The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability

The evolutionary conserved TOR complex 1 controls growth in response to the quality and quantity of nutrients such as carbon and amino acids. The protein kinase Sch9 is the main TORC1 effector in yeast. However, only few of its direct targets are known. In this study, we performed a genome-wide screening looking for mutants which require Sch9 function for their survival and growth. In this way, we identified multiple components of the highly conserved vacuolar proton pump (V-ATPase) which mediates the luminal acidification of multiple biosynthetic and endocytic organelles. Besides a genetic interaction, we found Sch9 also physically interacts with the V- ATPase to regulate its assembly state in response to glucose availability and TORC1 activity. Moreover, the interaction with the V-ATPase has consequences for ageing as it allowed Sch9 to control vacuolar pH and thereby trigger either lifespan extension or lifespan shortening. Hence, our results provide insights into the signaling mechanism coupling glucose availability, TORC1 signaling, pH homeostasis and longevity. As both Sch9 and the V-ATPase are highly conserved and implicated in various pathologies, these results offer fertile ground for further research in higher eukaryotes

The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability

The conserved protein kinase Sch9 is a central player in the nutrient-induced signaling network in yeast, although only few of its direct substrates are known. We now provide evidence that Sch9 controls the vacuolar proton pump (V-ATPase) to maintain cellular pH homeostasis and ageing. A synthetic sick phenotype arises when deletion of SCH9 is combined with a dysfunctional V-ATPase, and the lack of Sch9 has a significant impact on cytosolic pH (pHc) homeostasis. Sch9 physically interacts with, and influences glucose-dependent assembly/disassembly of the V-ATPase, thereby integrating input from TORC1. Moreover, we show that the role of Sch9 in regulating ageing is tightly connected with V-ATPase activity and vacuolar acidity. As both Sch9 and the V-ATPase are highly conserved in higher eukaryotes, it will be interesting to further clarify their cooperative action on the cellular processes that influence growth and ageing.status: publishe

Phenotypic overlap between <i>sch9Δ</i> and mutants in which V-ATPase function is impaired.

<p>Phenotypic overlap between <i>sch9Δ</i> and mutants in which V-ATPase function is impaired.</p

Hypothetical model depicting feedback regulation between Sch9 and the V-ATPase.

<p>Sch9 physically interacts with the V-ATPase to modulate its assembly state, thereby affecting vacuolar pH homeostasis. Moreover, Sch9 might also control pHv independently of the V-ATPase by affecting vacuolar proton exchangers. As protein hydrolysis and amino acid uptake are regulated by vacuolar acidity, a feedback mechanism onto Sch9 activity is provided through amino acid sensing by the EGO complex (EGOC) and subsequent regulation of TORC1 activity. Additionally, through modulation of V-ATPase (dis)assembly Sch9 has the ability to indirectly impact on PKA activity. For more details, see text.</p

Effects of <i>sch9Δ</i> and rapamycin on V-ATPase assembly and disassembly.

<p>Effects of <i>sch9Δ</i> and rapamycin on V-ATPase assembly and disassembly.</p

Sch9 physically interacts with the V-ATPase.

<p>(A, B) Physical interaction of Sch9 with Vma1 depends on glucose availability. Cells expressing HA₆-Sch9 were grown as in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.g006" target="_blank">Fig 6A</a></b>, followed by re-addition of 2% glucose (A) or 0.2% glutamine (B). Total lysates (input) and anti-Vma1 immunoprecipitates (IP) were analyzed by immunoblotting. (C, D) Sch9 regulates V-ATPase assembly downstream of TORC1. (C) WT and <i>sch9Δ</i> cells were grown to mid-log phase in YPD, pH 5. Half of the culture was treated with 200 nM rapamycin for 30 min and subsequently starved for glucose in the presence of rapamycin. The untreated half was further grown for 30 min and subsequently starved for glucose. (D) V-ATPase assembly was assessed in the <i>sch9Δ</i> strain expressing the empty vector (pRS416), the wild-type <i>SCH9</i> gene (Sch9^WT), or one of the <i>SCH9</i> mutant genes in which its TORC1 phosphorylation sites are mutated (Sch9^5A and Sch9^2D3E). The WT strain expressing the empty vector was taken as an additional control. Precultures were grown overnight in minimal medium lacking uracil buffered at pH 5 and inoculated in YPD medium (50 mM MES, pH 5). Once cells reached exponential phase, half of the culture was treated with 200 nM rapamycin (rapa) for 30 min. To quantify V-ATPase assembly, complexes were IPed with antibodies against Vma1 and Vph1. Results are depicted as mean values ± SEM from at least three independent experiments. One- or two-way ANOVA analyses were performed to determine statistical significances. Unless indicated otherwise, asterisks indicate a statistical significance compared to the WT strain grown in YPD without rapamycin. See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.t002" target="_blank">Table 2</a></b>.</p

Systematic analysis of genetic interaction partners of <i>SCH9</i>.

<p>(A) <i>SCH9</i> genetic interaction network. Osprey software was used to graphically display the relationships between genes identified in the SGA screening. Synthetic lethal interactions are connected by green lines; protein-protein interactions are shown in gray. Nodes are colored by process. Circles indicate well-defined protein complexes or group of genes that are functionally related. For clarity reasons, not all identified genes, nor all known interactions are shown. (B) Hypergeometric enrichment organized according to GO function, process and component. See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s011" target="_blank">S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s012" target="_blank">S2</a> Tables</b>.</p

Sch9 does not impact on vesicular trafficking.

<p>(A-E) Sorting and processing of vacuolar proteases is not impaired in exponentially growing <i>sch9Δ</i> cells. Processing of CPY (A), ALP (C) and Ape1 (E) was examined by Western blot. * represents cross-reacting band. Intracellular localization of CPY-GFP (B) and GFP-Pho8 (D) was examined by fluorescence microscopy. (F) Sch9 affects basal and non-specific autophagy. Exponentially growing cells expressing Pho8<i>Δ</i>60 were shifted to nitrogen starvation medium. Samples were taken at the indicated time points, proteins extracted, and specific activities determined. Results are normalized to the activity of a WT strain starved for 24h. Mean values ± SD are shown (unpaired t-test). See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s001" target="_blank">S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s002" target="_blank">S2</a> Figs</b>.</p

Function of Sch9 in regulating ageing is dependent on V-ATPase activity.

<p>With the exception of panel E, all chronological ageing data represent measurements performed on cells at day 8 in stationary phase. (A) Cell survival and (B) ROS determination of strains aged in non-buffered fully supplemented medium as determined by flow cytometry. (C) Cell survival and (D) ROS levels of strains aged in fully supplemented medium buffered at pH 5.5 as determined by flow cytometry. (E) Cell survival of strains grown in buffered medium at day 23 in stationary phase as determined by CFU counting. (F) pH of the culture medium of ageing cells grown in buffered medium. (G) Cell survival and (H) ROS determination of strains grown in medium containing the indicated concentration of methionine as determined by flow cytometry. Results depicted are mean values ± SD. (I) Sch9 affects pHv. Vacuolar pH was measured during exponential growth and during glucose starvation using the ratiometric fluorescent pH indicator BCECF-AM. Results depicted are mean values ± SEM of four independent experiments. All differences between strains and conditions are statistically significant unless stated as ns (not significant). A detailed statistical analysis is presented in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s013" target="_blank">S3 Table</a></b>. See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s007" target="_blank">S7</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s008" target="_blank">S8</a> Figs</b>.</p

Effects on colony size and growth by deletion of <i>VPH1</i>, <i>STV1</i> and/or <i>SCH9</i>.

<p>(A, B) A synthetic sick phenotype arises when deletion of <i>SCH9</i> is combined with a fully dysfunctional V-ATPase. (A) Tetrad dissection of the diploid strain JW 04 952 (<i>sch9Δ/SCH9 vph1Δ/VPH1 stv1Δ/STV1</i>). (B) Colony sizes were calculated, normalized relative to WT and are shown as mean values ± SD. Letters indicate groups of strains with a significant difference in colony size (p < 0.001, one-way ANOVA). (C, D) Strains combining deletion of <i>SCH9</i> with a fully dysfunctional V-ATPase show a deteriorated growth phenotype. OD_600nm was followed over time in fully supplemented medium without buffer (C) or buffered at pH 5 (D). A representative experiment with at least 4 independent colonies for each strain is shown. Error bars represent SD from the mean. See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s004" target="_blank">S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s005" target="_blank">S5</a> Figs</b>.</p