Binding of Vps4 to ESCRT-III is mainly mediated by Snf7 and Vps2. (A) The schematic representation shows ESCRT-III, the Snf7 homooligomer (blue dots), the MIM2 of Vps20 (green square), the MIM2 of Snf7 (blue square), the MIM1 of Vps24 (violet circle), the MIM1 of Vps2 (red circle), and its interaction with the MIT domain (green) of Vps4. N, N terminus; C, C terminus. (B, C, E, and F) Experiments were analyzed by SDS-PAGE and Western blotting. (B) Total cell lysates from WT cells and the vps20*, snf7*, vps24*, vps2* quadruple mutant. (C) Immunoprecipitation (IP) of Vps4-HA from cell lysates of WT cells and the indicated mutants. #, unspecific signal. (D) Quantification (MaxQuant) of mixed Vps4-HA immunoprecipitates from WT cells (labeled with [¹³C₆/¹⁵N₂] l-lysine) and from vps2* mutants. Heavy (H) to light (L) ratios of the ESCRT-III proteins and Vps4. Heavy/light = 1, equal peptides from WT (heavy) and vps2* (light); heavy/light > 1, more peptides from the WT (heavy). (E) Membrane fraction (M) and cytoplasmic fraction (C) of WT cells and the indicated mutants expressing Vps4-E233Q. (F) Immunoprecipitation of Vps4-E233Q-HA from cell lysates of the indicated MIM mutants. IN, input; n.a., not annotated.