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

Nature Structural & Molecular Biology volume 28, pages 418–425 (2021)Cite this article

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Abstract

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.

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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.

References

  1. Budenholzer, L., Cheng, C. L., Li, Y. & Hochstrasser, M. Proteasome structure and assembly. J. Mol. Biol. 429, 3500–3524 (2017).

    Article  CAS  Google Scholar 

  2. Rousseau, A. & Bertolotti, A. Regulation of proteasome assembly and activity in health and disease. Nat. Rev. Mol. Cell Biol. 19, 697–712 (2018).

    Article  CAS  Google Scholar 

  3. Dahlqvist, J. et al. A single-nucleotide deletion in the POMP 5′ UTR causes a transcriptional switch and altered epidermal proteasome distribution in KLICK genodermatosis. Am. J. Hum. Genet. 86, 596–603 (2010).

    Article  CAS  Google Scholar 

  4. Frentzel, S., Pesold-Hurt, B. & Seelig, A. 20S proteasomes are assembled via distinct precursor complexes. Processing of LMP2 and LMP7 proproteins takes place in 13-16S preproteasome complexes. J. Mol. Biol. 236, 975–981 (1994).

    Article  CAS  Google Scholar 

  5. Schmidtke, G., Schmidt, M. & Kloetzel, P.-M. Maturation of mammalian 20S proteasome: purification and characterization of 13 S and 16 S proteasome precursor complexes. J. Mol. Biol. 268, 95–106 (1997).

    Article  CAS  Google Scholar 

  6. Li, X., Kusmierczyk, A. R., Wong, P., Emili, A. & Hochstrasser, M. β-Subunit appendages promote 20S proteasome assembly by overcoming an Ump1-dependent checkpoint. EMBO J. 26, 2339–2349 (2007).

    Article  CAS  Google Scholar 

  7. Yashiroda, H. et al. Crystal structure of a chaperone complex that contributes to the assembly of yeast 20S proteasomes. Nat. Struct. Mol. Biol. 15, 228–236 (2008).

    Article  CAS  Google Scholar 

  8. Takagi, K. et al. Pba3–Pba4 heterodimer acts as a molecular matchmaker in proteasome α-ring formation. Biochem. Biophys. Res. Commun. 450, 1110–1114 (2014).

    Article  CAS  Google Scholar 

  9. Hirano, Y. et al. Dissecting β-ring assembly pathway of the mammalian 20S proteasome. EMBO J. 27, 2204–2213 (2008).

    Article  CAS  Google Scholar 

  10. Jaeger, S., Groll, M., Huber, R., Wolf, D. H. & Heinemeyer, W. Proteasome β-type subunits: unequal roles of propeptides in core particle maturation and a hierarchy of active site function. J. Mol. Biol. 291, 997–1013 (1999).

    Article  Google Scholar 

  11. Groll, M. et al. Structure of 20S proteasome from yeast at 2.4Å resolution. Nature 386, 463–471 (1997).

    Article  CAS  Google Scholar 

  12. Gerlinger, U. M., Gückel, R., Hoffmann, M., Wolf, D. H. & Hilt, W. Yeast cycloheximide-resistant CRL mutants are proteasome mutants defective in protein degradation. Mol. Biol. Cell 8, 2487–2499 (1997).

    Article  CAS  Google Scholar 

  13. Gueckel, R., Enenkel, C., Wolf, D. H. & Hilt, W. Mutations in the yeast proteasome-type subunit Pre3 uncover position-dependent effects on proteasomal peptidase activity and in vivo function. J. Biol. Chem. 273, 19443–19452 (1998).

    Article  CAS  Google Scholar 

  14. Kock, M. et al. Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1–Pba2 chaperone. Nat. Commun. 6, 6123 (2015).

    Article  CAS  Google Scholar 

  15. Sá-Moura, B. et al. Biochemical and biophysical characterization of recombinant yeast proteasome maturation factor Ump1. Comput. Struct. Biotechnol. J. 7, e201304006 (2013).

    Article  Google Scholar 

  16. le Tallec, B. et al. 20S proteasome assembly is orchestrated by two distinct pairs of chaperones in yeast and in mammals. Mol. Cell 27, 660–674 (2007).

    Article  Google Scholar 

  17. Chen, P. & Hochstrasser, M. Autocatalytic subunit processing couples active site formation in the 20S proteasome to completion of assembly. Cell 86, 961–972 (1996).

    Article  CAS  Google Scholar 

  18. Arendt, C. S. & Hochstrasser, M. Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly. EMBO J. 18, 3575–3585 (1999).

    Article  CAS  Google Scholar 

  19. Ramos, P. C., Marques, A. J., London, M. K. & Dohmen, R. J. Role of C-terminal extensions of subunits β2 and β7 in assembly and activity of eukaryotic proteasomes. J. Biol. Chem. 279, 14323–14330 (2004).

    Article  CAS  Google Scholar 

  20. Hirano, Y. et al. A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes. Nature 437, 1381–1385 (2005).

    Article  CAS  Google Scholar 

  21. Stadtmueller, B. M. et al. Structure of a proteasome Pba1–Pba2 complex implications for proteasome assembly, activation, and biological function. J. Biol. Chem. 287, 37371–37382 (2012).

    Article  CAS  Google Scholar 

  22. Wani, P. S., Rowland, M. A., Ondracek, A., Deeds, E. J. & Roelofs, J. Maturation of the proteasome core particle induces an affinity switch that controls regulatory particle association. Nat. Commun. 6, 6123 (2015).

    Article  Google Scholar 

  23. Eisele, M. R. et al. Expanded coverage of the 26S proteasome conformational landscape reveals mechanisms of peptidase gating. Cell Rep. 24, 1301–1315.e5 (2018).

    Article  CAS  Google Scholar 

  24. Ramos, P. C., Hoeckendorff, J., Johnson, E. S., Varshavsky, A. & Dohmen, J. R. Ump1p is required for proper maturation of the 20S proteasome and becomes its substrate upon completion of the assembly. Cell 92, 489–499 (1998).

    Article  CAS  Google Scholar 

  25. Leggett, D. S. et al. Multiple associated proteins regulate proteasome structure and function. Mol. Cell 10, 495–507 (2002).

    Article  CAS  Google Scholar 

  26. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  Google Scholar 

  27. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  Google Scholar 

  28. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  Google Scholar 

  29. Wagner, T. et al. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun. Biol. 2, 218 (2019).

    Article  Google Scholar 

  30. Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  CAS  Google Scholar 

  31. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. CryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  Google Scholar 

  32. Morin, A. et al. Collaboration gets the most out of software. Elife 2, e01456 (2013).

    Article  Google Scholar 

  33. Raman, S. et al. Structure prediction for CASP8 with all-atom refinement using Rosetta. Proteins 77, 89–99 (2009).

    Article  CAS  Google Scholar 

  34. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  35. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Struct. Biol. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  36. Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D Struct. Biol. 74, 519–530 (2018).

    Article  CAS  Google Scholar 

  37. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystrallogr. D Struct. Biol. 75, 861–877 (2019).

    Article  CAS  Google Scholar 

  38. Kleijnen, M. F. et al. Stability of the proteasome can be regulated allosterically through engagement of its proteolytic active sites. Nat. Struct. Mol. Biol. 14, 1180–1188 (2007).

    Article  CAS  Google Scholar 

  39. Roelofs J., Suppahia A., Waite K. A. & Park S. in The Ubiquitin Proteasome System. Methods in Molecular Biology Vol. 1844 (eds Mayor, T. & Kleiger, G.) 237–260 (Humana Press, 2018).

Download references

Acknowledgements

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.).

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Author notes

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

Authors and Affiliations

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

    Helena M. Schnell, Meera K. Bhanu, Angel Guerra-Moreno & John Hanna

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

    Richard M. Walsh Jr & Shaun Rawson

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

    Richard M. Walsh Jr & Shaun Rawson

  4. Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA

    Mandeep Kaur & Jeroen Roelofs

  5. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Geng Tian, Miguel A. Prado, Joao A. Paulo, Steven P. Gygi & Daniel Finley

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Contributions

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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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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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.

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Source data

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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 28, 418–425 (2021). https://doi.org/10.1038/s41594-021-00583-9

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