Figure 4. Bulk autophagosome formation under nutrient-rich conditions is facilitated by Atg11.

(A) atg13∆ pho8∆60, atg13∆ atg17∆ pho8∆60, atg11∆ atg13∆ pho8∆60, and atg13∆ atg19∆ pho8∆60 cells containing plasmid expressed Atg13^wt or Atg13^44A were grown in log phase and treated with hydroxyurea (HU) for 4 h. Pho8∆60 alkaline phosphatase activity was measured in three independent experiments. The values of each replicate (circles) and mean (bars) were plotted. All values were normalized to the mean Pho8Δ60 alkaline phosphatase activity of cells expressing Atg13^44A. (B) atg13∆ pho8∆60 cells containing plasmid expressed Atg13^wt, Atg13^44A, or an empty plasmid were grown to log phase, and cell extracts were prepared by TCA precipitation. Samples were analyzed by anti-Ape1 western blotting. One out of two independent biological replicates is shown. (C) Atg1-protA Atg17-myc atg13∆ cells containing plasmid expressed Atg13^wt, Atg13^46A, Atg13^46D, Atg13^44A, Atg13^44D or an empty plasmid were grown in log phase. Atg1-protA was immunoprecipitated using IgG beads, and immunoprecipitates and input extracts were analyzed by anti-protein A (PAP), anti-Atg13, anti-myc, and anti-Atg11 western blotting. Asterisk: non-specific band. One out of four independent biological replicates is shown. (D) Atg13^wt, atg13∆ and Atg13^44A cells were starved for 0 or 14 days and spotted in serial dilutions onto YPD plates. One representative experiment out of three is shown. (E) Atg13^wt pho8∆60, Atg13^44A pho8∆60, and atg13∆ pho8∆60 cells were grown in log phase and starved for 4 h, 8 h, 12 h, and 24 h as indicated. Pho8∆60 alkaline phosphatase activity was measured in three independent experiments. The values of each replicate (circles) and mean (bars) were plotted. All values were normalized to the mean Pho8Δ60 alkaline phosphatase activity of Atg13^wt pho8∆60 cells after 4 h of starvation. Source data are available online for this figure.