Dissociation of Atg2, Atg5, Atg11, and Atg17 from accumulated APs is defective in snf7Δ and vps4Δ mutant cells. (A–D) Endogenous Atg2 (A), Atg5 (B), Atg11 (C), and Atg17 (D) were tagged with GFP at their C terminus and Atg8 was tagged with mCherry at its N terminus in WT and mutant cells. Cells were grown in YPD medium and autophagy was induced by starvation (SD-N for starvation for 2 h; WT for 30 min). The colocalization of AtgX-GFP with mCherry-Atg8 was monitored using live-cell fluorescence microscopy (bottom, number of red puncta used for quantification). Arrows indicate AtgX-Atg8 colocalization; arrowheads indicate Atg8 puncta that do not colocalize with AtgX. Scale bar, 2 µm. (E) Quantification for AtgX-Atg8 colocalization (%) from A–D. In WT cells, a single dot per cell contains AtgX and Atg8 and represents a pre-autophagosomal structure or APs. More than 200 mCherry-Atg8 dots per strain were examined. Columns represent mean and error bars represent SD. Results represent three independent experiments. **P < 0.01.