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W2989403090 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Material and methods References Decision letter Author response Article and author information Metrics Abstract The degradation and recycling of cellular components is essential for cell growth and survival. Here we show how selective and non-selective lysosomal protein degradation pathways cooperate to ensure cell survival upon nutrient limitation. A quantitative analysis of starvation-induced proteome remodeling in yeast reveals comprehensive changes already in the first three hours. In this period, many different integral plasma membrane proteins undergo endocytosis and degradation in vacuoles via the multivesicular body (MVB) pathway. Their degradation becomes essential to maintain critical amino acids levels that uphold protein synthesis early during starvation. This promotes cellular adaptation, including the de novo synthesis of vacuolar hydrolases to boost the vacuolar catabolic activity. This order of events primes vacuoles for the efficient degradation of bulk cytoplasm via autophagy. Hence, a catabolic cascade including the coordinated action of the MVB pathway and autophagy is essential to enter quiescence to survive extended periods of nutrient limitation. https://doi.org/10.7554/eLife.07736.001 eLife digest Yeast and other organisms have evolved to survive extended periods of starvation by digesting their own proteins and other cell materials and thereby recycle them into new proteins and structures. One way in which these cell materials can be destroyed is by a process called autophagy. A membrane forms around the cell material to isolate it from the rest of the cell. In yeast, the resulting structure fuses with a cell compartment called the vacuole, which contains enzymes that break down the cargo into smaller molecules that can be re-used by the cell. When cells experience starvation, autophagy is not very selective in what it destroys and so it is tightly controlled to avoid damaging important structures in healthy cells. Alongside autophagy, specific proteins in the membrane surrounding a yeast cell can be targeted for destruction by another process called the MVB pathway. Certain membrane proteins are tagged with a small protein called ubiquitin, which leads them to being selectively incorporated into cell compartments called MVBs that then go on to fuse with the vacuole. However, it is not clear how the MVB pathway and autophagy may cooperate to enable the cell to survive periods of starvation. Here, Müller et al. monitored the changes in the proteins present in yeast cells during a period of starvation. The experiments show that many different membrane proteins in the yeast cells were destroyed via the MVB pathway within three hours of the removal of their food source. This was essential to allow the cells to carry on producing new proteins at this early stage in starvation. These new proteins included the enzymes found in vacuoles, which increased the ability of the cells to break down the proteins and other cell materials that were transported there via autophagy. These findings show how the MVB pathway and autophagy are co-ordinated to allow cells to survive periods of starvation. The next challenge is to work out how the MVB pathway is regulated at the molecular level in response to fluctuations in nutrient availability. https://doi.org/10.7554/eLife.07736.002 Introduction Evolutionary conserved selective and non-selective protein degradation pathways are essential for cell growth and survival. The ubiquitin-proteasome system (UPS) mediates selective poly-ubiquitination of cytoplasmic proteins and their degradation at 26S proteasomes for regulatory and quality control functions. Mis-folded proteins in the endoplasmic reticulum (ER) are also ubiquitinated, extracted by the ER-associated protein degradation system (ERAD) and degraded at 26S proteasomes in the cytoplasm (Vembar and Brodsky, 2008). Macro-autophagy (hereafter autophagy) non-selectively transports bulk cytoplasm into lysosomes. Therefore the induction of autophagy is tightly controlled: under normal growth conditions autophagy operates on a basal level because it is suppressed by signaling from the target of rapamycin complex 1 (TORC1) (Kamada et al., 2000; Loewith and Hall, 2011; Zoncu et al., 2011b). In response to cellular stress, such as nutrient depletion (e.g., of amino acids), TORC1 is inactivated (Loewith and Hall, 2011) and autophagy is strongly induced. Deregulation of autophagic processes is implicated in metabolic and infectious diseases as well as in cancer or neurodegeneration (Rubinsztein et al., 2012). Once induced, the autophagic machinery begins to sequester cytoplasmic components, ribosomes and organelles within a large double-membrane compartment termed the autophagosome (Yang and Klionsky, 2010; Kraft and Martens, 2012; Mizushima et al., 2011). In addition, some core components of the autophagic machinery such as LC3/Atg8 are transcriptionally induced (Kirisako et al., 1999). Direct fusion of autophagosomes with lysosomes delivers autophagic bodies and the sequestered cargo into the lysosomal lumen. Alternatively, autophagosomes can first fuse with multivesicular bodies (MVBs) to form so-called amphisomes, before they fuse with lysosomes (Seglen et al., 1991). Finally, the breakdown of autophagic bodies and the efficient degradation of autophagic cargo inside lysosomes is required to recycle amino acids, nucleotides, carbohydrates and lipids back to the cytoplasm. The recycling of these key metabolic building blocks protects cells from their fatal depletion and thus maintains cellular homeostasis to survive nutrient limitation (Onodera and Ohsumi, 2005; Vabulas and Hartl, 2005; Jones et al., 2012; Suraweera et al., 2012). Therefore evolutionary conserved starvation programs in mammalian cells and yeast expand and strengthen this intracellular recycling system by enhancing the de novo synthesis of vacuolar/lysosomal hydrolases (Gasch et al., 2000; Sardiello et al., 2009; Settembre et al., 2011; Shen and Mizushima, 2014). In addition to autophagy, TORC1 also regulates ubiquitin-mediated endocytosis of integral plasma membrane proteins. On the one hand, TORC1 signaling was required to promote the endocytosis of certain plasma membrane proteins (MacGurn et al., 2011). On the other hand, inactivation of TORC1 either by rapamycin or starvation, triggered the endocytosis of other plasma membrane proteins that were subsequently degraded in an ESCRT (endosomal sorting complex required for transport)-dependent manner via the MVB pathway (Schmidt et al., 1998; Jones et al., 2012; Lang et al., 2014). The extent to which starvation induces plasma membrane remodeling has yet to be determined. Furthermore, how the subsequent ubiquitin-dependent degradation of membrane proteins via the MVB pathway helps to meet the specific metabolic and energetic demands of cells during nutrient limitation is not fully understood. Therefore it is also not clear how selective (MVB) and non-selective (autophagy) lysosomal proteolysis pathways cooperate to mediate cell survival during nutrient limitation. To comprehensively address these questions we have used quantitative proteomics. Our results demonstrate that within the first 3 hr of amino acid starvation many integral plasma membrane proteins, including high-affinity amino acid permeases, glucose transporters and G-protein coupled receptors, were selectively removed from the cell surface by endocytosis and subsequently targeted into vacuoles via the ESCRT-dependent MVB pathway and degraded, while others remained stable or were up-regulated (e.g., the general amino acid permease, Gap1). This comprehensive and selective remodeling of the plasma membrane appeared to be completed within 3–4 hr of starvation. Autophagy was also immediately activated upon starvation and remained active throughout starvation. Surprisingly, early during starvation the selective degradation of membrane proteins via the MVB pathway was mainly responsible to maintain critical levels of free intracellular amino acids that were sufficient to uphold protein synthesis and promote the corresponding adaptation of the proteome. Most notably this included the de novo synthesis of vacuolar hydrolases, which boosted the proteolytic activity of vacuoles to support the efficient degradation of autophagic cargo. The continuous delivery and degradation of autophagic cargo further enhanced intracellular amino acid recycling and was ultimately essential to restore intracellular amino acid pools of cells during extended starvation. These findings reveal an unexpected role for the MVB pathway in maintaining intracellular amino acid homeostasis and thereby promoting the up-regulation of vacuolar hydrolases early during starvation, which is tightly coordinated with autophagy. This catabolic cascade is ultimately required to allow starving cells to complete their cell division cycle and enter a quiescent state for survival. Results Starvation induces selective and non-selective protein degradation pathways To understand how the MVB pathway, autophagy and proteasomal degradation cooperate during nutrient limitation, we first analyzed the starvation-induced degradation of model proteins in yeast. To assess selective membrane protein degradation via the MVB pathway, we followed the ubiquitin-dependent endocytosis of the plasma membrane methionine permease, Mup1-GFP and its transport into the vacuole in response to starvation (for amino acids and nitrogen sources) (Beck et al., 1999; Menant et al., 2006; Jones et al., 2012). Under rich growth conditions Mup1-GFP is mainly found at the plasma membrane and very little is degraded (Figure 1A,B). Yet, within 3 hr after the onset of starvation the majority of Mup1-GFP was removed from the cell surface, delivered into vacuoles and degraded (Figure 1A,B). The proteolytic degradation of Mup1-GFP inside vacuoles released free GFP, which remained stable and was monitored by western blotting (Figure 1A). The starvation-induced delivery of Mup1-GFP into the vacuole was dependent on the ESCRT machinery but was not affected in an autophagy (atg8∆) mutant (Figure 1B). In an ESCRT (vps4∆) mutant, the MVB pathway was blocked and Mup1-GFP was not delivered into the vacuole but instead accumulated on the class E compartment and at the plasma membrane (Figure 1B). Figure 1 with 1 supplement see all Download asset Open asset 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. https://doi.org/10.7554/eLife.07736.003 To define the timing of starvation-induced degradation of Mup1-GFP in the context of eukaryotic starvation programs, we compared it to the delivery of bulk cytoplasm via autophagy. Therefore we determined the degradation of highly abundant selective (ribosomes) and non-selective (Fba1) autophagic cargoes. Growing yeast cells contain about 200,000 ribosomes that occupy up to 30–40% of the cytoplasmic volume (Warner, 1999). Upon starvation, otherwise stable ribosomes are among the first autophagic cargoes and rapidly degraded by selective (ribophagy) and non-selective autophagy (Kraft et al., 2008; Ossareh-Nazari et al., 2014). We monitored the release of free GFP from two different ribosomal proteins by western blotting: Rpl25-GFP (large subunit) and Rps2-GFP (small subunit). Both are fully functional GFP fusion proteins that incorporate into ribosomes (Kraft et al., 2008). When equal amounts of cell lysates were subjected to western-blot analysis, the protein levels of full length Mup1-GFP and the GFP-tagged ribosomal subunits were comparable (Figure 1A, lanes 6, 16). After 3 hr, at a time when the majority of full length Mup1-GFP was already degraded, free GFP from Rpl25 was first detected, showing that autophagy was also delivering cytoplasmic contents into the vacuole (Figure 1A, lane 8). During subsequent starvation the protein levels of free GFP from both ribosomal subunits increased. Monitoring the autophagy-dependent degradation of Fba1-GFP, one of the most abundant cytoplasmic proteins with approximately 1.000.000 molecules/cell (Ghaemmaghami et al., 2003), yielded similar results. Free GFP was first detected after 3 hr of starvation and the protein levels free GFP strongly increased during subsequent starvation (Figure 1—figure supplement 1A). To determine the earliest possible starvation-induced autophagic activity, we monitored the transport and degradation of fully functional GFP-Atg8. Atg8 is a core component of the autophagic machinery that remains conjugated to the inner membrane of all selective and non-selective autophagosomes, including cytoplasm to vacuole targeting (cvt)-vesicles. Therefore Atg8 is degraded together with autophagic cargo inside vacuoles. To be able to compare the degradation of GFP-Atg8 to Mup1-GFP, 10 times more lysate of cells expressing GFP-Atg8 was subjected to western blot analysis (Figure 1A). Small amounts of free GFP released from GFP-Atg8 inside vacuoles could be readily detected by western blot analysis 1 hr after the onset of starvation and the levels of free GFP strongly increased at 3 hr of starvation (Figure 1A, lane 27–30). These findings are consistent with the strong increase of endogenous Atg8 levels during starvation (Figure 1—figure supplement 1B) as observed earlier (Kirisako et al., 1999). Previous work also demonstrated that Atg8 protein levels control the size of autophagosomes but not the frequency (about 9 autophagosomes/hour) by which they are formed (Abeliovich et al., 2000; Xie et al., 2008). Hence, the increase in Atg8 protein levels during the first 4 hr of starvation would result in the formation of bigger (but not more) autophagosomes that could capture larger volumes of cytoplasm later during starvation. Our results for the early degradation of GFP-Atg8 and the continuous increase in autophagic degradation of highly abundant selective as well as non-selective cargoes throughout starvation are fully consistent with this model. This idea was further supported using the Pho8∆60 assay, a sensitive method to measure bulk autophagy (Noda et al., 1995). Pho8∆60 activity was low under rich conditions, began to increase during the first 3 hr of starvation and continuously increased during extended periods of starvation (Figure 1—figure supplement 1C). These results show that autophagy is immediately activated upon starvation and delivers increasing volumes of cytoplasmic material into the vacuole with ongoing starvation (Figure 1). Additionally, we investigated how cytoplasmic proteins were degraded at proteasomes upon starvation. Therefore, we employed a ubiquitin-GFP (Ub-GFP) fusion protein, which is an established reporter for proteasomal activity (Johnson et al., 1992; Vabulas and Hartl, 2005). It is detected at low levels in proliferating cells reflecting the equilibrium between its rapid degradation and its synthesis. Upon starvation, Ub-GFP was rapidly degraded at 26S proteasomes. The degradation of the reporter was exclusively dependent on proteasomal degradation but did not require autophagy or the MVB pathway (Figure 1C, lanes 5–7; Figure 1D). Overall, these findings indicate that starvation triggered protein degradation by different selective and non-selective degradation pathways: the constitutive protein degradation via the proteasome was active from the onset of starvation and was previously suggested to play a key role upon acute nutrient restriction (Vabulas and Hartl, 2005). Our results further suggest that both autophagy and starvation induced-endocytosis were simultaneously activated early during starvation. Autophagy continuously delivered ever-increasing volumes of cytoplasm into vacuoles, whereas the starvation-induced degradation of membrane proteins was completed within 3 hr. These findings suggest an important role for the MVB pathway early during starvation. The MVB pathway and autophagy contribute differentially to maintain free amino acid levels and protein synthesis during starvation While protein degradation at 26S proteasomes provides an immediate amino acid pool for protein synthesis already within minutes of acute starvation (Vabulas and Hartl, 2005) and autophagy is required to supply amino acids during extended periods of starvation (Onodera and Ohsumi, 2005), the relative contribution of the MVB pathway to overall amino acid homeostasis was not clear. Therefore we next measured the intracellular levels of 18 different amino acids in isogeneic WT cells, MVB (vps4∆) or autophagy (atg8∆) mutants as well as double mutants (vps4∆, atg8∆) by liquid chromatography (Altmann, 1992). These strains were auxotrophic for the amino acids lysine and leucine. When grown in synthetic medium supplemented with amino acids (rich), the intracellular free amino acid levels were comparable in WT cells and autophagy mutants (atg8∆) (Figure 2A), but slightly lower in vps4∆ mutants, which was mainly due to reduced lysine and arginine levels (Figure 2A,B). Figure 2 with 1 supplement see all Download asset Open asset 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. https://doi.org/10.7554/eLife.07736.005 1 hr after starvation in synthetic medium without amino acids and ammonium salts, the total free amino acid pool decreased to similar levels in WT cells and all mutant strains (Figure 2A,B). In WT cells the levels of most amino acids continued to decrease for another hour. Interestingly, at around 4 hr of starvation the overall levels of amino acids almost fully recovered, suggesting strong amino acid recycling. However, the levels of arginine and lysine, which were among the most abundant free amino acids, decreased further. The levels of glutamine, threonine and glycine did not recover very well, while the levels of other amino acids (particularly of glutamate) increased. The recovery of amino acid levels after 4 hr of starvation was strongly dependent on autophagy. These results are at large consistent with previous findings, where an approximately threefold reduction in intracellular amino acid levels was detected during the first 2 hr of starvation and autophagy was required for the partial recovery of amino acid levels from 3 to 6 hr of starvation (Onodera and Ohsumi, 2005). In our strain background the levels of amino acids were generally lower under rich growth and we observed an approximately twofold reduction in intracellular amino acids during the first 2 hr of starvation. At 4 hr of starvation amino acid levels fully recovered in an autophagy dependent manner (Figure 2A,B). In addition, our findings showed that the MVB pathway essentially contributed to maintain the overall levels of free intracellular amino acids. 1 hr after starvation, the amino acids levels decreased similar in vps4∆ mutants, autophagy mutants (atg8∆) and WT cells. However, after 2 hr the overall amino acid levels were lower in vps4∆ mutants compared to WT cells and autophagy mutants. The levels of 14 individual amino acids were lower in vps4∆ mutants when compared to WT cells or autophagy mutants. Moreover the amino acid levels failed to recover during extended starvation (Figure 2A). From 2 hr onwards, the amino acid levels were always lowest in the double mutants (vps4∆, atg8∆) (Figure 2A,B). To exclude effects contributed by amino acid auxotrophies, the same analysis was performed in a different genetic background with fully prototrophic WT cells and the respective vps4∆ and atg8∆ single mutants (Mülleder et al., 2012). During the first 2 hr of starvation, the amino acid levels initially declined in the prototrophic WT cells, but not as strongly as in auxotrophic strains, and recovered at around 4 hr of starvation, which was dependent on autophagy (Figure 2—figure supplement 1A,B). In prototrophic vps4∆ mutants, the levels of most amino acids (12) were lower after 2 hr of starvation when compared to WT cells or autophagy mutants (atg8∆), as observed in the auxotrophic strains (Figure 2A,B; Figure 2—figure supplement 1A,B). These results showed that the MVB pathway was essential to maintain the levels of most free intracellular amino acids within the first 2 hr of starvation, while autophagy was essential to restore intracellular amino acids later during starvation. Based on these results we next tested how the MVB pathway would contribute to uphold protein synthesis during starvation. Therefore we measured 35S-methionine/cysteine incorporation into newly synthesized proteins. Under rich conditions, 35S-label incorporation was comparable in WT cells, atg8∆ and vps4∆ single or double mutants (Figure 2C,D), although the methionine permease Mup1 was more abundant in ESCRT mutants. Already 1 hr after starvation, 35S-label incorporation was reduced to 40% in WT cells. During the next 3 hr of starvation, WT cells managed to maintain protein synthesis at this level (Figure 2C, lanes 1, 5, 9,13, Figure 2D). In the autophagy-deficient atg8∆ mutant, 35S-label incorporation was initially similar to WT cells for up to 2 hr, but began to decline after 4 hr of starvation (Figure 2C, lanes 3, 7, 11, 15, Figure 2D), which is consistent with the key role of autophagy in amino acid recycling. In ESCRT mutants (vps4∆), protein synthesis declined faster when compared to WT cells or autophagy mutants, which seems consistent with the more rapid decline of intracellular amino acids (Figure 2C, lanes 2, 6, 10, 14, Figure 2D). vps4∆, atg8∆ double mutants showed an additive effect, since even less 35S-label was incorporated upon starvation compared to the single deletion mutants (Figure 2C,D). Taken together these findings suggest that (i) the MVB pathway is essential to maintain a critical pool of free amino acids for protein synthesis early during starvation. (ii) In the absence of the MVB pathway autophagy can only partially uphold amino acids levels and protein synthesis (iii) The MVB pathway and autophagy cooperate to maintain intracellular amino acids during starvation, potentially in a consecutive manner. The ESCRT machinery is not required for the induction of autophagy, the formation of autophagosomes and the delivery of autophagosomes into the vacuole Recent reports have shown an important role for the ESCRT machinery in higher eukaryotic cells in regulating autophagy at the stage of amphisomes fusing with lysosomes (Nara et al., 2002; Filimonenko et al., 2007; Lee et al., 2007; Rusten et al., 2007; Metcalf and Isaacs, 2010; Spitzer et al., 2015). Therefore we next carefully examined the role of the ESCRT machinery in distinct steps of autophagy in yeast. The induction of autophagy is tightly controlled by TORC1. Under nutrient rich growth conditions, TORC1 was active and its direct targets Sch9 and Atg13 were phosphorylated in WT cells, vps4∆ and atg8∆ mutants (Figure 3A,B, lane 1, 3, 5) (Kamada et al., 2000; Urban et al., 2007). When autophagy and the MVB pathway were simultaneously disrupted (vps4∆, atg8∆), TORC1 signaling appeared to be reduced under rich growth conditions, but not completely switched off (Figure 3A, lane 7). Upon starvation TORC1 signaling was efficiently turned off and the autophagy core component Atg13 was dephosphorylated in all strains, which is a prerequisite for the induction of autophagy (Figure 3B) (Kamada et al., 2000). Figure 3 Download asset Open asset 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. https://doi.org/10.7554/eLife.07736.007 To assess the formation and the delivery of autophagosomes into vacuoles, we followed the transport of GFP-tagged Atg8 using live cell fluorescence microscopy. Upon starvation, GFP-Atg8 was efficiently transported into the lumen of vacuoles in WT cells and vps4∆ mutants (Figure 3C), indicating that the autophagic machinery was fully operational and independent of the ESCRT machinery. This conclusion was further strengthened using the Pho8∆60 autophagy-reporter assay (Noda et al., 1995). Pho8∆60 activity increased after 4 hr of starvation in WT and vps4∆ mutants, but not in atg8∆ cells (Figure 3D). Collectively, these results show that in yeast autophagosomes together with their cargo were delivered into the vacuoles of vps4∆ mutants in response to starvation, which is consistent with earlier reports (Reggiori et al., 2004). Next, we analyzed autophagic processes further downstream and examined the lysis of autophagic bodies. This is a prerequisite for the subsequent proteolytic breakdown of autophagic cargo (Takeshige et al., 1992; Yang et al., 2006) and depends on vacuolar acidification, the catabolic activity of Pep4 and Prb1 and the lipase Atg15 (Teter et al., 2000; Epple et al., 2001). To determine the breakdown of autophagic bodies in living cells, we generated a functional pHluorin-Atg8 chimera. The fluorescence of pHluorin-Atg8 is detectable at cytosolic pH but not at the lower pH within the vacuole (Prosser et al., 2010). In WT cells the fluorescence of pHluorin-Atg8 was efficiently quenched in the lumen of vacuoles upon starvation (>90% of cells, n = 157). In contrast, in mutants that are either deficient in vacuolar peptidases (prb1∆, prc1∆, pep4∆) or vacuolar acidification (vma4∆) pHluorin-Atg8 was not quenched (<10% of cells, n = 198 and n = 40, respectively) and pHluorin-Atg8 positive vesicular structures were detected inside their vacuoles, suggesting that autophagic bodies were not efficiently lysed (Figure 3E,F). In the vast majority of vps4∆ mutants (>90% of cells, n = 121), the fluorescence of pHluorin-Atg8 was quenched in the vacuoles similar to WT cells, but occasionally few perivacuolar pHluorin-Atg8 positive structures were observed (<17% of cells, n = 121). Overall, it seemed that autophagic bodies were efficiently lysed in acidified vacuoles of vps4∆ mutants (Figure 3E,F). These findings emphasize that in yeast the autophagic machinery, the fusion of autophagosomes with the vacuole per se and the lysis of autophagic bodies is not impaired in ESCRT mutants. The MVB pathway is required for early proteome remodeling during starvation Next we determined in detail how the MVB pathway would contribute to the starvation program of yeast. Therefore we measured how the proteome of WT (vps4∆ complemented with VPS4) cells changed within the first 3 hr of starvation using stable isotope labeling with amino acids in cell culture (SILAC) (de Godoy et al., 2008). WT cells were grown under rich conditions with heavy 13C615N2-lysine or light 12C614N2-lysine, and light cells were subsequently starved for 3 hr. Equal cell numbers were mixed prior to lysis and mass spectrometry (MS) analysis (Figure 4—figure supplement 1A, upper panel). In total 2941 proteins were quantified (peptide count ≥ 2), comprising 58% of the characterized yeast ORFs (Figure 4A, Figure 4—figure supplement 1A, upper panel, Supplementary file 1). In this early phase of starvation, the yeast proteome already underwent extensive remodeling and 264 proteins significantly changed in abundance (MaxQuant significance B) (Cox and Mann, 2008). 101 proteins were significantly down- and 163 proteins significantly up-regulated. Figure 4 with 2 supplements see all Download asse" @default.
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W2989403090 title "Author response: The coordinated action of the MVB pathway and autophagy ensures cell survival during starvation" @default.
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