The R-SNARE Ykt6 acts on the autophagosome to promote autophagosome–vacuole fusion. (A) Left: The indicated strains expressing GFP-Atg8 were grown to logarithmic phase (rich) and were subsequently starved for 4 h (SD-N). TCA extracts were prepared, and GFP-Atg8 cleavage was monitored by anti-GFP Western blotting. One representative experiment out of three is shown. Right: Pho8Δ60 assay of nyv1Δ cells. Indicated cells were grown to mid–log phase and starved for 4 h where indicated. Pho8Δ60-specific alkaline phosphatase activity was measured in three independent experiments, and the mean was plotted normalized to starved pho8Δ60 alkaline phosphatase activity. (B and C) Left: The indicated strains expressing GFP-Atg8 were grown at 24°C to logarithmic phase. Cultures were split and incubated for 4 h at the permissive (24°C) or restrictive (37°C) temperature, with (SD-N) or without starvation (rich) as indicated. TCA extracts were prepared, and GFP-Atg8 cleavage was monitored by anti-GFP Western blotting. One representative experiment out of three is shown. Right: Pho8Δ60 assay. The indicated strains were grown at 24°C to logarithmic phase. Cultures were split and incubated for 4 h at the permissive (24°C) or restrictive (37°C) temperature, with (SD-N) or without starvation (rich) as indicated. Pho8Δ60-specific alkaline phosphatase activity was measured in three independent experiments, and the mean was plotted normalized to starved pho8Δ60 alkaline phosphatase activity at the respective temperature. Molecular masses are given in kilodaltons. (D and E) Fusion assay as in Fig. 2 D. Cytosol, autophagosomes, and vacuoles were prepared from the indicated deletion or ts strains at 24°C. Fusion reactions were incubated at the restrictive temperature (30°C) for 2 h. All graphs show the mean from at least three independent experiments. Error bars are SD.