Figure 4. The PI3-kinase complex I is... | Rockefeller University Press

Figure 4.

$Figure 4. The PI3-kinase complex I is required for the recruitment of Ypt7 to autophagosomes to promote autophagosome–vacuole fusion. (A) Fusion assay as in Fig. 2 D. Cytosol was prepared from the indicated deletion strains. (B) Fusion assay as in Fig. 2 D. Cytosol was prepared from atg14Δ or vps34Δ cells or from cells overexpressing the FYVE domain. (C) Fusion assay as in Fig. 2 D. Fusion reactions were either mock treated or incubated without an ATP regeneration system or with 10 mM GDP. (D) Fusion assay as in Fig. 2 D. Cytosol and autophagosomes were prepared from the indicated deletion strains. The FYVE domain was overexpressed where indicated. (E) Autophagosomes were isolated from the indicated strains as in Fig. 2 A. The presence of Ypt7 in the autophagosome-enriched pellet was monitored by Western blotting. One representative experiment out of three is shown. (F) Autophagosomes were isolated from the indicated strains as in Fig. 2 A, incubated with cytosolic fractions as indicated, and again isolated by centrifugation. The binding of Ypt7 to the autophagosomes was assessed by Western blotting. One representative experiment out of three is shown. Molecular masses are given in kilodaltons. (G) Fusion assay as in Fig. 2 D. Cytosol, autophagosomes, and vacuoles were prepared from the indicated deletion or ts strains at 24°C. Fusion reactions were incubated at the restrictive temperature (30°C) for 2 h. All graphs show the mean from at least three independent experiments. Error bars are SD.$

The PI3-kinase complex I is required for the recruitment of Ypt7 to autophagosomes to promote autophagosome–vacuole fusion. (A) Fusion assay as in Fig. 2 D. Cytosol was prepared from the indicated deletion strains. (B) Fusion assay as in Fig. 2 D. Cytosol was prepared from atg14Δ or vps34Δ cells or from cells overexpressing the FYVE domain. (C) Fusion assay as in Fig. 2 D. Fusion reactions were either mock treated or incubated without an ATP regeneration system or with 10 mM GDP. (D) Fusion assay as in Fig. 2 D. Cytosol and autophagosomes were prepared from the indicated deletion strains. The FYVE domain was overexpressed where indicated. (E) Autophagosomes were isolated from the indicated strains as in Fig. 2 A. The presence of Ypt7 in the autophagosome-enriched pellet was monitored by Western blotting. One representative experiment out of three is shown. (F) Autophagosomes were isolated from the indicated strains as in Fig. 2 A, incubated with cytosolic fractions as indicated, and again isolated by centrifugation. The binding of Ypt7 to the autophagosomes was assessed by Western blotting. One representative experiment out of three is shown. Molecular masses are given in kilodaltons. (G) 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.