Autophagy cargos that accumulate in single and double ESCRT mutant cells are completely sensitive to protease. (A and B) SNF7 is epistatic to YPT7. (C and D) VPS4 is epistatic to YPT7. (E and F) VPS4 is epistatic to PEP4. Lysates of WT and single- and double-mutant cells expressing GFP-Atg8 were subjected to the conventional protease (PK)-protection assay combined with immunoblot analysis (see Materials and methods). Levels of Ape1 (A, C, and E; black bars) and GFP-Atg8 (B, D, and F; gray bars) were determined before (0) and after addition of protease (PK), with or without addition of detergent (TX). Protease protection of cargos can be seen when PK is added to membranes without detergent (middle of each lane triplet). Nondegraded cargos: prApe1 and GFP-Atg8; degraded cargos: mApe1 and GFP. Bottom: Bar graph showing percentage of nondegraded cargos; columns represent mean and error bars represent SD. Results represent three independent experiments. ***P < 0.001.