rpt proteins in proximity to the CP.
Figure 4: Evolutionary conservation of the proteasome assembly pathway.


  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. Park, S. et al. Hexameric assembly of the proteasomal ATPases is templated through their C termini. Nature 10.1038/nature08065 (this issue)

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

    Article  CAS  Google Scholar 

  4. 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 

  5. Wang, X. et al. Mass spectrometric characterization of the affinity-purified human 26S proteasome complex. Biochemistry 46, 3553–3565 (2007)

    Article  CAS  Google Scholar 

  6. Dawson, S., Higashitsuji, H., Wilkinson, A. J., Fujita, J. & Mayer, R. J. Gankyrin: a new oncoprotein and regulator of pRb and p53. Trends Cell Biol. 16, 229–233 (2006)

    Article  CAS  Google Scholar 

  7. Guerrero, C., Milenkovic, T., Przulj, N., Kaiser, P. & Huang, L. Characterization of the proteasome interaction network using a QTAX-based tag-team strategy and protein interaction network analysis. Proc. Natl Acad. Sci. USA 105, 13333–13338 (2008)

    Article  ADS  CAS  Google Scholar 

  8. Park, Y. et al. Proteasomal ATPase-associated factor 1 negatively regulates proteasome activity by interacting with proteasomal ATPases. Mol. Cell. Biol. 25, 3842–3853 (2005)

    Article  CAS  Google Scholar 

  9. Lassot, I. et al. The proteasome regulates HIV-1 transcription by both proteolytic and nonproteolytic mechanisms. Mol. Cell 25, 369–383 (2007)

    Article  CAS  Google Scholar 

  10. Collins, G. A. & Tansey, W. P. The proteasome: a utility tool for transcription? Curr. Opin. Genet. Dev. 16, 197–202 (2006)

    Article  CAS  Google Scholar 

  11. Imai, J., Maruya, M., Yashiroda, H., Yahara, I. & Tanaka, K. The molecular chaperone Hsp90 plays a role in the assembly and maintenance of the 26S proteasome. EMBO J. 22, 3557–3567 (2003)

    Article  CAS  Google Scholar 

  12. 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 

  13. 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 

  14. Kusmierczyk, A. R. & Hochstrasser, M. Some assembly required: dedicated chaperones in eukaryotic proteasome biogenesis. Biol. Chem. 389, 1143–1151 (2008)

    Article  CAS  Google Scholar 

  15. 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 

  16. 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 

  17. 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 

  18. 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 

  19. 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 

  20. Ammelburg, M., Frickey, T. & Lupas, A. N. Classification of AAA+ proteins. J. Struct. Biol. 156, 2–11 (2006)

    Article  CAS  Google Scholar 

  21. 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 

  22. Nakamura, Y. et al. Structure of the oncoprotein gankyrin in complex with S6 ATPase of the 26S proteasome. Structure 15, 179–189 (2007)

    Article  CAS  Google Scholar 

  23. Zhang, F. et al. Structural insights into the regulatory particle of the proteasome from Methanocaldococcus jannaschii . Mol. Cell (in the press)

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

    Article  ADS  CAS  Google Scholar 

  25. 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 

  26. 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 

  27. Ellis, R. J. Molecular chaperones: assisting assembly in addition to folding. Trends Biochem. Sci. 31, 395–401 (2006)

    Article  CAS  Google Scholar 

  28. Hirano, Y. et al. Dissecting beta-ring assembly pathway of the mammalian 20S proteasome. EMBO J. 27, 2204–2213 (2008)

    Article  CAS  Google Scholar 

  29. Effantin, G., Rosenzweig, R., Glickman, M. & Steven, A. C. Electron microscopic evidence in support of α-solenoid models of proteasomal subunits Rpn1 and Rpn2. J. Mol. Biol. 386, 1204–1211 (2009)

    Article  CAS  Google Scholar 

  30. 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 

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We thank C. Mann for Rpt1 (also known as Cim5) and Rpt6 (Cim3) antibodies, K. Kubota for preparing HeLa lysates and especially L. Huang for the human Rpn11-HTBH-expressing cell line. We also thank J. Hanna, M. Schmidt and members of the Finley laboratory, in particular S. Elsasser, for critically reading the manuscript. This work was supported by the NIH (GM043601 to D.F. and GM67945 to S.P.G.), a NIH NRSA postdoctoral fellowship (5F32GM75737-2 to S.P.) and an EMBO long-term fellowship (to J.R.).

Author Contributions J.R., Y.H. and F.E.M. performed the experiments. W.H., F.E.M. and S.P.G. performed mass spectrometry. G.T. performed modelling. F.Z. and Y.S. contributed to modelling. B.-H.L. purified human proteasomes. J.R., S.P. and D.F. interpreted data and developed the model. J.R. and D.F. planned the studies and wrote the manuscript. All authors commented on the manuscript.

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Correspondence to Daniel Finley.

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Roelofs, J., Park, S., Haas, W. et al. Chaperone-mediated pathway of proteasome regulatory particle assembly. Nature 459, 861–865 (2009).

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