Figure 3.

Autophagosome–vacuole fusion depends on time, temperature, and cytosol concentration and requires energy. (A) Vacuoles containing or lacking Atg15 as indicated were analyzed in an in vitro fusion assay as described in Fig. 2 D. (B) Samples as described in Fig. 2 D were incubated for the time indicated, and autophagosome–vacuole fusion was quantified. (C) Fusion reactions were performed in the presence and absence of an ATP regeneration system and with or without the prior incubation of the cytosolic fraction for 5 min at 95°C (boil) or RNase treatment. Dash indicates that no cytosol was added. (D) The amount of cytosol added to the fusion reaction was titrated as indicated. (E) Fusion reactions were performed at different temperatures as indicated. (F) Comparison of a 20,000 g and a 100,000 g cytosolic supernatant in the fusion reaction. (G) Comparison of cytosol prepared from logarithmically growing cells (rich) with cytosol from cells that had been treated with 220 nm rapamycin for 4 h before harvesting (rapa). All graphs show the mean from at least three independent experiments. Error bars are SD.