The coordinated action of the MVB pathway and autophagy ensures cell survival during starvation
- Medical University of Innsbruck, Austria
- University of Vienna, Austria
- University of Natural Resources and Applied Biosciences, Austria
- Austrian Drug Screening Institute, Austria
Figures
Figure 1 with 1 supplement
Starvation induces selective and non-selective protein degradation pathways.
(A) WT cells expressing Mup1-GFP, Rpl25-GFP, Rps2-GFP or GFP-Atg8 were grown in rich medium (0 hr) or starved as indicated. Cell lysates were analyzed by SDS-PAGE and western blot (WB) using the indicated antibodies. *residual anti-GFP signal after re-probing the membrane with anti-Pgk1 antibody. (B) Fluorescence microscopy of Mup1-GFP in WT cells, vps4∆ mutants and atg8∆ mutants growing under rich or starvation conditions. (V)acuoles, (P)lasma (M)embrane and class (E) compartments. Scale bar = 5 µm. (C, D) Whole cell lysates of WT cells or the indicated mutants grown under rich conditions or starved for the indicated times were separated by SDS-PAGE and analyzed by western blot using the indicated antibodies. (C) pdr5∆ cells were treated with the proteasome inhibitor MG132 (50 µM) or vehicle (DMSO) during starvation.
Figure 1—figure supplement 1
Induction of autophagy.
(A) SDS-PAGE and western blot analysis of WT cells grown under rich conditions or starved using the indicated antibodies. (B) Vacuolar hydrolase-deficient cells (pep4∆, prb1∆, prc1∆) analyzed as in (A). (C) Pho8∆60-specific alkaline phosphatase activity was measured in WT, and atg8∆ cells under rich conditions and after starvation (n = 6, mean ± SD). WT Pho8∆60 activity after 20 hr of starvation was set to 100%.
Figure 2 with 1 supplement
Changes in free amino acid levels and protein synthesis during starvation.
(A) Cells were grown to mid-log phase (rich) and starved as indicated. Free amino acids were extracted and analyzed by liquid chromatography. Data are represented as the sum of free amino acids (mg) per gram of dry yeast. Mean ± SD, n ≥ 3. (B) Changes in individual amino acids from (A) normalized to maximal values. Mean ± SD, n ≥ 3. (C, D) Cells grown under the indicated conditions were incubated for 5 min with 35S-labeled Met and Cys. (C) 35S-incorporation was analyzed by SDS-PAGE and digital autoradiography. Coomassie staining shows equal protein loading. (D) Quantification of 35S-incorporation under rich conditions and after 1, 2 and 4 hr of starvation by liquid scintillation counting. Incorporation under rich conditions was set to 100%. Mean ± SEM, n = 3.
Figure 2—figure supplement 1
Changes in free amino acids levels during starvation in prototrophic yeast.
(A) Prototrophic cells were grown to mid-log phase (rich) and starved as indicated. Free amino acids were extracted and analyzed by liquid chromatography. Data are represented as the sum of free amino acids (mg) per gram of dry yeast. Mean ± SD, n = 3. (B) Changes in individual amino acids from (A) were normalized to maximal values. Mean ± SD, n = 3.
Figure 3
Autophagy in ESCRT mutants.
(A, B) SDS-PAGE and western blot analysis of total cell lysates from WT cells and vps4∆, atg8∆ single and double mutants grown in rich medium (0 hr) or during starvation using the indicated antibodies. (C) Live-cell fluorescence microscopy of WT cells and vps4∆ mutants expressing GFP-Atg8 (green) and mCherry-CPS (red) under rich conditions or 4 hr after starvation. (D) Pho8∆60-specific alkaline phosphatase activity was measured in WT, vps4∆ and atg8∆ cells under rich conditions and after 4 hr of starvation (n = 8, ±SD). WT Pho8∆60 activity under rich conditions was normalized to 100%. (E) Fluorescence microscopy of pHluorin-Atg8 (green) and mCherry-CPS (red) in WT cells and indicated mutants under rich conditions or after 4 hr of starvation. (F) Quantification of quenching of vacuolar pHluorin-Atg8 from E. (C, E) (V)acuoles and class (E) compartments. Scale bar = 5 µm.
Figure 4 with 2 supplements
The MVB is required for starvation induced proteome remodeling.
(A) Schematic presentation of proteome remodeling in WT cells during starvation. Starvation induced changes in protein levels were measured using SILAC based quantitative proteomics (see also Figure 4—figure supplement 1A,B, Supplementary files 1, 2). The major changes in WT cells under starvation as indicated by Gene Ontology (GO) analysis of significantly changed proteins are shown. Green: up-regulated; red: down-regulated under starvation. (B) Correlation of changes in protein abundance in WT cells and vps4∆ mutants during starvation (see also Figure 4—figure supplement 1A, Supplementary file 3). WT and vps4∆ mutant protein ratios (log2 [starved/rich]) are plotted against each other. Green: significantly regulated in both datasets; blue: significantly regulated only in vps4∆; purple: significantly regulated only in WT. Grey: not significantly regulated. (C) Density plot showing log2-transformed protein ratio distributions in the three quantitative proteome datasets. The significant protein changes are excluded. Blue: WT (starved)/WT(rich); green: vps4∆(starved)/vps4∆(rich); red: vps4∆(rich)/WT(rich) (see also Figure 4—figure supplement 1, Supplementary file 4). p-value according to Kolmogorov–Smirnov <6 × 10−16 (D) The 135 significantly differentially regulated proteins during starvation between WT cells (black bars) and vps4∆ mutants (white bars) (see also Supplementary file 5). (E) Enrichment of GO terms in 135 significantly differentially regulated proteins (from D). Data are represented as fold-enrichment over whole genome frequency (see also Supplementary file 6). * significantly represented GO terms.
Figure 4—figure supplement 1
Quantitative proteomics and GO analysis.
(A, C) Representation of SILAC based quantitative proteomic changes. Protein ratio is plotted against signal intensity. Proteins that were significantly (significance B) down-regulated (red dots) or up-regulated (green dots) during 3 hr of starvation are shown. Non-regulated proteins are shown as grey bars. (A) Upper panel: protein ratios of WT cells starved/rich (labeled with heavy lysine); lower panel: protein ratios of vps4∆ mutants starved/rich (labeled with heavy lysine). (C) Protein ratios of vps4∆(rich)/WT(rich, labeled with heavy lysine). (B) GO terms (cellular processes and components) that were significantly up- or down-regulated in the proteomic analysis of WT cells under starvation. Data are represented as enrichment over whole genome frequency for each GO term. (Bold: more than 1.5-fold enriched). (D) SDS-PAGE and western blot analysis of cells grown under rich conditions using the indicated antibodies.
Figure 4—figure supplement 2
Starvation induced endocytosis.
(A) Fluorescence microscopy of Ste2-GFP, Can1-GFP, Fur4-GFP, and Pma1-GFP in WT and vps4∆ cells growing under rich conditions or after starvation. Vacuole (V), class E compartment (E) and plasma membrane (PM) are labeled. Scale bar = 5 µm. (B) Fluorescence microscopy of Gap1-GFP in WT cells growing under rich conditions or after starvation. Plasma membrane (PM) is labeled. Scale bar = 5 µm. (C) WT and vps4∆ cells expressing Gap1-GFP were grown in rich medium or starved as indicated. Cell lysates were analyzed by SDS-PAGE and western blot (WB) using the indicated antibodies.
Figure 5 with 1 supplement
Boosting the catabolic activity of vacuoles during starvation requires the MVB pathway.
(A) Starvation-induced changes in the protein levels of various vacuolar hydrolases based on SILAC data in WT (black) and vps4∆ mutants (white). (B) Indicated yeast strains were grown in rich medium (0 hr) or starved for 3 hr. Cell lysates were subjected to SDS-PAGE and western blot analysis with the indicated antibodies. p, precursor form; m, mature form; s, soluble form. (C, D) Soluble Pho8 (sPho8) activity in rich medium and upon starvation of the indicated strains (mean ± SEM, n = 4). (E) Fluorescence microscopy of Pep4-GFP (green) in WT cells and vps4∆ mutants growing under rich or starvation conditions. (V)acuoles (FM4-64, red) and class (E) compartments. Scale bar = 5 µm. vps10∆ + VPS10 (2 µ) cells were used as the isogenic WT control in (D) and (E).
Figure 5—figure supplement 1
Boosting the catabolic activity of vacuoles requires membrane protein degradation via the MVB pathway.
(A) Soluble vacuolar alkaline phosphatase (sPho8) activity in WT and ESCRT mutants (vps36∆; vps23∆). Data are represented as fold increase in activity after 3 hr starvation over rich growth condition (n = 3; mean ± SEM). (B) Fluorescence microscopy of CPY(1–50)-mRFP and Mup1-GFP in WT cells, vps4∆ mutants and vps4∆ mutants over-expressing Vps10 growing under rich or starvation conditions. (V)acuoles and class (E) compartments. (C) Indicated yeast strains were grown in rich medium (0 hr) or starved for 3 hr. Cell lysates were subjected to SDS-PAGE and western blot analysis with the indicated antibodies. p, precursor form; m, mature form; s, soluble form; *unspecific background band. (D) WT cells, vps4∆ mutants and vps4∆ mutants over-expressing Vps10 were grown to mid-log phase (rich) and starved as indicated. Free amino acids were extracted and analyzed by liquid chromatography. Data are represented as the sum of free amino acids (mg) per gram of dry yeast. Mean ± SD, n = 3. (E) 35S-Met/Cys incorporation into proteins of WT (vps10∆ + VPS10, 2 µ), vps4∆ mutants and vps4∆ mutants over-expressing Vps10 under rich growth conditions and during starvation, was analyzed by SDS-PAGE and digital autoradiography. Coomassie staining shows equal protein loading. (F) Quantification of 35S-incorporation under rich conditions and after 1, 2 and 4 hr of starvation by liquid scintillation counting. Incorporation under rich conditions was set to 100%. Mean ± SEM, n = 3.
Figure 6 with 1 supplement
Boosting the catabolic activity of vacuoles is essential for the efficient degradation of autophagic cargo.
(A, B, C) SDS-PAGE and western blot analysis of total cell lysates from WT cells and vps4∆ mutants grown in rich medium or during starvation using the indicated antibodies. p(ro)Ape1, m(ature)Ape1. *residual anti-GFP signal after re-probing the membrane with anti-Pgk1 antibody. (D) Fluorescence microscopy of Rpl25-GFP (green) and mCherry-CPS (red) in WT cells and vps4∆ mutants under rich conditions or after starvation. Dashed lines indicate the vacuolar membrane. (V)acuoles and class (E) compartments. Scale bar = 5 µm.
Figure 6—figure supplement 1
Proteolytic processing of autophagic cargo.
(A) SDS-PAGE and western blot analysis of total cell lysates from WT cells and the indicated mutants starved for 4 hr using the indicated antibodies. p, precursor form; m, mature form; Ψ-m, pseudo-mature form generated in prb1∆; *residual anti-GFP signal after re-probing the membrane with anti-Pgk1 antibody. (B) SDS-PAGE and western blot analysis of total cell lysates from vps4∆ mutants and vps4∆ mutants overexpressing Vps10 grown in rich medium or during starvation using the indicated antibodies.
Figure 7 with 1 supplement
The coordinated function of the MVB pathway and autophagy is required to enter quiescence upon starvation.
(A) Growth of WT cells and the indicated mutants after shift from logarithmic growth in rich medium (0 hr) to starvation measured with OD600nm. Mean ± SEM, n = 4. (B) Quantification of unbudded cells (G1/G0 arrested) under rich conditions (0 hr) or after indicated time of starvation. (C) Cells were starved for the indicated times and equal amounts of cells in serial dilutions were placed on rich medium (YPD). (D) Model representing the coordinated action of lysosomal protein degradation pathways for amino acid maintainance and recycling as well as protein synthesis during starvation.
Figure 7—figure supplement 1
Cell growth and entry into quiescence upon starvation.
(A) Growth of WT cells and the indicated mutants overexpressing Vps10 after shift from logarithmic growth in rich medium to starvation measured with OD600nm. Mean ± SEM, n = 4. (B) Quantification of unbudded cells (G1/G0 arrested) under rich growth or after 4 hr or 24 hr of starvation. n > 210.
Additional files
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Supplementary file 1
Quantification of proteome changes in WT cells during amino acid starvation.
- https://doi.org/10.7554/eLife.07736.017
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Supplementary file 2
GO Analysis of significantly up- or down-regulated proteins under starvation.
- https://doi.org/10.7554/eLife.07736.018
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Supplementary file 3
Quantification of proteome changes in WT cells and vps4Δ mutants during starvation.
- https://doi.org/10.7554/eLife.07736.019
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Supplementary file 4
Quantitative proteomics comparing WT and vps4Δ under rich growth conditions.
- https://doi.org/10.7554/eLife.07736.020
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Supplementary file 5
135 most differentially regulated proteins in WT and vps4Δ mutants during starvation.
- https://doi.org/10.7554/eLife.07736.021
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Supplementary file 6
GO Analysis of proteins that are differentially regulated under starvation.
- https://doi.org/10.7554/eLife.07736.022
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Supplementary file 7
Yeast strains and plasmids.
- https://doi.org/10.7554/eLife.07736.023
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Supplementary file 8
Primer sequences.
- https://doi.org/10.7554/eLife.07736.024
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The coordinated action of the MVB pathway and autophagy ensures cell survival during starvation
eLife 4:e07736.
https://doi.org/10.7554/eLife.07736
