Fig. 4: Loss of Vac8 prevents PAS and vacuole recruitment of the PI3K complex.

a Model of hierarchical recruitment of selective PAS factors based on the literature. See also¹ for details. Circles containing numbers correspond to the respective Atg proteins. PE, phosphatidylethanolamine. b The indicated strains containing GFP-Atg8 were grown to mid-log phase. The percentage of cells with GFP-Atg8 puncta was analyzed in three independent biological replicates. For each condition and replicate at least 100 cells were analyzed. The values of each replicate (circle) and the mean (bars) were plotted. Statistical analysis using two-tailed unpaired t-tests. Significance is indicated with asterisks: ***p < 0.001, **p < 0.01, *p < 0.05, n.s. (not significant) p > 0.05. Exact numerical values are reported in the source data. See Supplementary Fig. 5a for representative fluorescence microscopy images. WT wild-type. c The indicated strains containing Atg14-3xGFP were grown to mid-log phase. The percentage of cells with Atg14-3xGFP puncta was analyzed in three independent biological replicates. For each condition and replicate at least 100 cells were analyzed. The values of each replicate (circle) and the mean (bars) were plotted. Statistical analysis using two-tailed unpaired t-tests. Significance is indicated with asterisks: ***p < 0.001, **p < 0.01, *p < 0.05, n.s. (not significant) p > 0.05. Exact numerical values are reported in the source data. See Supplementary Fig. 5b for representative fluorescence microscopy images. d The indicated Atg9-GFP strains containing mTagBFP2-Ape1 were grown to mid-log phase. The percentage of GFP positive BFP-Ape1 puncta was analyzed in three independent biological replicates. For each condition and replicate at least 60 BFP puncta were analyzed. The values of each replicate (circle) and the mean (bars) were plotted. Statistical analysis using two-tailed unpaired t-tests. Significance is indicated with asterisks: ***p < 0.001, **p < 0.01, *p < 0.05, n.s. (not significant) p > 0.05. Exact numerical values are reported in the source data. See Supplementary Fig. 5e for representative fluorescence microscopy images. e Vacuolar localization of Atg14-3xGFP was monitored in atg8∆ atg19∆ or atg8∆ vac8∆ cells. Vacuoles were stained with FM4–64. Cells were grown to mid-log phase. Representative fluorescence microscopy images of one out of two independent experiments are shown. Dashed lines indicate the contour of individual cells (see also Supplementary Fig. 5f). f The indicated Atg14-3xGFP strains were subjected to cell lyses and subsequent fractionation. Supernatant and pellet fractions were separated at 20,000 × g, and the distribution of Atg14-3xGFP in different fractions was monitored by anti-GFP western blotting. Wild-type cells without GFP-tagged Atg14 were used as a control. Pgk1 was used as a cytosolic marker, Tom20 was used as membrane marker. One representative experiment out of two independent experiments is shown. In, input; S, supernatant; P, pellet fraction. g Vacuolar localization of Vps15-GFP was monitored in Vph1-4xmCherry wild-type and vac8∆ cells. Cells were grown to mid-log phase. Representative fluorescence microscopy images of one out of two independent experiments are shown. Dashed lines indicate the contour of individual cells (see also Supplementary Fig. 5g). h The indicated Vps15-GFP, Vps34-GFP or wild-type strains containing Atg14-TAP or an empty plasmid were grown to mid-log phase. Cell extracts were subjected to GFP immunoprecipitation using GFP-trap affinity resin, and the amount of precipitated protein and coprecipitated Atg14-TAP was analyzed by anti-GFP and anti-protein A western blotting. One representative experiment out of three independent experiments is shown.