Abstract
Protein aggregation is mostly viewed as deleterious and irreversible causing several pathologies. However, reversible protein aggregation has recently emerged as a novel concept for cellular regulation. Here, we characterize stress-induced, reversible aggregation of yeast pyruvate kinase, Cdc19. Aggregation of Cdc19 is regulated by oligomerization and binding to allosteric regulators. We identify a region of low compositional complexity (LCR) within Cdc19 as necessary and sufficient for reversible aggregation. During exponential growth, shielding the LCR within tetrameric Cdc19 or phosphorylation of the LCR prevents unscheduled aggregation, while its dephosphorylation is necessary for reversible aggregation during stress. Cdc19 aggregation triggers its localization to stress granules and modulates their formation and dissolution. Reversible aggregation protects Cdc19 from stress-induced degradation, thereby allowing cell cycle restart after stress. Several other enzymes necessary for G1 progression also contain LCRs and aggregate reversibly during stress, implying that reversible aggregation represents a conserved mechanism regulating cell growth and survival.
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Acknowledgements
We thank S. Alberti (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany), M. Ralser (Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK) and K. Weis (Institute of Biochemistry, Department of Biology, ETH Zürich, Zürich, Switzerland) for providing reagents; S. McKnight, D. Frantz and L. Berchowitz for help with b-isox experiments; A. Timofiiva for help with microscopy; and T. Mayor, A. Smith, P. Kimmig and members of the Peter laboratory for helpful discussions and comments on the manuscript. This work was funded by the Swiss National Science Foundation (SNF, project grants 31003A_166513 to R.D. and 310030B_160312 to M.P.), the European Research Council (ERC, Rubinet) and ETH Zürich.
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Conceptualization: S.S., M.P. and R.D.; investigation: S.S., G.C. and Y.F.; formal analysis: R.D.; writing—original draft preparation: S.S. and R.D.; writing—review and editing: S.S., G.C., Y.F., P.P., M.P. and R.D.; visualization: S.S. and R.D.; supervision: P.P., M.P. and R.D.; funding acquisition: M.P. and R.D.
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Supplementary Figure 1 Cdc19 forms reversible aggregates upon stress and tetrameric Cdc19ΔPEP is unable to support cell growth in yeast.
(a,b) Major protein degradation pathways do not significantly contribute to reversibility of Cdc19 aggregates. Cells expressing Cdc19-GFP and the indicated mutations were grown to stationary phase, loaded into a microfluidic chip and reversibility of aggregates was scored upon glucose readdition in the presence of (a) PMSF (prevents vacuolar degradation) or (b) MG132 to inhibit proteasomal degradation. Note that expression of the Pdr1-Cyc8 fusion necessary for efficient inhibition of proteasomal activity by MG132. The reason for faster solubilisation of aggregates in this strain is unclear. Arrowheads indicate frames with near-complete solubilisation of detectable aggregates. Time relative to glucose readdition is indicated. Data shown are representative of 3 independent experiments. (c) Cdc19-aggregates in stationary phase are solid-like. Cells were grown to stationary phase and Cdc19-GFP aggregates were subjected to FRAP analysis as described in Fig. 1. Quantification of fluorescence recovery for all analyzed cells is shown as the mean of the relative fluorescence intensity as a function of time (red line) together with the individual cell traces (light and dark gray) (n = 3 independent experiments). (d) Cells harboring the tetO7-CDC19 allele expressing wild-type or mutant Cdc19 or an empty vector were grown in the presence or absence of doxycycline (10 μg/ml, added at t = 0h) and growth was analyzed using a BioLector (m2p-labs). Accumulation of biomass is shown as a function of time. Data shown are representative of 2 independent experiments. (e) Cytoplasmic acidification is not required for the formation of Cdc19 aggregates. Cells were grown as in Fig. 1a and representative images of Cdc19-GFP upon glucose starvation in C-starvation media (pH 4.6 or pH 7.4) or PBS (pH 7.4) from 3 independent experiments are shown. In all images, the scale bar represents 3 μm. Statistical source data for c can be found in Supplementary Table 4.
Supplementary Figure 2 Cdc19 colocalizes with RNA containing stress granules and failure of recruitment renders Cdc19 unstable.
(a) Cdc19 aggregates colocalize with the stress granule marker Pub1. Cells expressing Cdc19-GFP and Pub1-RFP were starved and localization of Cdc19 aggregates relative to Pub1 was analyzed. Data shown are representative of 2 independent experiments. (b) Cdc19 aggregates colocalize with the stress granule marker Pab1, but not with the P-body marker Edc3. Cells expressing Cdc19-GFP, Pab1-CFP and Edc3-RFP were grown as in Fig. 1 and timing of the appearance of P-bodies and stress granules and their localization was analyzed relative to Cdc19 aggregation. Data shown are representative of 3 independent experiments. Time relative to glucose readdition is indicated. Note that Cdc19-GFP was imaged using YFP filter sets to avoid any spectral mixing in cells expressing Pab1-CFP. (c) Cells expressing wild-type Cdc19-TAP or Cdc19ΔPEP-TAP and Hxk2-TAP as control were grown in SD media and UV treated. Cross-linked protein-mRNA complexes were isolated using poly-dT and analyzed by western-blot. Data shown are representative of 3 independent experiments. (d) Cells expressing wild-type or mutant Cdc19-GFP expressing Pab1-CFP were starved for glucose and localization of Cdc19 and Pab1 was analyzed at the indicated time-points. Note that Cdc19-GFP was imaged using YFP filter sets to avoid any spectral mixing in cells expressing Pab1-CFP. Data shown are representative of 3 independent experiments. (e) Cells expressing wild-type or mutant Cdc19 were grown as in Fig. 3g and relative abundance of Cdc19 was determined by mass-spectrometry and plotted as the mean ± S.E.M. of three technical replicates. In all images, the scale bar represents 3 μm. Uncropped blot is shown in Supplementary Fig. 5.
Supplementary Figure 3 Cdc19 forms Thioflavin-T positive aggregates in vitro and LCRs might generally contribute to aggregation.
(a) Wild-type and mutant Cdc19 incubated at the indicated temperatures were mixed with ThT as in Fig. 4e and emission spectra were recorded. Data shown are representative of 3 independent experiments. (b) Sbp1 and Ssa2 colocalize with stress granules upon glucose starvation. Cells expressing Sbp1-GFP or Ssa2-GFP and Pab1-CFP were grown as in Fig. 1 and scored for localization of the indicated proteins. Data shown are representative of 3 independent experiments. Note that Sbp1-GFP and Ssa2-GFP were imaged using YFP filter sets to avoid any spectral mixing in cells expressing Pab1-CFP. The scale bar represents 3 μm.
Supplementary Figure 4 Dephosphorylation of the Cdc19 LCR is required for reversible aggregation.
(a) Schematic representation of the MS-workflow to determine the fraction of phosphorylated peptides. Unphosphorylated peptides were measured with or without phosphatase treatment (PPase) and abundance relative to a control peptide was determined. (b) The LCR of Cdc19 is part of the subunit interface in Cdc19 tetramers. Crystal structure of Cdc19 showing a part of the subunit interface of Cdc19 tetramers. Blue: ribbon representation of Cdc19 with the region corresponding to the LCR shown in yellow. Thr372, Thr376, Ser377 and Ser385 are shown in space filling representation. Other subunits of the Cdc19 tetramer (light gray, dark-gray, red) are shown as surface representations. Note that Ser385 forms intersubunit contacts (hydrogen bond via side chain-OH), strongly suggesting that phosphorylation at this site would be incompatible with phosphorylation. (c) Cells expressing wild-type or mutant Cdc19-GFP expressing Pab1-CFP were starved for glucose and localization of Cdc19 and Pab1 was analyzed at the indicated time-points. Note that Cdc19-GFP was imaged using YFP filter sets to avoid any spectral mixing in cells expressing Pab1-CFP. Data shown are representative of 3 independent experiments. (d) Cells as in Fig. 7a were grown and analyzed for aggregate formation upon heat shock (30 min, 42 °C) and after recovery at 30 °C for 60 min. Representative images of 3 independent experiments for the indicated conditions are shown. In all images, the scale bar represents 3 μm.
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Saad, S., Cereghetti, G., Feng, Y. et al. Reversible protein aggregation is a protective mechanism to ensure cell cycle restart after stress. Nat Cell Biol 19, 1202–1213 (2017). https://doi.org/10.1038/ncb3600
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DOI: https://doi.org/10.1038/ncb3600
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