α-Syn and synphilin-1 equally enhance cell death in aged yeast cells.

<p>A: Quantification of ROS accumulation using DHE staining at different times during growth of yeast strains transformed with an empty plasmid (□, Ctrl.) or constructs allowing for expression of α-Syn (▪), SY^WT (Δ), SY^R621C (▴),α-Syn and SY^WT (○) or α-Syn and SY^R621C (•). B: Quantification of the number of cells that display phosphatidylserine externalization or loss of membrane integrity using annexinV/propidium iodide (PI) co-staining at 36 h of growth in the strains used in A. C: Quantification of viable cells present in the strains used in A at 36 h of growth as determined by their ability to form colonies. D: Fluorescence microscopic visualization of cells expressing combinations of α-Syn, SY^WT or SY^R621C as indicated and stained with DHE (upper panels) or co-stained with annexinV and PI (lower panels) after 36 h of growth. E and F: Quantification of viable cells (E) and cells producing ROS (F) in the strains used in A when kept in culture for two weeks. All data represent mean ± SEM of six independent transformants. Significance of the data was determined by t-tests (* = p<0.05; ** = p<0.01; *** = p<0.001).</p

Co-expression of synphilin-1 increases α-Syn S129-phosphorylation.

<p>A: Phosphorylation of α-Syn at S129 in the BY4741 wild-type strain when the expression of α-Syn was combined with an empty plasmid or constructs allowing for co-expression of SY^WT or SY^R621C as indicated. The panel on the left represents the average S129-phosphorylation as determined by immunodetection using a P-S129 specific monoclonal antibody, shown in the right panel, and quantified relative to intensity obtained for immunodetection with a polyclonal α-Syn antibody. B: The left panel shows the average number of H4 neuroglioma cells containing inclusions formed by α-Syn–EGFP when expressed alone or in combination with SY^WT as determined by fluorescence microscopic visualization, for which a representative picture is shown in the right panel. C: Phosphorylation of α-Syn at S129 in H4 neuroglioma cells as detected by immunodetection using a P-S129 specific monoclonal antibody and quantified relative to the intensity obtained for immunodetection with a polyclonal α-Syn antibody. The panel on the left represents the relative average phosphorylation, the panel on the right a corresponding Western blot analysis. All data represent the mean ± SEM of at least three independent experiments. Significance was assayed using a 1-way ANOVA (A) or t-test (B and C)(* = p<0.05; ** = p<0.01; *** = p<0.001).</p

Synphilin-1 forms aggresomes in cells approaching stationary phase.

<p>A: Fluorescence microscopy pictures of post-diauxic wild-type and <i>sir2Δ</i> cells with large inclusions formed by dsRed-SY^WT and stained with DAPI to visualize the nucleus. B: Fluorescence microscopy pictures of post-diauxic wild-type and <i>sir2Δ</i> cells expressing either EGFP or EGFP-SY^WT, as indicated, and stained with DHE to visualize ROS producing cells (left panels) or with PI to discriminate death cells (right panels).</p

Synphilin-1 induces inclusion formation of α-Syn in yeast.

<p>A: Fluorescence microscopic visualization and intracellular localization of the α-Syn–EGFP (upper panels), dsRed-SY^WT (middle panels) or dsRed-SY^R621C (lower panels) fusion proteins expressed separately in the BY4741 wild-type yeast strain. The panels display cells without (left) or with (right) aggregates. The percentages refer to the number of cells with or without inclusions in an exponential culture. Cells expressing native EGFP or dsRed served as controls and showed a dispersed cytoplasmic localization. B: Fluorescence microscopic visualization and intracellular localization of α-Syn–EGFP and dsRed-SY^WT upon combined expression in the BY4741 wild-type yeast strain. The upper panels display cells where both fusion proteins co-localize, the middle panels cells with intracellular inclusions of synphilin-1 and plasma membrane localized α-Syn, and the lower panels cells with peripheral inclusions of α-Syn and a dispersed cytoplasmic distribution of synphilin-1. C: Western blot analysis of strains transformed with an empty plasmid (Ctrl.) or a construct allowing the expression of dsRed-SY^WT or dsRed-SY^R621C. Immunodetection was performed using primary antibodies directed against synphilin-1 or Adh2 as indicated on the left. Molecular weight markers are indicated on the right. D and E: The percentage of cells containing inclusions of wild-type or mutant synphilin-1 (D) or α-Syn (E) in exponential cultures. Data represent the combined results of at least three independent experiments. Error bars represent the variation between different counts. Significance was assayed on the total amount of cells counted using a t-test (*** = p<0.001).</p

Transport of synphilin-1 inclusions along actin cables.

<p>A: Fluorescence microscopy images of late exponential <i>sir2Δ</i> cells expressing dsRed-SY^WT and α-Syn-EGFP showing that daughter cells inherit cytosolic synphilin-1 inclusions and plasma membrane associated α-Syn. B: Fluorescence microscopy images of late exponential wild-type cells expressing dsRed-SY^WT stained with Alexa Fluor 488 phalloidin to visualize actin patches and actin fibers and with Calcofluor to visualize the cell wall. Shown are the pictures obtained with the fluorescent proteins or dyes as well as the corresponding merges. C: Assessment of growth of wild-type cells with or without expression of native SY^WT or SY^R621C when plated on media supplemented with either Latranculin-B, Benomyl or the solvent DMSO.</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

Sir2 mediates synphilin-1 toxicity in yeast.

<p>A: Relative quantification of viable cells as determined by their ability to form colonies at different times after inoculation of the wild-type strain or the isogenic <i>sir2Δ</i> mutant transformed with empty plasmids or constructs allowing for expression of α-Syn or SY^WT, either alone or in combination as indicated. The number of viable cells in samples taken after 24 h of growth of the two strains transformed with the empty plasmids was set at 100%. B and C: Quantification of viable cells (B) and cells producing ROS (C) during chronological ageing of the wild-type strain transformed with an empty plasmid (□) or expressing SY^WT (▪) and the isogenic <i>sir2Δ</i> mutant transformed with an empty plasmid (○) or expressing SY^WT (•). All data represent mean ± SEM of six independent transformants. Significance of the data was determined by t-tests (* = p<0.05; ** = p<0.01; *** = p<0.001).</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

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