snf7Δand vps4Δ ESCRT mutant cells exhibit selective and general autophagy defects. (A) ESCRT mutants exhibit a complete block in Ape1 processing during normal growth. WT and mutant cells were grown in rich medium (YPD) to mid-log phase. Processing of pre-Ape1 (prApe1) to mature Ape1 (mApe1) was determined in cell lysates using immunoblot analysis with anti-Ape1; percentage of processed cargo in each lane is shown under the blot. (B and C) ESCRT mutants exhibit a severe loss of viability under nitrogen starvation. WT and mutant cells grown in YPD to mid-log phase were shifted to SD-N. Cell viability, shown as percentage of cell viability at day zero, was tested by trypan blue staining (B) and ability to form colonies on YPD plates (C) at time zero and after the indicated number of days in SD-N. (D) ESCRT mutant cells are defective in general autophagy measured by the Pho8Δ60 alkaline phosphatase assay. Alkaline phosphatase activity was determined in lysates of WT and mutant cells grown in YDP or SD-N (4 h). (E) ESCRT mutants exhibit a partial defect in GFP-Atg8 (top) and Ape1 (bottom) processing under starvation. WT and mutant cells expressing GFP-Atg8 were grown in YPD and shifted to starvation medium (SD-N for 2 h). Processing was determined in cell lysates by immunoblot analysis using anti-GFP and anti-Ape1 antibodies, respectively; percentage of processed cargo in each lane (GFP and mApe1) is shown under the blot. (F) ESCRT mutants are partially defective in GFP-Atg8 and Ape1 processing during rapamycin-induced autophagy. WT and mutant cells were grown to mid-log phase in YPD, and autophagy was induced by the addition of rapamycin (10 ng/ml for 4 h). GFP-Atg8 (top) and Ape1 (bottom) processing was determined and presented as in E. Error bars and +/− represent SD. Results represent three independent experiments. **P < 0.01; ***P < 0.001.