Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A multimeric assembly factor controls the formation of alternative 20S proteasomes

Nature Structural & Molecular Biology volume 15, pages 237–244 (2008)Cite this article

Abstract

The proteasome is the central regulatory protease of eukaryotic cells. Heteroheptameric α-subunit and β-subunit rings stack to form the 20S proteasome, which associates with a 19S regulatory particle (RP). Here we show that two yeast proteins, Pba3 and Pba4, form a previously unidentified 20S proteasome–assembly chaperone. Pba3–Pba4 interacts genetically and physically with specific proteasomal α subunits, and loss of Pba3–Pba4 causes both a reduction and a remodeling of cellular proteasomes. Notably, mutant cells accumulate proteasomes in which a second copy of the α4 subunit replaces α3. 20S proteasome–assembly defects also are associated with altered RP assembly; this unexpected result suggests that the 20S proteasome can function as an RP-assembly factor in vivo. Our data demonstrate that Pba3–Pba4 orchestrates formation of a specific type of proteasome, the first example of a trans-acting factor that controls assembly of alternative proteasomal complexes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: pba3Δ and pba4Δ strains have properties of partially defective proteasome mutants.
Figure 2: Pba3 and Pba4 form a heteromultimer.
Figure 3: Pba3–Pba4 binds to specific α subunits.
Figure 4: A proteasome-assembly defect in pba3Δ and pba4Δ mutants.
Figure 5: Pba4 ensures incorporation of α3 into the 20S proteasome.
Figure 6: Fully assembled 26S proteasomes from pba4Δ cells contain α4-α4 rings.
Figure 7: Model for Pba3–Pba4 action.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Hochstrasser, M. Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30, 405–439 (1996).

    Article  CAS  Google Scholar 

  2. Glickman, M.H. & Ciechanover, A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, 373–428 (2002).

    Article  CAS  Google Scholar 

  3. DeMartino, G.N. & Slaughter, C.A. The proteasome, a novel protease regulated by multiple mechanisms. J. Biol. Chem. 274, 22123–22126 (1999).

    Article  CAS  Google Scholar 

  4. Heinemeyer, W., Ramos, P.C. & Dohmen, R.J. The ultimate nanoscale mincer: assembly, structure and active sites of the 20S proteasome core. Cell. Mol. Life Sci. 61, 1562–1578 (2004).

    Article  CAS  Google Scholar 

  5. 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 

  6. Arendt, C.S. & Hochstrasser, M. Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation. Proc. Natl. Acad. Sci. USA 94, 7156–7161 (1997).

    Article  CAS  Google Scholar 

  7. Heinemeyer, W., Fischer, M., Krimmer, T., Stachon, U. & Wolf, D.H. The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing. J. Biol. Chem. 272, 25200–25209 (1997).

    Article  CAS  Google Scholar 

  8. Kruger, E., Kloetzel, P.M. & Enenkel, C. 20S proteasome biogenesis. Biochimie 83, 289–293 (2001).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Nandi, D., Woodward, E., Ginsburg, D.B. & Monaco, J.J. Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor beta subunits. EMBO J. 16, 5363–5375 (1997).

    Article  CAS  Google Scholar 

  11. Ramos, P.C., Hockendorff, J., Johnson, E.S., Varshavsky, A. & Dohmen, R.J. 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 

  12. Hirano, Y. et al. Cooperation of multiple chaperones required for the assembly of mammalian 20S proteasomes. Mol. Cell 24, 977–984 (2006).

    Article  CAS  Google Scholar 

  13. 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 

  14. Yuan, X., Miller, M. & Belote, J.M. Duplicated proteasome subunit genes in Drosophila melanogaster encoding testes-specific isoforms. Genetics 144, 147–157 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. De, M. et al. Beta2 subunit propeptides influence cooperative proteasome assembly. J. Biol. Chem. 278, 6153–6159 (2003).

    Article  CAS  Google Scholar 

  16. Velichutina, I., Connerly, P.L., Arendt, C.S., Li, X. & Hochstrasser, M. Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast. EMBO J. 23, 500–510 (2004).

    Article  CAS  Google Scholar 

  17. Enyenihi, A.H. & Saunders, W.S. Large-scale functional genomic analysis of sporulation and meiosis in Saccharomyces cerevisiae. Genetics 163, 47–54 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Dohmen, R.J., Willers, I. & Marques, A.J. Biting the hand that feeds: Rpn4-dependent feedback regulation of proteasome function. Biochim. Biophys. Acta 1773, 1599–604 (2007).

    Article  CAS  Google Scholar 

  19. Pan, X. et al. A DNA integrity network in the yeast Saccharomyces cerevisiae. Cell 124, 1069–1081 (2006).

    Article  CAS  Google Scholar 

  20. Chen, P. & Hochstrasser, M. Biogenesis, structure, and function of the yeast 20S proteasome. EMBO J. 14, 2620–2630 (1995).

    Article  CAS  Google Scholar 

  21. Varshavsky, A. Regulated protein degradation. Trends Biochem. Sci. 30, 283–286 (2005).

    Article  CAS  Google Scholar 

  22. Ravid, T., Kreft, S.G. & Hochstrasser, M. Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways. EMBO J. 25, 533–543 (2006).

    Article  CAS  Google Scholar 

  23. Krogan, N.J. et al. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440, 637–643 (2006).

    Article  CAS  Google Scholar 

  24. Isono, E. et al. The assembly pathway of the 19S regulatory particle of the yeast 26S proteasome. Mol. Biol. Cell 18, 569–580 (2006).

    Article  Google Scholar 

  25. Schmidt, M. et al. The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle. Nat. Struct. Mol. Biol. 12, 294–303 (2005).

    Article  CAS  Google Scholar 

  26. Altschul, S.F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    Article  CAS  Google Scholar 

  27. 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  CAS  Google Scholar 

  28. Groll, M. et al. A gated channel into the proteasome core particle. Nat. Struct. Biol. 7, 1062–1067 (2000).

    Article  CAS  Google Scholar 

  29. Brennan, R.J. & Schiestl, R.H. Cadmium is an inducer of oxidative stress in yeast. Mutat. Res. 356, 171–178 (1996).

    Article  Google Scholar 

  30. Inai, Y. & Nishikimi, M. Increased degradation of oxidized proteins in yeast defective in 26 S proteasome assembly. Arch. Biochem. Biophys. 404, 279–284 (2002).

    Article  CAS  Google Scholar 

  31. Lee, S.C. & Shaw, B.D. A novel interaction between N-myristoylation and the 26S proteasome during cell morphogenesis. Mol. Microbiol. 63, 1039–1053 (2007).

    Article  CAS  Google Scholar 

  32. Rivett, A.J., Bose, S., Brooks, P. & Broadfoot, K.I. Regulation of proteasome complexes by γ-interferon and phosphorylation. Biochimie 83, 363–366 (2001).

    Article  CAS  Google Scholar 

  33. Liu, C.W., Corboy, M.J., DeMartino, G.N. & Thomas, P.J. Endoproteolytic activity of the proteasome. Science 299, 408–411 (2003).

    Article  CAS  Google Scholar 

  34. Guthrie, C. & Fink, G.R. Guide to Yeast Genetics and Molecular Biology (Academic Press, San Diego, 1991).

    Google Scholar 

  35. Ferreras, M., Gavilanes, J.G. & Garcia-Segura, J.M. A permanent Zn2+ reverse staining method for the detection and quantification of proteins in polyacrylamide gels. Anal. Biochem. 213, 206–212 (1993).

    Article  CAS  Google Scholar 

  36. Verma, R. et al. Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol. Biol. Cell 11, 3425–3439 (2000).

    Article  CAS  Google Scholar 

  37. Elsasser, S., Schmidt, M. & Finley, D. Characterization of the proteasome using native gel electrophoresis. Methods Enzymol. 398, 353–363 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Finley (Harvard University, Boston) and W. Heinemeyer (University of Stuttgart, Germany) for antibodies to proteasome subunits. This work was supported by grants from the US National Institutes of Health (NIH) to M.H. A.R.K. was supported in part by a postdoctoral fellowship from the Canadian Institutes of Health Research. M.J.K. was supported by NIH training grant GM007223.

Author information

Authors and Affiliations

  1. Department of Molecular Biophysics & Biochemistry, Yale University, 266 Whitney Avenue, New Haven, 06520-8114, Connecticut, USA

    Andrew R Kusmierczyk, Mary J Kunjappu, Minoru Funakoshi & Mark Hochstrasser

Authors
  1. Andrew R Kusmierczyk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mary J Kunjappu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Minoru Funakoshi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mark Hochstrasser
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.R.K. and M.H. developed the experimental approach. A.R.K., M.J.K., M.F. and M.H. carried out experiments. A.R.K. and M.H. wrote the paper.

Corresponding author

Correspondence to Mark Hochstrasser.

Supplementary information

Supplementary Text and Figures

Supplementary figures 1–3 and Tables 1 and 2 (PDF 570 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kusmierczyk, A., Kunjappu, M., Funakoshi, M. et al. A multimeric assembly factor controls the formation of alternative 20S proteasomes. Nat Struct Mol Biol 15, 237–244 (2008). https://doi.org/10.1038/nsmb.1389

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1389

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing