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Structures of chaperone-associated assembly intermediates reveal coordinated mechanisms of proteasome biogenesis


The proteasome mediates most selective protein degradation. Proteolysis occurs within the 20S core particle (CP), a barrel-shaped chamber with an α7β7β7α7 configuration. CP biogenesis proceeds through an ordered multistep pathway requiring five chaperones, Pba1–4 and Ump1. Using Saccharomyces cerevisiae, we report high-resolution structures of CP assembly intermediates by cryogenic-electron microscopy. The first structure corresponds to the 13S particle, which consists of a complete α-ring, partial β-ring (β2–4), Ump1 and Pba1/2. The second structure contains two additional subunits (β5–6) and represents a later pre-15S intermediate. These structures reveal the architecture and positions of Ump1 and β2/β5 propeptides, with important implications for their functions. Unexpectedly, Pba1’s N terminus extends through an open CP pore, accessing the CP interior to contact Ump1 and the β5 propeptide. These results reveal how the coordinated activity of Ump1, Pba1 and the active site propeptides orchestrate key aspects of CP assembly.

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Fig. 1: Biochemical characterization of CP mutants.
Fig. 2: Structures of the 13S and pre-15S CP assembly intermediates.
Fig. 3: Structures of the β2 and β5 propeptides.
Fig. 4: Pba1 transits through the open CP pore to contact Ump1 and the β5 propeptide.

Data availability

Cryo-EM maps and atomic model coordinates have been deposited in the Electron Microscopy Data Bank and Research Collaboratory for Structural Bioinformatics, respectively: 13S (EMD-23508, PDB 7LSX), Pre-15S (EMD-23503, PDB 7LS6) and Pre3-1 20S (EMD-23502, PDB 7LS5). Additional structures referenced here include PDB 4G4S, PDB 1RYP, PDB 2Z5C and PDB 6FVY. Source data are available with this paper.


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Cryo-EM data were collected at the Harvard Cryo-Electron Microscopy Center for Structural Biology at Harvard Medical School. This work was supported by National Institutes of Health grant nos. DP5-OD019800 (to J.H.), R01-GM043601 (to D.F.), R01-GM67945 (to S.P.G.), R01-GM132129 (to J.A.P.), P20-GM103418 (to J.R.) and R01-GM118660 (to J.R.).

Author information




H.M.S., J.H., M.B. and A.G.-M. performed the biochemical aspects of the work. R.M.W. and S.R. performed cryo-EM sample preparation, data collection, data processing, model building and refinement, while R.M.W., S.R. and H.M.S. performed the data analysis. M.K. and J.R. performed the experiments in Fig. 4f. G.T. helped with size exclusion chromatography, while M.A.P. performed mass spectrometry with supervision from J.A.P., S.P.G. and D.F. H.M.S., J.H., S.R. and R.M.W. prepared the figures. J.H. wrote the paper with assistance from H.M.S., R.M.W. and S.R. and with input from all authors.

Corresponding author

Correspondence to John Hanna.

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The authors declare no competing interests.

Additional information

Peer review information Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Cryo-EM classification of CP species.

Processing scheme for classification and refinement of proteasome species. ‘Junk’ classes throughout colored grey – identifiable species colored by species. All 3D classification steps other than the subtracted β5-6 classification were carried out in cryoSPARC.

Extended Data Fig. 2 Cryo-EM data analysis for CP species.

a, Representative micrograph of proteasome particles embedded in vitreous ice (scale bar = 500 Å). A total of 21,000 micrographs were collected from a single multi day experiment. b, Selected 2D class averages of 20S and 13S particles (scale bar = 200 Å). c, Proteasome reconstructions filtered and colored by local resolution (left), gold-standard Fourier shell correlation (FSC) curves from cryoSPARC (center) and viewing direction distribution plots (right). Resolution determined at FSC = 0.143.

Extended Data Fig. 3 Structure of 20S CP from pre3-1 mutant.

Cryo-EM density of the pre3-1 20S species (2.7 Å) modeled onto the crystal structure of wild-type mature 20S (PDB: 1RYP). The position of Pre3 (β1) is indicated in red in the middle panel. Pre3-1 harbors a G34D mutation13 and the position of G34 in 1RYP is shown in yellow in the right panel.

Extended Data Fig. 4 Confirmation of the assignment of Ump1 to the novel central density within 13S and pre-15S structures.

The Ump1 model is shown overlaid onto the primary cryo-EM map density. The four boxed panels show close-up views confirming that the density precisely matches the modeled amino acid side chains of Ump1.

Extended Data Fig. 5 Extensive contacts between Ump1 and the CP.

a, Multiple views of Ump1’s contacts with α-subunits and Pba1. b, Multiple views of β-subunits. In both panels, contacts were determined using PDBePISA (see Supplementary Table 1 for details).

Extended Data Fig. 6 Potential steric clash between Ump1 and Pba4.

Surface of the α-ring with the associated Ump1 density. Pba3 and Pba4 (PDB: 2Z5C) have been modeled onto this structure, and Pba4 (yellow) shows extensive clash with Ump1 (red) in the vicinity of α4.

Extended Data Fig. 7 Comparison of β2’s N-terminal propeptide and C-terminal loop in mature CP and pre-15S structures.

Relationship between β2 and β3 in the wild-type mature 20S (purple; PDB: 1RYP) and the pre-15S structure (green). Multiple views are shown. The propeptide is absent in mature 20S, while the C-terminal loop is largely unresolved in the maturing CP.

Extended Data Fig. 8 Identification of N-terminal β5 propeptide helix.

a, Ump1 hinge region showing clear density assigned to β5 propeptide (orange) in pre-15S reconstruction. b, Corresponding region of the 13S reconstruction shows no density. c, Low resolution map of 13S + β5 reconstruction showing density is restored. Surrounding density in all panels hidden for clarity using a 2–3 Å carve radius.

Supplementary information

Supplementary Information

Supplementary Tables 1–3 and Extended Data Set 1.

Reporting Summary

Source data

Source Data Fig. 1

Unprocessed gels and blots.

Source Data Fig. 4

Unprocessed blot and gels.

Source Data Fig. 4

Raw data.

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Schnell, H.M., Walsh, R.M., Rawson, S. et al. Structures of chaperone-associated assembly intermediates reveal coordinated mechanisms of proteasome biogenesis. Nat Struct Mol Biol (2021).

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