(D) Effect of Atg1-Atg13 subcomplex on autophagosome-vacuole fusion in vitro. The vam3∆ strain carrying pRS413 plasmid encoding GAL1pr-YKT6 (wild-type or mutant), and expressing Atg9-3xFLAG and GFP-Atg8, were starved for 3 h at 30°C before purifying autophagosomes. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes in the presence or absence of 50 nM Atg1-Atg13 subcomplex at 26°C for 1 h with or without ATP (E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified. 