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Structure of the preholoproteasome reveals late steps in proteasome core particle biogenesis

Nature Structural & Molecular Biology volume 30, pages 1516–1524 (2023)Cite this article

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Abstract

Assembly of the proteasome’s core particle (CP), a barrel-shaped chamber of four stacked rings, requires five chaperones and five subunit propeptides. Fusion of two half-CP precursors yields a complete structure but remains immature until active site maturation. Here, using Saccharomyces cerevisiae, we report a high-resolution cryogenic electron microscopy structure of preholoproteasome, a post-fusion assembly intermediate. Our data reveal how CP midline-spanning interactions induce local changes in structure, facilitating maturation. Unexpectedly, we find that cleavage may not be sufficient for propeptide release, as residual interactions with chaperones such as Ump1 hold them in place. We evaluated previous models proposing that dynamic conformational changes in chaperones drive CP fusion and autocatalytic activation by comparing preholoproteasome to pre-fusion intermediates. Instead, the data suggest a scaffolding role for the chaperones Ump1 and Pba1/Pba2. Our data clarify key aspects of CP assembly, suggest that undiscovered mechanisms exist to explain CP fusion/activation, and have relevance for diseases of defective CP biogenesis.

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Fig. 1: Proposed consequences of the pre1-1 and pre4-1 mutations.
Fig. 2: Biochemical and structural analysis of pre1-1, 4-1 proteasomes.
Fig. 3: Subunit contacts and overall positioning of Ump1 within the preholoproteasome.
Fig. 4: The β2 propeptide and C-terminal extension.
Fig. 5: Comparison of the position and conformation of Pba1/2 over the course of CP assembly.
Fig. 6: Summary of high-resolution structural analysis of CP assembly.

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Data availability

Cryo-EM maps and atomic model coordinates have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank, respectively: pre1-1, 4-1 preholoproteasome (EMD-40838, PDB 8T08) and pre1-1, 4-1 20S proteasome (EMD-40944, PDB 8T0M). Additional PDB structures referenced here include 5CZ4, 7LSX, and 7LS6; additional EMD structures include 23508, 23503 and 23502. Source data are provided with this paper.

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Acknowledgements

We thank D. Finley for assistance with size exclusion chromatography. This work was supported by NIH grant R01-GM144367 (to J.H. and R.W.).

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Author notes

  1. These authors contributed equally: Richard M. Walsh, Jr., Shaun Rawson.

Authors and Affiliations

  1. Harvard Cryo-Electron Microscopy Center for Structural Biology, Harvard Medical School, Boston, MA, USA

    Richard M. Walsh Jr. & Shaun Rawson

  2. Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Richard M. Walsh Jr. & Shaun Rawson

  3. Department of Pathology, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA, USA

    Helena M. Schnell, Benjamin Velez, Tamayanthi Rajakumar & John Hanna

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Contributions

J.H., H.M.S., B.V. and T.R. performed the biochemical aspects of the work. R.W. and S.R. performed cryo-EM sample preparation, data collection, data processing, model building and refinement, and data analysis. R.W., S.R. and J.H. prepared the figures. J.H., R.W. and S.R. wrote the paper with input from all authors.

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Correspondence to John Hanna.

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The authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Youdong Mao, Jeroen Roelofs and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Sara Osman and Dimitris Typas were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Protein Degradation Defects in the pre1-1, 4-1 Mutant.

The rate of degradation of a short-lived cytoplasmic protein (Tmc1; panel a) and an ERAD substrate (CPY*; panel b) was determined by cycloheximide chase assay. Cycloheximide (100 μg/ml) was added at time zero, and whole cell extracts were prepared at the indicated time points. SDS-PAGE was performed, followed by immunoblot analysis with antibodies against Tmc1, CPY*, and Hog1 (loading control). Similar results were seen in two experiments for each of the two substrates.

Source data

Extended Data Fig. 2 Cryo-EM Classification of CP Species.

Shown is the processing scheme for the classification and refinement of proteasome species. ‘Junk’ classes are colored grey. Identifiable species are colored as follows: 20 S, pink; preholoproteasome, cyan; Blm10-CP, purple; and sub-20S species, yellow.

Extended Data Fig. 3 Cryo-EM Data Analysis for CP Species.

a, Representative micrograph of proteasome particles embedded in vitreous ice. Scale bar=500 Å. b, Selected 2D class averages of preholoproteasome particles. c, Reconstructions of preholoproteasome (top panels) and 20 S CP (bottom panels) filtered and colored by local resolution (left panels), gold-standard Fourier shell correlation (FSC) curves from cryoSPARC (middle panels), and viewing direction distribution plots (right panels). Resolution determined at FSC = 0.143.

Extended Data Fig. 4 Detailed Comparison of the 20S Cryo-EM Density and Molecular Model.

Representative sections of the 20S molecular model for two CP subunits (α1, panel a; β2, panel b) are shown superimposed onto the primary cryo-EM map densities, confirming that the model precisely matches the density. The relevant protein residues are listed for each example. The density is displayed with a map contour of 0.8 and a Zone radius of 3.0 Å.

Extended Data Fig. 5 Comparison of the Preholoproteasome and 20S Structures.

a, Molecular model of the 20S CP. b-c, Primary cryo-EM densities of the 20S CP (panel B) and preholoproteasome (panel C). The boxed region in the 20S CP shows excellent resolution at the β4/β5 interface, in contrast to the preholoproteasome where there is marked loss of resolution which likely reflects subunit flexibility. d, The β5 catalytic triad appears largely intact in the 20S CP, suggesting that other, potentially dynamic aspects, of catalytic function are impaired in the pre1-1, 4-1 mutant. The cryo-EM density in panel D is displayed with a map contour of 0.67 and a Zone radius of 3.0 Å.

Supplementary information

Source data

Source Data Fig. 2a,b,e

Unprocessed gel and blots.

Source Data Fig. 2D

Raw data.

Source Data Extended Data Fig. 1a,b

Unprocessed blots.

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Walsh, R.M., Rawson, S., Schnell, H.M. et al. Structure of the preholoproteasome reveals late steps in proteasome core particle biogenesis. Nat Struct Mol Biol 30, 1516–1524 (2023). https://doi.org/10.1038/s41594-023-01081-w

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