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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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.).
The authors declare no competing interests.
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.
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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.
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. 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.
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).
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.
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.
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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). https://doi.org/10.1038/s41594-021-00583-9
Nature Structural & Molecular Biology (2021)