Figure 5.
Vacuole acidity depends on Atg18 and its binding to PI3,5P2. (A) Representative still images and v-SEP/mCherry ratios of mother (MV) and daughter vacuoles (DV) in WT (MHY283) and atg18∆ (MHY277) cells. Data shown are from two independent experiments. (B) Analysis of vacuolar pH via time-lapse microscopy in atg18∆ cells (MHY277). Selected time points from the time series are shown. The graph represents the mean of v-SEP/mCherry ratios of mother and daughter vacuoles as a function of time. Data from individual cells are aligned with respect to the vacuole segregation. Error bars show the standard error of the mean. (C) Analysis of vacuolar pH in log-phase culture of WT (MHY283), atg18∆ (MHY277), Atg18-Sloop (MHY315), and Atg18-FGGG (MHY317) cells. Data come from two independent experiments. (D) Atg18-GFP vacuolar localization in cells with and without Vph1-GBP. Representative still images and the percentage of cells in which Atg18-GFP localizes on both vacuoles are shown (MHY223 and SEY082). Graph shows the mean of three independent experiments. Approximately 100 cells were counted per strain per experiment. Error bars: standard deviation. ****P < 0.0001 according to unpaired two-tailed t test. (E) Analysis of SnxA-mCherry vacuole periphery localization in cells carrying Vph1-GFP and Atg18-GFP Vph1-GBP (MHY285 and MHY297). (F) Effect of constitutive Atg18 targeting to both vacuoles (Atg18-GFP/GBP) on vacuolar pH (MHY283, MHY297, MHY291, and MHY312). Data shown come from two independent experiments. In box-and-whisker graphs, boxes show the 25th and 75th percentiles, whiskers indicate the 10th and 90th percentiles, and the line in the boxes represents the median. In A, C, E, and F, ordinary one-way ANOVA was performed with Fisher’s LSD test. ****P < 0.0001, **P ≤ 0.0093, ***P = 0.0004, and ns: non-significant with P ≥ 0.6580. Scale bars: 3 μm. BF: bright field. (G) Working model. PI(3,5)P2 accumulates on the vacuoles of the daughter cell while it is depleted from the vacuoles of the mother cell during mid-to-late mitosis. Concomitantly, the daughter cell’s vacuole becomes acidified and the acidity of the mother cell’s vacuole declines. Thus, the presence of PI(3,5)P2 in the daughter vacuole plays a role in the daughter cell’s rejuvenation, and the absence of PI(3,5)P2 from the mother vacuole leads to the mother cell’s aging.

Vacuole acidity depends on Atg18 and its binding to PI3,5P2. (A) Representative still images and v-SEP/mCherry ratios of mother (MV) and daughter vacuoles (DV) in WT (MHY283) and atg18∆ (MHY277) cells. Data shown are from two independent experiments. (B) Analysis of vacuolar pH via time-lapse microscopy in atg18∆ cells (MHY277). Selected time points from the time series are shown. The graph represents the mean of v-SEP/mCherry ratios of mother and daughter vacuoles as a function of time. Data from individual cells are aligned with respect to the vacuole segregation. Error bars show the standard error of the mean. (C) Analysis of vacuolar pH in log-phase culture of WT (MHY283), atg18∆ (MHY277), Atg18-Sloop (MHY315), and Atg18-FGGG (MHY317) cells. Data come from two independent experiments. (D) Atg18-GFP vacuolar localization in cells with and without Vph1-GBP. Representative still images and the percentage of cells in which Atg18-GFP localizes on both vacuoles are shown (MHY223 and SEY082). Graph shows the mean of three independent experiments. Approximately 100 cells were counted per strain per experiment. Error bars: standard deviation. ****P < 0.0001 according to unpaired two-tailed t test. (E) Analysis of SnxA-mCherry vacuole periphery localization in cells carrying Vph1-GFP and Atg18-GFP Vph1-GBP (MHY285 and MHY297). (F) Effect of constitutive Atg18 targeting to both vacuoles (Atg18-GFP/GBP) on vacuolar pH (MHY283, MHY297, MHY291, and MHY312). Data shown come from two independent experiments. In box-and-whisker graphs, boxes show the 25th and 75th percentiles, whiskers indicate the 10th and 90th percentiles, and the line in the boxes represents the median. In A, C, E, and F, ordinary one-way ANOVA was performed with Fisher’s LSD test. ****P < 0.0001, **P ≤ 0.0093, ***P = 0.0004, and ns: non-significant with P ≥ 0.6580. Scale bars: 3 μm. BF: bright field. (G) Working model. PI(3,5)P2 accumulates on the vacuoles of the daughter cell while it is depleted from the vacuoles of the mother cell during mid-to-late mitosis. Concomitantly, the daughter cell’s vacuole becomes acidified and the acidity of the mother cell’s vacuole declines. Thus, the presence of PI(3,5)P2 in the daughter vacuole plays a role in the daughter cell’s rejuvenation, and the absence of PI(3,5)P2 from the mother vacuole leads to the mother cell’s aging.

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