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Reconfiguration of the proteasome during chaperone-mediated assembly

Nature volume 497, pages 512–516 (2013)Cite this article

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Abstract

The proteasomal ATPase ring, comprising Rpt1–Rpt6, associates with the heptameric α-ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt carboxy-terminal tails inserting into pockets of the α-ring1,2,3,4. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit5,6,7,8,9,10. Here we report that the base subassembly of the Saccharomyces cerevisiae proteasome, which includes the Rpt ring, forms a high-affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6 and Rpn14. Chaperone-mediated dissociation was abrogated by a non-hydrolysable ATP analogue, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α-pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3-pocket. Although the Rpt6 tail is not visualized within an α-pocket in mature proteasomes2,3,4, it inserts into the α2/α3-pocket in the base–CP complex and is important for complex formation. Thus, the Rpt–CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme.

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Figure 1: Chaperones inhibit base–CP assembly.
Figure 2: Base–CP association is nucleotide dependent.
Figure 3: Difference maps reveal binding sites of Rpt C-terminal peptides to CP α-pockets.
Figure 4: Rpt6 C-terminal tail promotes formation of base–CP complex.
Figure 5: Three-dimensional reconstruction of base–CP complex reveals an asymmetric interaction between the Rpt ring and the α-ring of the CP.

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Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Data have been deposited in the Electron Microscopy Data Bank under the following accession numbers: free CP, EMD-5593; Rpt1–CP, EMD-5611; Rpt2–CP, EMD-5612; Rpt3–CP, EMD-5613; Rpt4–CP, EMD-5614; Rpt5–CP, EMD-5615; Rpt6–CP, EMD-5616; and base1–CP: EMD-5617. For the crystal structures, data have been deposited in the Protein Data Bank under accessions 4FP7 (Hsm3) and 4JPO (Hsm3–Rpt1 C domain).

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Acknowledgements

We thank M. Schmidt, T. Walz, C. Chen, and Finley laboratory members for suggestions, and C. Mann for antibodies. This work was supported in part by grants from the National Institutes of Health (NIH; R01GM082893 and 1S10RR026814-01), the University of California San Francisco Program for Breakthrough Biomedical Research (New Technology Award) to Y.C.; the Johnson Cancer Research Center, the National Center for Research Resources (5P20RR017708 and P20 RR016475) and NIH (8 P20 GM103420 and P20 GM103418) to J.R.; and grants from the NIH to P.C. (R01GM045335) and D.F. (R37GM043601). S.P. was supported by the Charles A. King Trust Postdoctoral Research Fellowship Program of the Medical Foundation. Use of IMCA-CAT was supported by the Industrial Macromolecular Crystallography Association though a contract with the Hauptman-Woodward MRI. Use of the Advanced Photon Source was supported by the US Department of Energy (contract no. DE-AC02-06CH11357).

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Author notes

  1. Ho Min Kim

    Present address: Present address: Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.,

  2. Soyeon Park, Xueming Li and Ho Min Kim: These authors contributed equally to this work.

Authors and Affiliations

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

    Soyeon Park, Geng Tian & Daniel Finley

  2. MCD Biology, University of Colorado Boulder, Boulder, 80309, Colorado, USA

    Soyeon Park

  3. Department of Biochemistry and Biophysics, The W.M. Keck Advanced Microscopy Laboratory, University of California San Francisco, 600 16th Street, San Francisco, California 94158, USA,

    Xueming Li, Ho Min Kim & Yifan Cheng

  4. Division of Biology, Kansas State University, 338 Ackert Hall, Manhattan, Kansas 66506, USA,

    Chingakham Ranjit Singh & Jeroen Roelofs

  5. Department of Microbiology and Immunology, University of California San Francisco, 513 Parnassus Avenue, San Francisco, California 94143, USA,

    Martin A. Hoyt & Philip Coffino

  6. Protein Structure Laboratory, Del Shankel Structural Biology Center, University of Kansas, Lawrence, 66047, Kansas, USA

    Scott Lovell

  7. IMCA-CAT Hauptman-Woodward Medical Research Institute, 9700 South Cass Avenue, Building 435A, Argonne, Illinois 60439, USA,

    Kevin P. Battaile

  8. Department of Biochemistry, Kansas State University, 176 Chalmers Hall, Manhattan, Kansas 66506, USA,

    Michal Zolkiewski

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Contributions

S.P. performed reconstitution of the base–CP complex and holoenzyme stability. X.L. performed all cryoEM experiments and analysis. H.M.K. and C.R.S. generated yeast strains. H.M.K. purified GST-fused CP, and participated in cryoEM experiments and analysis. C.R.S. performed purifications, and M.Z. performed ultracentrifugation. K.P.B. and S.L. determined crystal structures, J.R. and G.T. performed structural analysis and modelling. M.A.H., H.M.K. and P.C. performed phenotypic and native gel analysis of Rpt6 mutations. J.R. wrote the supplement with contributions from all authors. The manuscript was drafted by D.F. and Y.C., and modified by all authors.

Corresponding authors

Correspondence to Jeroen Roelofs, Yifan Cheng or Daniel Finley.

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The authors declare no competing financial interests.

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Supplementary Information

This file contains Supplementary Figures 1-21, Supplementary Tables 1-3, Supplementary Methods and Supplementary References. (PDF 12079 kb)

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Park, S., Li, X., Kim, H. et al. Reconfiguration of the proteasome during chaperone-mediated assembly. Nature 497, 512–516 (2013). https://doi.org/10.1038/nature12123

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