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

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

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

Dynamic localization of Sch9 and its regulation of V-ATPase disassembly.

<p>(A) Vacuolar membrane enrichment of Sch9 is regulated by glucose availability. Cells expressing GFP-Sch9 were grown to exponential phase in minimal medium buffered at pH 5 and stained with FM4-64. Next, cells were washed in starvation medium and deprived of either glucose or nitrogen for 30 min. Alternatively, cells were treated with 200 nM rapamycin. For each condition, vacuolar membrane localization of GFP-Sch9 was assessed for at least 750 cells from three to four independent experiments. Mean values ± SD are shown. A one-way ANOVA analysis was performed to designate statistical differences. (B) V-ATPase activity does not mediate Sch9 localization. Cells of the indicated genotype co-expressing GFP-Sch9 and mCherry-Pho8 were grown to exponential phase in minimal medium buffered at pH 5 and their intracellular localization was analyzed by fluorescence microscopy. Vacuolar membrane localization of GFP-Sch9 was assessed in at least 600 cells from two to three independent experiments. Mean values ± SD are shown. (C-F) Sch9 regulates V-ATPase disassembly in response to glucose availability. WT (C) and <i>sch9Δ</i> (D) cells co-expressing Vma5-RFP and Vph1-GFP were grown as in <b>Fig 6A</b> and their intracellular localization was analyzed by fluorescence microscopy. (E, F) Combined fluorescence intensity profile plots of Vma5-RFP (red) and Vph1-GFP (green) measured along the line displayed in the merged panel for WT (E) and <i>sch9Δ</i> (F) cells. The x-axis depicts the distance along the line in pixels, while the y-axis indicates the relative RFP or GFP signal intensities. The Pearson’s coefficient (R) ± SD was calculated using the ImageJ plugin JACoP. See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s009" target="_blank">S9 Fig</a></b>.</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

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