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Chaperone-mediated pathway of proteasome regulatory particle assembly

Nature volume 459, pages 861–865 (2009)Cite this article

Abstract

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.

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Acknowledgements

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

    Jeroen Roelofs, Soyeon Park, Wilhelm Haas, Geng Tian, Fiona E. McAllister, Ying Huo, Byung-Hoon Lee, Steven P. Gygi & Daniel Finley

  2. Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey 08544, USA,

    Fan Zhang & Yigong Shi

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Correspondence to Daniel Finley.

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This file contains Supplementary Figures 1-9 with Legends, Supplementary Tables 1-4, Supplementary Methods and Supplementary References. (PDF 3849 kb)

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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). https://doi.org/10.1038/nature08063

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