Atg17 and Snf7 colocalize in a Vps21-dependent manner and forcing their interaction bypasses the requirement for Vps21 GTPase in autophagy. (A and B) Atg17 and Snf7 colocalize in a Vps21-dependent manner. WT and mutant cells expressing Atg17-GFP and Snf7-mCherry were grown to mid-log phase, shifted to SD-N for 30 min, and analyzed by live-cell fluorescence microscopy (A; right, number of green dots used for quantification in B). (C) Coexpression of Atg17-GFP and Snf7-GBP-mCherry results in their colocalization even in vps21Δ mutant cells. WT and vps21Δ mutant cells expressing from their endogenous loci Atg17-GFP (left), Snf7-GBP-mCherry (middle), or both (right) were grown, starved, and visualized as described for A. Bar graph showing percentage of red dots colocalizing with green dots in WT and vps21Δ mutant cells (bottom). (D) Forcing the Atg17–Snf7 interaction using GFP and GBP suppresses the autophagy defect of vps21Δ mutant cells. Processing of Ape1 in lysates from cells used in C was determined by immunoblot analysis; bar graph shows the percentage of processed mApe1 in the different cells. (A and C) Scale bar, 5 µm; arrows point to colocalizing puncta. (B and D) Bars represent mean ± SD of each variable from three independent experiments. Results represent three independent experiments. (E) Diagram of Rab5 GTPase-regulated and ESCRT III–catalyzed AP closure. We propose that a Rab5 GTPase module, which includes a GEF and downstream effectors (e.g., Vps8 and Pep12), controls the recruitment of ESCRT to unsealed APs through an interaction of the ESCRT subunit Snf7 with Atg17 on open APs. ESCRT machinery then catalyzes AP closure followed by Atgs dissociation (including Atg17) to yield mature APs, which, in turn, fuse with the lysosome in a Ypt7-regulated manner. Following fusion, ABs are deposited to the lysosomal lumen and autophagy cargos can be degraded by proteases (e.g., Pep4). n.s., not significant; ***P < 0.001.