Reconstitution of the initial step of starvation-specific PAS assembly. (A) Localization of Atg17-GFP in the wild-type (WT) and MKO (ATG17-GFP) strains. The WT (ATG17-GFP) strain; the MKO (ATG17-GFP) strain transformed with vector (pRS415), a plasmid expressing Atg1 (pATG1(415)), Atg13 (pATG13(415)), or both Atg1 and Atg13 (pATG1-ATG13(415)); and the MKO (ATG11 ATG17-GFP) strain transformed with a plasmid expressing Atg1 or Atg13 were grown in selective SMD medium to mid-log phase and shifted to SD-N for 2 h for starvation. Samples were taken from both growing (mid-log phase) and starvation conditions for fluorescence microscopy analyses. Atg17-GFP displayed a punctate structure in WT cells in both growing and starvation conditions; some cells had more than one punctum in starvation conditions. In the MKO (ATG17-GFP) strain, Atg17 was cytosolic in both conditions; its localization pattern changed to punctate structures in starvation only when Atg1 and Atg13 were coexpressed. Coexpression of Atg1 and Atg11 or Atg13 and Atg11 could not redistribute Atg17 from the cytosol to punctate structures. Representative images are shown. DIC, differential interference contrast. (B) Perivacuolar localization of Atg17-GFP in the MKO (ATG17-GFP) strain. The MKO (ATG17-GFP) strain transformed with a plasmid expressing both Atg1 and Atg13 (pATG1-ATG13(415)) was grown to mid-log phase, stained with the vacuolar dye FM 4–64 as described in Materials and methods, and shifted to SD-N for 2 h before imaging. When both Atg1 and Atg13 were expressed, Atg17-GFP puncta localized at perivacuolar sites under starvation conditions. Bars, 2.5 μm. (C) The number of cells that contained Atg17-GFP PAS puncta in MKO strains (HCY107, HCY113, and HCY151) with coexpression of various combinations of Atg proteins was quantified under vegetative (open bars) and starvation (closed bars) conditions. Approximately 100–250 cells for each strain were analyzed for scoring the percentage of cells with fluorescent PAS puncta.