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:

An evolutionarily conserved pathway controls proteasome homeostasis

Nature volume 536, pages 184–189 (2016)Cite this article

Subjects

Abstract

The proteasome is essential for the selective degradation of most cellular proteins, but how cells maintain adequate amounts of proteasome is unclear. Here we show that there is an evolutionarily conserved signalling pathway controlling proteasome homeostasis. Central to this pathway is TORC1, the inhibition of which induced all known yeast 19S regulatory particle assembly-chaperones (RACs), as well as proteasome subunits. Downstream of TORC1 inhibition, the yeast mitogen-activated protein kinase, Mpk1, acts to increase the supply of RACs and proteasome subunits under challenging conditions in order to maintain proteasomal degradation and cell viability. This adaptive pathway was evolutionarily conserved, with mTOR and ERK5 controlling the levels of the four mammalian RACs and proteasome abundance. Thus, the central growth and stress controllers, TORC1 and Mpk1/ERK5, endow cells with a rapid and vital adaptive response to adjust proteasome abundance in response to the rising needs of cells. Enhancing this pathway may be a useful therapeutic approach for diseases resulting from impaired proteasomal degradation.

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: TORC1 inhibition induces the proteasome assembly chaperone Adc17 and increases proteasome levels.
Figure 2: The MAPK Mpk1 is a master regulator of the stress-inducible proteasome assembly chaperone Adc17.
Figure 3: Mpk1 coordinates the expression of all yeast RACs to control proteasome abundance.
Figure 4: Post-transcriptional control of RAC and proteasome subunit abundance by Mpk1.
Figure 5: Mpk1 adjusts proteasome degradation to match the needs of the cell.
Figure 6: Evolutionary conservation of the pathway controlling RACs and proteasome abundance.

Similar content being viewed by others

References

  1. Goldberg, A. L. Functions of the proteasome: from protein degradation and immune surveillance to cancer therapy. Biochem. Soc. Trans. 35, 12–17 (2007)

    Article  CAS  PubMed  Google Scholar 

  2. Finley, D. Recognition and processing of ubiquitin–protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Tanaka, K., Mizushima, T. & Saeki, Y. The proteasome: molecular machinery and pathophysiological roles. Biol. Chem. 393, 217–234 (2012)

    Article  CAS  PubMed  Google Scholar 

  4. Tomko, R. J. Jr & Hochstrasser, M. Molecular architecture and assembly of the eukaryotic proteasome. Annu. Rev. Biochem. 82, 415–445 (2013)

    Article  CAS  PubMed  Google Scholar 

  5. Le Tallec, B., Barrault, M. B., Guérois, R., Carré, T. & Peyroche, A. Hsm3/S5b participates in the assembly pathway of the 19S regulatory particle of the proteasome. Mol. Cell 33, 389–399 (2009)

    Article  CAS  PubMed  Google Scholar 

  6. Saeki, Y., Toh-E, A., Kudo, T., Kawamura, H. & Tanaka, K. Multiple proteasome-interacting proteins assist the assembly of the yeast 19S regulatory particle. Cell 137, 900–913 (2009)

    Article  CAS  PubMed  Google Scholar 

  7. Funakoshi, M., Tomko, R. J. Jr, Kobayashi, H. & Hochstrasser, M. Multiple assembly chaperones govern biogenesis of the proteasome regulatory particle base. Cell 137, 887–899 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Roelofs, J. et al. Chaperone-mediated pathway of proteasome regulatory particle assembly. Nature 459, 861–865 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kaneko, T. et al. Assembly pathway of the mammalian proteasome base subcomplex is mediated by multiple specific chaperones. Cell 137, 914–925 (2009)

    Article  CAS  PubMed  Google Scholar 

  10. Hanssum, A. et al. An inducible chaperone adapts proteasome assembly to stress. Mol. Cell 55, 566–577 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wiseman, R. L., Haynes, C. M. & Ron, D. SnapShot: The unfolded protein response. Cell 140, 590–590.e2 (2010)

    Article  PubMed  Google Scholar 

  12. Venters, B. J. et al. A comprehensive genomic binding map of gene and chromatin regulatory proteins in Saccharomyces. Mol. Cell 41, 480–492 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Marion, R. M. et al. Sfp1 is a stress- and nutrient-sensitive regulator of ribosomal protein gene expression. Proc. Natl Acad. Sci. USA 101, 14315–14322 (2004)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Jorgensen, P. et al. A dynamic transcriptional network communicates growth potential to ribosome synthesis and critical cell size. Genes Dev. 18, 2491–2505 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lempiäinen, H. et al. Sfp1 interaction with TORC1 and Mrs6 reveals feedback regulation on TOR signaling. Mol. Cell 33, 704–716 (2009)

    Article  PubMed  CAS  Google Scholar 

  16. Takahara, T. & Maeda, T. Transient sequestration of TORC1 into stress granules during heat stress. Mol. Cell 47, 242–252 (2012)

    Article  CAS  PubMed  Google Scholar 

  17. Soulard, A. & Hall, M. N. SnapShot: mTOR signaling. Cell 129, 434.e1–434.e2 (2007)

    Article  Google Scholar 

  18. Loewith, R. & Hall, M. N. Target of rapamycin (TOR) in nutrient signaling and growth control. Genetics 189, 1177–1201 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zoncu, R., Efeyan, A. & Sabatini, D. M. mTOR: from growth signal integration to cancer, diabetes and ageing. Nature Rev. Mol. Cell Biol. 12, 21–35 (2011)

    Article  CAS  Google Scholar 

  20. Bonilla, M. & Cunningham, K. W. Mitogen-activated protein kinase stimulation of Ca2+ signaling is required for survival of endoplasmic reticulum stress in yeast. Mol. Biol. Cell 14, 4296–4305 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Krause, S. A. & Gray, J. V. The protein kinase C pathway is required for viability in quiescence in Saccharomyces cerevisiae. Curr. Biol. 12, 588–593 (2002)

    Article  CAS  PubMed  Google Scholar 

  22. Torres, J., Di Como, C. J., Herrero, E. & De La Torre-Ruiz, M. A. Regulation of the cell integrity pathway by rapamycin-sensitive TOR function in budding yeast. J. Biol. Chem. 277, 43495–43504 (2002)

    Article  CAS  PubMed  Google Scholar 

  23. Babour, A., Bicknell, A. A., Tourtellotte, J. & Niwa, M. A surveillance pathway monitors the fitness of the endoplasmic reticulum to control its inheritance. Cell 142, 256–269 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Levin, D. E. Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway. Genetics 189, 1145–1175 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  26. 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  PubMed  Google Scholar 

  27. Xie, Y. & Varshavsky, A. RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: a negative feedback circuit. Proc. Natl Acad. Sci. USA 98, 3056–3061 (2001)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Suraweera, A., Münch, C., Hanssum, A. & Bertolotti, A. Failure of amino acid homeostasis causes cell death following proteasome inhibition. Mol. Cell 48, 242–253 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Hiller, M. M., Finger, A., Schweiger, M. & Wolf, D. H. ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science 273, 1725–1728 (1996)

    Article  ADS  CAS  PubMed  Google Scholar 

  30. Medicherla, B., Kostova, Z., Schaefer, A. & Wolf, D. H. A genomic screen identifies Dsk2p and Rad23p as essential components of ER-associated degradation. EMBO Rep. 5, 692–697 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Asano, S. et al. A molecular census of 26S proteasomes in intact neurons. Science 347, 439–442 (2015)

    Article  ADS  CAS  PubMed  Google Scholar 

  32. Truman, A. W. et al. Expressed in the yeast Saccharomyces cerevisiae, human ERK5 is a client of the Hsp90 chaperone that complements loss of the Slt2p (Mpk1p) cell integrity stress-activated protein kinase. Eukaryot. Cell 5, 1914–1924 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhang, Y. et al. Coordinated regulation of protein synthesis and degradation by mTORC1. Nature 513, 440–443 (2014)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zhao, J., Zhai, B., Gygi, S. P. & Goldberg, A. L. mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy. Proc. Natl Acad. Sci. USA 112, 15790–15797 (2015)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Albert, V. & Hall, M. N. mTOR signaling in cellular and organismal energetics. Curr. Opin. Cell Biol. 33, 55–66 (2015)

    Article  CAS  PubMed  Google Scholar 

  36. Gietz, R. D. & Woods, R. A. Yeast transformation by the LiAc/SS carrier DNA/PEG method. Methods Mol. Biol. 313, 107–120 (2006)

    CAS  PubMed  Google Scholar 

  37. Zhang, T. et al. An improved method for whole protein extraction from yeast Saccharomyces cerevisiae. Yeast 28, 795–798 (2011)

    Article  CAS  PubMed  Google Scholar 

  38. von der Haar, T. Optimized protein extraction for quantitative proteomics of yeasts. PLoS One 2, e1078 (2007)

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  39. Urban, J. et al. Sch9 is a major target of TORC1 in Saccharomyces cerevisiae. Mol. Cell 26, 663–674 (2007)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  41. Knutson, B. A. & Hahn, S. Domains of Tra1 important for activator recruitment and transcription coactivator functions of SAGA and NuA4 complexes. Mol. Cell. Biol. 31, 818–831 (2011)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Y. Lee and M. Hochstrasser for the kind gift of Nas2, Nas6, Hsm3 and Rpn14 antibodies; D. H. Wolf for CPY*–HA and Δss-CPY*–GFP constructs; T. Maeda for the P-Sch9 antibody; and members of the Bertolotti laboratory for discussion. A.B. is an honorary fellow of the University of Cambridge Clinical Neurosciences Department. This work was supported by the Medical Research Council (UK) MC_U105185860. A.R. is supported by an EMBO long-term fellowship.

Author information

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, CB2 0QH, Cambridge, UK

    Adrien Rousseau & Anne Bertolotti

Authors
  1. Adrien Rousseau
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anne Bertolotti
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.R. designed, performed and analysed all experiments, prepared the figures and helped with the manuscript. A.B. designed and supervised the study and wrote the manuscript.

Corresponding author

Correspondence to Anne Bertolotti.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks S. Murata and D. Sabatini and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Adc17 induction is increased in mrs6-DAmp cells and occurs when Sfp1 is cytosolic.

a, Immunoblots of the indicated proteins in lysates of wild-type and Mrs6-hypomorphic (mrs6-DAmP) yeast strains ± tunicamycin for 4 h. b, Representative images of yeast cells carrying a GFP-tagged SFP1 at the endogenous locus, ± tunicamycin for 4 h. Scale bar, 5 μm. Representative results of at least three independent experiments (biological replicates) are shown.

Extended Data Figure 2 Mpk1 is essential for tunicamycin and rapamycin survival and Adc17 induction.

a, mpk1 Δ cells transformed with wild-type MPK1 or a kinase-dead allele (MPK1-K52R) or empty vector were spotted in a sixfold dilution and grown on plates containing or lacking tunicamycin. b, Immunoblots of lysates of yeast strains shown in a, cultured for 4 h ± tunicamycin. c, Cells of the indicated genotype were spotted in a sixfold dilution and grown for 3 days at 30 °C on plates containing or lacking rapamycin. d, Immunoblots of lysates from wild-type and MAPK genetic deletion mutant yeast cells cultured for 4 h ± tunicamycin or rapamycin. e, Same as in a, using mpk1 Δ cells transformed with empty vector or a vector encoding MPK1 or HOG1. Representative results of at least three independent experiments (biological replicates) are shown.

Extended Data Figure 3 Mpk1 MAPK pathway is essential for stress-mediated RACs induction.

a, b, Immunoblots of the indicated proteins in lysates of wild-type yeast cells ± tunicamycin (a) or rapamycin (b) for the indicated time. c, g, Immunoblots of the indicated proteins in lysates of wild-type and bck1Δ cells cultured ± tunicamycin or rapamycin for 4 h. d, h, Immunoblots of the indicated proteins in lysates of wild-type and mkk1/2Δ cells cultured ± tunicamycin or rapamycin for 4 h. e, f, Immunoblots of the indicated proteins in lysates of wild-type or mpk1 Δ cells ± 50 μg ml−1 Congo red (CR) for 4 h. Representative results of at least three independent experiments (biological replicates) are shown.

Extended Data Figure 4 Induction of RACs under challenging conditions is an important function of Mpk1.

a, Wild-type cells or mpk1 Δ cells transformed with one or combinations of two or three RACs were spotted in a sixfold dilution and grown on plates containing or lacking tunicamycin, where indicated. b, Multiple-deletion yeast strains of different RACs were spotted in a sixfold dilution and grown for 3 days at 33 °C on plates containing or lacking rapamycin. Representative results of at least three independent experiments (biological replicates) are shown.

Extended Data Figure 5 Pba1 and Pba2 are induced by tunicamycin in a Mpk1-independent manner.

a–d, Immunoblots of the indicated proteins in lysates of wild-type yeast cells carrying a TAP-tagged Pba1 (a), Pba2 (b), Pba3 (c) and Pba4 (d) at the endogenous locus ± tunicamycin for 3 h. Representative results of at least three independent experiments (biological replicates) are shown.

Extended Data Figure 6 Mpk1 post-transcriptionally regulates proteasome subunits and RACs.

a, b, Immunoblots of the indicated proteins in lysates of wild-type (a) and rpn4 Δ (b) cells ± tunicamycin or rapamycin for 4 h. c, Immunoblots of the indicated proteins in lysates of wild-type yeast cells carrying a TAP-tagged RPN4 at the endogenous locus ± tunicamycin or rapamycin for 4 h. d, Immunoblots of the indicated proteins in lysates of wild-type and mpk1 Δ cells ± tunicamycin or rapamycin for 4 h. e, rpn4 Δ cells transformed with RPN4, MPK1, a kinase-dead allele of MPK1 (MPK1-K52R) or empty vector were spotted in a sixfold dilution and grown on plates containing or lacking tunicamycin. f, mpk1 Δ cells transformed with MPK1, RPN4 or empty vector were spotted in a sixfold dilution and grown on plates containing or lacking tunicamycin where indicated. g, Immunoblots of the indicated proteins in lysates of wild-type and mpk1 Δ cells carrying a TAP-tagged RPN4 at the endogenous locus ± tunicamycin or rapamycin for 4 h. h, i, Immunoblots of the indicated proteins in lysates of wild-type (h, i) and mpk1 Δ (i) cells treated with different combinations of drugs: 5 μg ml−1 tunicamycin, 0.2 μg ml−1 rapamycin and 35 μg ml−1 cycloheximide, where indicated for 4 h. Representative results of at least three independent experiments (biological replicates) are shown.

Extended Data Figure 7 Mpk1 maintains the adequate levels of proteasome required to sustain protein degradation.

a, c, Yeast cells of the indicated genotype expressing GFP-tagged Ura3-3 proteins were treated with cycloheximide and incubated at 37 °C for the indicated time. b, d, Quantifications from three independent experiments (biological replicates) such as the one shown in a and c. e, g, Cells of the indicated genotype expressing CPY*–HA (e) or Δss-CPY*–GFP (g) proteins were treated with tunicamycin for 4 h. f, h, Quantifications from three independent experiments (biological replicates) such as the one shown in e and g. i, k, Cells of the indicated genotype expressing CPY*–HA (i) or Δss-CPY*–GFP (k) proteins were treated with rapamycin for 4 h. j, l, Quantifications from three independent experiments (biological replicates) such as the one shown in i and k. b, d, f, h, j and l, Data are mean ± s.d. n = 3 biological replicates. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; NS, not significant (two-way ANOVA).

Source data

Extended Data Figure 8 Starvation inhibits TORC1 signalling, induces mammalian RACs and increases proteasome abundance.

a, b, Immunoblots (a) and quantification (b) of the indicated proteins in lysates of HeLa cells after EBSS (Earle’s balanced salt solution) treatment for the indicated time. c, HeLa cell extracts following EBSS treatment for the indicated time were resolved on native PAGE (4.2%) and monitored using the fluorogenic substrate Suc-LLVY–AMC or by immunoblots. d, Quantification of the 26S proteasome activity (RPCP and RP2CP) of experiments such as the one shown in c. b, d, Data are mean ± s.d. n = 3 biological replicates. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; NS, not significant (one-way ANOVA). Representative results of at least three independent experiments (biological replicates) are shown.

Source data

Extended Data Figure 9 TORC1 activation by nutrient replenishment decreases the abundance of RACs as well as 26S proteasome.

a, b, Immunoblots (a) and quantification (b) of the indicated proteins in lysates of HeLa cells after replenishment with rich complete medium for the indicated time. c, Native PAGE (4.2%) of cell extracts from HeLa cells following media replenishment as in a, monitored by the fluorogenic substrate Suc-LLVY–AMC or by immunoblots. d, Quantification of the 26S proteasome activity (RPCP and RP2CP) of experiments such as the one shown in c. b, d, Data are mean ± s.d. n = 3 biological replicates. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; NS, not significant (one-way ANOVA). Representative results of at least three independent experiments (biological replicates) are shown.

Source data

Extended Data Figure 10 mTORC1 inhibition by Torin-1 and rapamycin acutely induced the RACs.

a, b, Immunoblots (a) and quantification (b) of the indicated proteins in lysates of HeLa cells treated with 250 nM Torin-1 or 200 nM rapamycin for the indicated time. Data are mean ± s.d. n = 3 biological replicates. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; NS, not significant (two-way ANOVA). Representative results of at least three independent experiments (biological replicates) are shown.

Source data

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-2, listing the strains and the plasmids used in the study, respectively, and Supplementary Figure 1 containing full-scan gel images with size indications corresponding to Figures 1a-e, 1g-h, 2c, 2e, 2g, 3a-b, 3e-g, 5a, 5c, 5e, 6a, 6c, 6e-f, 6h, and Extended Data Figures 1a, 2b, 2d, 3a-h, 5a-d, 6a-d, 6g-i, 7a, 7c, 7e, 7g, 7i, 7k, 8a, 8c, 9a, 9c and 10a. (PDF 10128 kb)

PowerPoint slides

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rousseau, A., Bertolotti, A. An evolutionarily conserved pathway controls proteasome homeostasis. Nature 536, 184–189 (2016). https://doi.org/10.1038/nature18943

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature18943

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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