An evolutionarily conserved pathway controls proteasome homeostasis

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.

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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.

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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.

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Authors

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.

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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)

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

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