Reconfiguration of the proteasome during chaperone-mediated assembly

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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.

At a glance


  1. Chaperones inhibit base-CP assembly.
    Figure 1: Chaperones inhibit base–CP assembly.

    a, Purified base (160nM) and CP (80nM) were incubated with or without Rpn14, Nas6 and Hsm3 (trio, 1.6μM each), and resolved by native PAGE. Top, in-gel peptidase assay (0.02% SDS); bottom, Coomassie stain. For input protein see Supplementary Fig. 2. b, Base (5nM) and CP (2nM) were challenged with chaperone trio (amounts in molar excess of base; ATP at 2mM). In this and all real-time experiments, LLVY-AMC hydrolysis is expressed as relative fluorescence units (r.f.u.) and experiments were performed in triplicate with traces combined for presentation. c, Native gel analysis of base–CP formation as in a, after addition of chaperones to base (160nM) singly or in combination at tenfold molar excess of base. d, A yeast Rpt hexamer model was built, using the hexameric P97 D1 domain structure as a template (see Supplementary Methods). This model was fit into the EM map2 of yeast Rpt hexamer. Relative positions of Hsm3 (red) and Nas6 (yellow) on the Rpt ring (blue) were assessed by superimposing Hsm3–Rpt1C and Nas6–Rpt3C structures onto the Rpt ring model that had been fit into the EM map. A clipped view of the Rpt ring with bound chaperones and CP (green) is presented. Areas of overlap highlight steric clashes between chaperones and CP.

  2. Base-CP association is nucleotide dependent.
    Figure 2: Base–CP association is nucleotide dependent.

    a, CP (2 nM) activity stimulated by base (5nM) was monitored over time (2mM ATP, 50mM KCl). At 5.5min, chaperone trio or CP trap was added in molar excess of base or active CP, respectively. CP trap inhibits re-association of base with active CP. Right, hydrolysis rate (r.f.u.min−1) over time. b, Purified base (5nM) and CP (2nM) were assembled in the presence of ATPγS (0.1mM throughout). At 6min, chaperone trio or CP trap were added in molar excess. c, CP (15nM) was immobilized on IgG resin via ProA tag, and incubated with base (~80nM) and chaperone trio (160nM) in the presence of 2mM ATP or 0.5mM ATPγS. CP-bound proteins were washed with buffer (50mM KCl), then eluted with TEV protease while maintaining nucleotide concentration. Immunoblots (IBs) were probed with indicated antibodies. Images are from the same gel and exposure. d, CP (2 nM) activity (LLVY-AMC hydrolysis; r.f.u.) was monitored in the presence of base (5 nM) and 0.1 mM ATPγS for 5 min. Chaperone trio (50 nM) or buffer alone containing 0.1 mM ATPγS was then added. At 10 min, buffer containing either ATPγS or ATP plus ATPγS was added. Final nucleotide concentrations were either 0.1mM ATPγS or 10mM ATP plus 0.1mM ATPγS. See also Supplementary Fig. 12.

  3. Difference maps reveal binding sites of Rpt C-terminal peptides to CP [agr]-pockets.
    Figure 3: Difference maps reveal binding sites of Rpt C-terminal peptides to CP α-pockets.

    a, Top views of three-dimensional density maps of CP superimposed with difference densities corresponding to C-terminal peptides of each Rpt. Peptides were present at 0.5mM, CP at 1.6μM. The amount of each peptide bound is reflected by the size of black densities within each pocket. b, Summary of Rpt tail peptide-binding sites and relative intensities. Sizes of circles represent the volume of difference densities generated by peptides. Grey diagonals denote Rpt tail–α-pocket mapping of intact proteasomes by crosslinking22. Rpt4, Rpt5 and Rpt1 each crosslink to two α-pockets, suggesting an ambiguous register. c, Predominant tail–pocket interactions in yeast holoenzymes as determined by cryoEM2, 3, 4.

  4. Rpt6 C-terminal tail promotes formation of base-CP complex.
    Figure 4: Rpt6 C-terminal tail promotes formation of base–CP complex.

    a, Growth defects of C-terminal rpt6 mutants. Strains were spotted onto plates containing rich media (YPD) in fourfold serial dilutions. Plates were incubated at 30°C for 2 days. WT, wild type Capital letters indicate the last three amino acids of Rpt6 (LFK) or mutants thereof. b, Whole-cell extracts (100μg) from rpt6 mutants as in a were resolved by native PAGE and subjected to LLVY-AMC assay in 0.02% SDS. c, Role of Rpt6 tail in base–CP association. Assembly kinetics of wild-type or rpt6-Δ1 base with CP was measured using LLVY-AMC hydrolysis. Purified CP (2nM) was mixed with the indicated fold excess of base (2mM ATP). LLVY-AMC hydrolysis is indicated in r.f.u. d, Stability of proteasome holoenzyme (2nM) from wild-type or rpt6-Δ1 mutants was assessed in 2mM ATP by adding 50-fold molar excess of CP trap or buffer alone at 15min.

  5. Three-dimensional reconstruction of base-CP complex reveals an asymmetric interaction between the Rpt ring and the [agr]-ring of the CP.
    Figure 5: Three-dimensional reconstruction of base–CP complex reveals an asymmetric interaction between the Rpt ring and the α-ring of the CP.

    a, Three-dimensional reconstruction of the singly capped base–CP complex was determined by single-particle cryoEM to a resolution of ~10Å. CP subunits are rendered in different colours as indicated. A difference map was calculated between the original three-dimensional reconstruction and one rotated 180° around the two-fold CP symmetry axis. The positive difference density (grey) corresponds to base bound to CP. It shows prominent densities from C termini of Rpt6, Rpt2 and Rpt1, which are clustered on one side of the Rpt ring, bound to specific α-pockets. b, Each panel shows an α-pocket. Arrows indicate densities corresponding to Rpt C termini extending towards the pockets. Asterisks indicate pockets without detectable density from Rpt C termini. Thresholds of CP and base densities are set separately but are identical in all panels. C termini of Rpt6, Rpt2 and Rpt1 are seen to insert into α-pockets.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank


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Author information

  1. These authors contributed equally to this work.

    • Soyeon Park,
    • Xueming Li &
    • Ho Min Kim


  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, Colorado 80309, USA

    • Soyeon Park
  3. The W.M. Keck Advanced Microscopy Laboratory, Department of Biochemistry and Biophysics, 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, Kansas 66047, 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
  9. Present address: Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.

    • Ho Min Kim


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.

Competing financial interests

The authors declare no competing financial interests.

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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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  1. Supplementary Information (11.7 MB)

    This file contains Supplementary Figures 1-21, Supplementary Tables 1-3, Supplementary Methods and Supplementary References.

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