(A-D) Yeast cells were grown in SMD to mid-log phase and nitrogen starved for the indicated times. (A) WLY176 atg5∆ yeast cells expressed GFP-Atg8 through its endogenous promoter and plasmid-based Atg5WT-PA, Atg5E141D-PA or an empty vector. Protein extracts were analyzed for GFP-Atg8 processing by western blot. The ratio of free GFP to Dpm1 (loading control) is presented below the blots, and quantification is presented on the right (Student’s t test, n=4; *p < 0.05); the value for Atg5WT at 6 hr was set to 1.0 and other values were normalized. (B) WLY176 atg5∆ yeast cells expressed either plasmid-based Atg5WT-PA, Atg5E141D-PA or an empty vector. Protein extracts were used to measure autophagy through the Pho8Δ60 assay (Student’s t test, n=6; *p < 0.05). (C) WLY176 cells with genomic integrated Atg5WT or Atg5E141D were used to generate protein extracts and autophagy was monitored through the Pho8Δ60 assay (Student’s t test, n=3; *p < 0.05). (D) WLY176 atg5∆ yeast cells expressing plasmid-based Atg5WT-PA, Atg5E141D-PA or an empty vector were used to generate protein extracts. The ratio of Atg8–PE to total Atg8 is presented below the blots based on western blot using antiserum to Atg8. Dpm1 was used as a loading control. (E) MKO ATG3 (YCY137) cells were co-transformed with pATG8∆R-ATG7-ATG10(414), and either pATG5WT-HA-ATG12(416), pATG5E141D-HA-ATG12(416), pATG5WT-HA-ATG12-ATG16(416), or pATG5E141D-HA-ATG12-ATG16(416). Overnight cultures were diluted to OD=0.02 in SMD -Ura -Trp. The cells were incubated at 30°C for 18 hr to mid-log phase before they were shifted to SD-N for nitrogen starvation. Samples at the corresponding time points were collected, TCA precipitated and subsequently analyzed by western blot. S.E., short exposure; L.E., long exposure.