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Yeast PI31 inhibits the proteasome by a direct multisite mechanism

Abstract

Proteasome inhibitors are widely used as therapeutics and research tools, and typically target one of the three active sites, each present twice in the proteasome complex. An endogeneous proteasome inhibitor, PI31, was identified 30 years ago, but its inhibitory mechanism has remained unclear. Here, we identify the mechanism of Saccharomyces cerevisiae PI31, also known as Fub1. Using cryo-electron microscopy (cryo-EM), we show that the conserved carboxy-terminal domain of Fub1 is present inside the proteasome’s barrel-shaped core particle (CP), where it simultaneously interacts with all six active sites. Targeted mutations of Fub1 disrupt proteasome inhibition at one active site, while leaving the other sites unaffected. Fub1 itself evades degradation through distinct mechanisms at each active site. The gate that allows substrates to access the CP is constitutively closed, and Fub1 is enriched in mutant CPs with an abnormally open gate, suggesting that Fub1 may function to neutralize aberrant proteasomes, thereby ensuring the fidelity of proteasome-mediated protein degradation.

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Fig. 1: Enrichment of Fub1 in the α3Δ mutant.
Fig. 2: α3Δ CP shows an open gate due to aberrant α-ring composition.
Fig. 3: Cryo-EM structure of Fub1-bound CP.
Fig. 4: Interaction of Fub1 with proteasome active sites.
Fig. 5: Inhibition of individual CP active sites by Fub1.
Fig. 6: Fub1 evades degradation by distinct mechanisms at each active site.
Fig. 7: Fub1 is enriched in open gate CP mutants, and its ability to inhibit CP is evolutionarily conserved.

Data availability

Cryo-EM maps and atomic model coordinates have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank, respectively, as α3∆ 20S (EMD-25847, PDB 7TEJ) and α3∆ 20S + Fub1 complex (EMD-25848, PDB 7TEO). Additional structures referenced here include 3MG0, 5CZ4 and 7LS5. Source data are provided with this paper.

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Acknowledgements

We thank D. Waterman and D. Finley for early contributions to the project, and D. Finley for assistance with chromatography, helpful advice and comments on the manuscript. We thank J. Roelofs for the Pba1/2 antibody. This work was supported by National Institutes of Health (NIH) grants R01-GM144367 (to J.H.), DP5-OD019800 (to J.H.), R01-GM074830 (to L.H.) and R01-GM130144 (to L.H.).

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Authors and Affiliations

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Contributions

J.H., B.V., H.M.S., M.B., J.A. and M.K.B. 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, and R.M.W., S.R. and H.M.S. performed the data analysis. F.J. and L.H. performed the crosslinking experiments. 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., as well as input from all authors.

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Correspondence to John Hanna.

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Nature Structural and Molecular Biology thanks Youdong Mao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Florian Ullrich, in collaboration with the Nature Structural and Molecular Biology team.

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Extended data

Extended Data Fig. 1 Proteasome Activity in Whole Cell Extracts.

a, Proteasome activity in whole cell extracts from wild-type and fub1∆ cells, as determined using the fluorogenic substrate suc-LLVY-AMC. Open circles indicate individual data points from biologic replicates. b, Proteasome levels are comparable in whole cell extracts. Extracts were prepared and analyzed by SDS-PAGE followed by immunoblotting with indicated antibodies. Alpha5 is a CP subunit while Pgk1 represents a loading control. Uncropped images for panel b and data for panel a are available as source data.

Source data

Extended Data Fig. 2 Additional Analysis of Fub1 Binding to alpha3Δ CP.

a, Purified CP (1.4 µg) from the indicated strains was analyzed by native gel electrophoresis followed by immunoblotting with antibodies recognizing Fub1 and Alpha5. These results confirm that the Fub1-immunoreactive species do indeed represent Fub1. b, Purified CP (1.1 µg) from the indicated strains was analyzed by native gel electrophoresis followed by immunoblotting with antibodies recognizing Fub1 and Blm10. These results indicate that the two supra-20S species contain Blm10. The identity of the highest molecular weight species, however, remains uncertain as doubly Blm10-capped CP was not visualized by cryo-EM (see Extended Data Fig. 3, below). Similar results were obtained in two independent experiments.

Extended Data Fig. 3 Cryo-EM Classification of CP Species.

Processing scheme for classification and refinement of proteasome species. “Junk” classes are colored grey while identifiable species are colored by species. All 3D classification steps were carried out in cryoSPARC.

Extended Data Fig. 4 Cryo-EM Data Analysis for CP Species.

a, Representative micrograph of proteasome particles embedded in vitreous ice. Scale bar = 500 Å. A total of 12,834 micrographs were collected from a single multi-day experiment. b, Selected 2D class averages of 20 S particles. c, Local resolution slice through the α3Δ + Fub1 containing reconstruction. Local resolution values vary between ~3.0–5.0 Å in this region. CP density around Fub1 is shown for orientation. d, Reconstructions of Fub1-containing (top panels) and Fub1-lacking (bottom panels) CP filtered and colored by local resolution (left), gold-standard Fourier shell correlation (FSC) curves from cryoSPARC (middle), and viewing direction distribution plots (right). Resolution determined at FSC = 0.143.

Extended Data Fig. 5 Further Analysis of β2 Inhibition by Fub1 Peptides.

Mapping of the inhibitory effect of the β2 peptide. The structure of this part of Fub1 is shown above, although the last three residues were not resolved.

Extended Data Fig. 6 Additional Low Resolution Density in the α3Δ + Fub1 Structure. Shown.

Shown is the same low pass filtered map from Fig. 7d. In addition to the capping density shown in Fig. 7d, there is further density from difference maps (shown in green) within the CP barrel that extends through the α-ring and into the β-ring. Difference density has been further gaussian filtered and additional unconnected density omitted for clarity.

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Supplementary Tables 1–3 and Supplementary Fig. 1.

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Rawson, S., Walsh, R.M., Velez, B. et al. Yeast PI31 inhibits the proteasome by a direct multisite mechanism. Nat Struct Mol Biol 29, 791–800 (2022). https://doi.org/10.1038/s41594-022-00808-5

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