Figure S1.
Properties of SnxA-GFP and Atg18-mNG as in vivo PI(3,5)P2sensors. (A) SnxA-GFP expression levels in different strains. A representative immunoblot is shown in the bottom. Tub1 was used as a loading control. The graph on the top of each band shows the average of anti-GFP to anti-Tub1 band intensity ratios from four experiments. Error bars: standard error of the mean. Strains used: MHY286, MHY288, MHY293, MHY304, MHY305, MHY303, MHY302, and AAY007. (B) Box-and-whisker graph and representative images of vacuole diameters in wildtype (WT), vac7∆, and SnxA-GFP containing yeast cells (BBY070, MHY286, and SEY256). Data shown come from three independent experiments. Boxes show 25th and 75th percentiles, whiskers indicate 10th and 90th percentiles, and the line in the boxes represents the median. Ordinary one-way ANOVA was performed with Fisher’s least significant difference (LSD) test. ****P < 0.0001, ns: non-significant with P = 0.7079. Scale bars: 3 μm. BF: bright field. (C) Spot assays that show growth of cells with (+SnxA-GFP) or without SnxA-GFP. Serial dilutions of indicated strains (ESM356-1 and MHY286) were spotted on YPD plates and incubated at indicated temperatures. (D–F) Vacuolar localization patterns of SnxA. SnxA-GFP is shown in cyan, Vph1-3xmCherry is shown in magenta. (G) Analysis Atg18-mNG colocalization with Vph1-3xmCherry upon hyperosmotic shock. Atg18, Atg8-Sloop mutant, and Atg18-FGGG mutant C-terminally fused with mNG were analyzed (MUK089, MUK090, and MUK091). Atg18-FGGG mutant served as a negative control for vacuole periphery localization. The PCC value of 0.5, indicated with a red dashed line, was selected as a threshold for vacuole membrane localization based on the PCC data of Atg18-FGGG. Data come from two independent experiments. (H) Atg18-mNG vacuole localization reflects PI(3,5)P2 levels. PI(3,5)P2 levels were manipulated through the use of mutants that are deficient in PI(3,5)P2 (vac7∆ and vac14∆), as well as through the use of fab1-ha and 0.9 M NaCl treatment in combination with wildtype and vac7∆ and vac14∆ mutants. PCC values of mNG and Vph1-3xmCherry colocalization were plotted, and representative images were also shown. Analyzed strains: MUK089, MUK098, MUK081, MUK113, MUK080, and MUK110. 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. In G and H, ordinary one-way ANOVA was performed with Fisher’s LSD test. ****P < 0.0001, and ns: non-significant with P = 0.9908. Scale bars are 3 μm. Source data are available for this figure: SourceData FS1.

Properties of SnxA-GFP and Atg18-mNG as in vivo PI(3,5)P 2 sensors. (A) SnxA-GFP expression levels in different strains. A representative immunoblot is shown in the bottom. Tub1 was used as a loading control. The graph on the top of each band shows the average of anti-GFP to anti-Tub1 band intensity ratios from four experiments. Error bars: standard error of the mean. Strains used: MHY286, MHY288, MHY293, MHY304, MHY305, MHY303, MHY302, and AAY007. (B) Box-and-whisker graph and representative images of vacuole diameters in wildtype (WT), vac7∆, and SnxA-GFP containing yeast cells (BBY070, MHY286, and SEY256). Data shown come from three independent experiments. Boxes show 25th and 75th percentiles, whiskers indicate 10th and 90th percentiles, and the line in the boxes represents the median. Ordinary one-way ANOVA was performed with Fisher’s least significant difference (LSD) test. ****P < 0.0001, ns: non-significant with P = 0.7079. Scale bars: 3 μm. BF: bright field. (C) Spot assays that show growth of cells with (+SnxA-GFP) or without SnxA-GFP. Serial dilutions of indicated strains (ESM356-1 and MHY286) were spotted on YPD plates and incubated at indicated temperatures. (D–F) Vacuolar localization patterns of SnxA. SnxA-GFP is shown in cyan, Vph1-3xmCherry is shown in magenta. (G) Analysis Atg18-mNG colocalization with Vph1-3xmCherry upon hyperosmotic shock. Atg18, Atg8-Sloop mutant, and Atg18-FGGG mutant C-terminally fused with mNG were analyzed (MUK089, MUK090, and MUK091). Atg18-FGGG mutant served as a negative control for vacuole periphery localization. The PCC value of 0.5, indicated with a red dashed line, was selected as a threshold for vacuole membrane localization based on the PCC data of Atg18-FGGG. Data come from two independent experiments. (H) Atg18-mNG vacuole localization reflects PI(3,5)P2 levels. PI(3,5)P2 levels were manipulated through the use of mutants that are deficient in PI(3,5)P2 (vac7∆ and vac14∆), as well as through the use of fab1-ha and 0.9 M NaCl treatment in combination with wildtype and vac7∆ and vac14∆ mutants. PCC values of mNG and Vph1-3xmCherry colocalization were plotted, and representative images were also shown. Analyzed strains: MUK089, MUK098, MUK081, MUK113, MUK080, and MUK110. 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. In G and H, ordinary one-way ANOVA was performed with Fisher’s LSD test. ****P < 0.0001, and ns: non-significant with P = 0.9908. Scale bars are 3 μm. Source data are available for this figure: SourceData FS1.

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