pH calibration curves and the effect of carbon source, amino acids, and Pho85 on SnxA localization. (A–C) pH sensitivity of v-SEP (A), mCherry (B), and pH calibration curve for v-SEP/mCherry (C). Pink shaded areas are where v-SEP intensity and v-SEP/mCherry ratios linearly correlate with pH. Green shaded area in C marks the v-SEP/mCherry ratios within the linear range. Data come from two independent experiments. In each experiment, at least 100 vacuoles were quantified per different pH. Error bars are standard deviation. Fitted lines were obtained with a simple linear regression model in prism. (D–F) Analysis of SnxA-GFP localization at the vacuole periphery with respect to the carbon source (D), amino acid supplementation (E), and the presence of Pho85 (F). Representative still images were shown and PCC values were plotted. Data come from three independent experiments. In box-and-whiskers graphs boxes show the 25th and 75th percentiles, whiskers point to the 10th and 90th percentile, and the line in the boxes represents the median. Ordinary one-way ANOVA was performed with Fisher’s LSD test. ****P < 0.0001. Scale bars are 3 μm. DV: daughter vacuole, MV: mother vacuole.