The interaction of Vps4 with Snf7 and Vps2 is essential for efficient ESCRT-III disassembly and MVB sorting. (A–C) Experiments were analyzed by SDS-PAGE and Western blotting. (A) Solubilized membrane fractions (13,000 g pellet) of WT cells and the indicated MIM mutants were subjected to velocity sedimentation. (B) Membrane fractions (M) and cytoplasmic fractions (C) of WT cells and the indicated mutants. (C) Semi–in vitro disassembly assay with membrane fractions isolated from vps4Δ mutants in combination with the indicated MIM mutations. Membrane fractions were incubated with ATP and 100 nM of recombinant Vps4 for 0, 30, or 60 s. Membrane-associated proteins (13,000 g pellet [P]) and released proteins (13,000 g supernatant [S]) were separated by centrifugation. (D) Live-cell fluorescence microscopy of WT cells and the indicated mutants expressing GFP-CPS, FM4-64, vacuole (V), and the class E compartment (E). Bar, 5 µm. Schematic presentation of GFP-CPS–sorting phenotypes. DIC, differential interference contrast. (E) GFP-CPS–sorting phenotypes of all ESCRT-III MIM mutant combinations. (F) Quantification of the LUCID assay in WT cells and the indicated mutants. Ratios of the Sna3-FLuc reporter activity to the cytoplasmic Renilla luciferase (RLuc) activity normalized with SDs are shown; n = 6. (G) Dilution series of WT cells and the indicated mutants were grown on yeast nitrogen base plates at 26 or 37°C. **, P < 0.01; ***, P < 0.001.