snf7Δand vps4Δ ESCRT mutant cells accumulate GFP-Atg8 clusters under nitrogen starvation. (A–C) Accumulation of Atg8 clusters and class E compartment in snf7Δ and vps4Δ single-mutant cells and in combination with the atg1Δ mutation. (A) Cells expressing GFP-Atg8 and the class E compartment marker 3×tagBFP-Pho8 were grown in YPD (top) and shifted to SD-N (bottom) as in Fig. 1 E. FM4-64 was used to label the vacuolar membrane before cells were visualized by live-cell microscopy (right, number of cells used for quantification in B and C). Arrowheads point to class E compartments; arrows indicate GFP-Atg8 clusters. Scale bar, 2 µm. (B) Atg8 clusters accumulate in snf7Δ and vps4Δ single-mutant cells only under starvation and not when combined with the atg1Δ mutation. Results from A were quantified and the bar graph shows percentage of cells with an Atg8 cluster. (C) Class E compartment accumulates in snf7Δ and vps4Δ single-mutant cells regardless of the growth conditions and the atg1Δ mutation. Results from A were quantified and percentage of cells with class E compartment is shown. (D and E) Epistasis analysis of GFP-Atg8 accumulation in single and double vps4Δ and pep4Δ mutant cells under nitrogen starvation. (D) Cells expressing GFP-Atg8 were grown in SD-N and FM4-64 was used to label the vacuolar membrane before visualization by live-cell microscopy (right, number of cells used for quantification in E). Arrows point to GFP-Atg8 clusters. Scale bar, 2 µm. (E) Whereas WT and pep4Δ mutant cells accumulate GFP-Atg8 inside the vacuole, vps4Δ and vps4Δ pep4Δ mutant cells accumulate GFP-Atg8 clusters outside the vacuole. Results from D were quantified, and percentage of cells with an Atg8 (AP) cluster outside the vacuole (black bars) or as ABs inside the vacuole (gray bar) are shown. In B–D: >800 cells per strain were counted; columns represent mean, error bars represent SD. Results in this figure represent three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.