Figure 6. Atg11 contributes to PAS formation and bulk autophagy function.

(A) atg1∆ pho8∆60 or atg1∆ atg13∆ pho8∆60 cells containing plasmid expressed Atg1, Atg1-13^wt, Atg1-13^MD, Atg1-13^44D or an empty plasmid were starved 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 Atg1. The mutants in this experiment were analyzed in parallel with the mutants shown in Fig. 5E. Therefore, the data for the control samples and the Atg1-13^wt fusion are the same. (B) atg1∆ atg13∆ pho8∆60 or atg1∆ atg11∆ atg13∆ pho8∆60 cells containing plasmid expressed Atg1, Atg1-13^wt, Atg1-13^MD, Atg1-13^44D or an empty plasmid were starved 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 Atg1-13^wt. Dashed rectangles represent the mean Pho8Δ60 alkaline phosphatase activity of the respective mutant measured in atg1∆ ATG11 atg13∆ pho8∆60 cells shown in panel (A). (C) Wild-type (wt), atg11∆ and atg19∆ cells containing an empty plasmid and atg1∆ atg13∆, atg1∆ atg11∆ atg13∆, and atg1∆ atg13∆ atg19∆ cells containing plasmid expressed Atg1-13^wt, Atg1-13^MD, and Atg1-13^44D were grown to log phase and starved for 4 h, and cell extracts were prepared by TCA precipitation. Ape 1 processing was analyzed by anti-Ape1 western blotting. One out of two independent biological replicates is shown. (D) atg1∆ atg13∆ atg19∆ or atg1∆ atg11∆ atg13∆ atg19∆ cells containing plasmid expressed Atg1-13^wt-GFP, Atg1-GFP, Atg1-13^MD-GFP, or Atg1-13^44D-GFP were starved for 30 min. The percentage of cells with GFP puncta was quantified in three independent experiments. For each strain and replicate at least 100 cells were analyzed. Scale bar: 2 µm. (E) atg13∆ atg19∆ or atg1∆ atg13∆ atg19∆ cells containing plasmid expressed Atg13^wt-GFP, Atg1-13^wt-GFP, Atg1-13^MD-GFP or Atg1-13^44D-GFP were starved for 30 min and treated with 1,6-hexanediol or mock treated with digitonin for 3 min, and analyzed by fluorescence microscopy. The percentage of cells with GFP puncta was quantified in three or four independent experiments. For each strain and replicate at least 100 cells were analyzed. Scale bar: 2 µm. Source data are available online for this figure.