Figure 2. Reconstitution of... | Rockefeller University Press

Figure 2.

$Figure 2. Reconstitution of autophagosome–vacuole fusion in vitro. (A) GFP-Atg8 ypt7Δ cells were starved for 16 h. After cell lysis, autophagosomes were enriched in a 20,000 g pellet and analyzed by anti-Atg1, anti-Ape1, anti-Pgk1, and anti-GFP Western blotting. atg1Δypt7Δ cells that cannot form autophagosomes served as a control. One representative experiment out of three is shown. (B) Samples from A were subjected to proteinase K (ProtK) and Triton X-100 (TX100) treatment as indicated and analyzed by anti-Atg1, anti-Ape1, and anti-GFP Western blotting. Ape1*, proteinase K–resistant fragment of Ape1. One representative experiment out of three is shown. Molecular masses are given in kilodaltons. (C) Samples from A were subjected to proteinase K and Triton X-100 treatment as indicated and analyzed by fluorescence microscopy. Arrows point to autophagosomes. One representative experiment out of three is shown. Bar, 10 µm. (D) The autophagosomal, vacuolar, and cytosolic fractions from Figs. 1 (C and D) and 2 A were coincubated together with an energy regeneration system for 2 h. Fusion was monitored by fluorescence microscopy and judged by the appearance of a mobile green dot in the vacuole. Shown are stills of a 12-s time-lapse video (Video 1). Bar, 0.5 µm.$

Reconstitution of autophagosome–vacuole fusion in vitro. (A) GFP-Atg8 ypt7Δ cells were starved for 16 h. After cell lysis, autophagosomes were enriched in a 20,000 g pellet and analyzed by anti-Atg1, anti-Ape1, anti-Pgk1, and anti-GFP Western blotting. atg1Δypt7Δ cells that cannot form autophagosomes served as a control. One representative experiment out of three is shown. (B) Samples from A were subjected to proteinase K (ProtK) and Triton X-100 (TX100) treatment as indicated and analyzed by anti-Atg1, anti-Ape1, and anti-GFP Western blotting. Ape1*, proteinase K–resistant fragment of Ape1. One representative experiment out of three is shown. Molecular masses are given in kilodaltons. (C) Samples from A were subjected to proteinase K and Triton X-100 treatment as indicated and analyzed by fluorescence microscopy. Arrows point to autophagosomes. One representative experiment out of three is shown. Bar, 10 µm. (D) The autophagosomal, vacuolar, and cytosolic fractions from Figs. 1 (C and D) and 2 A were coincubated together with an energy regeneration system for 2 h. Fusion was monitored by fluorescence microscopy and judged by the appearance of a mobile green dot in the vacuole. Shown are stills of a 12-s time-lapse video (Video 1). Bar, 0.5 µm.