Binding of Vps4 to Snf7 and Vps2 is required for ILV neck constriction. (A) Schematic representation of ILV budding profiles as detected in tomographic reconstructions. (B) MVB of WT cells and MVB-like structure from snf7*, vps2* double mutants. The arrows indicate ILV necks. (C) Mean membrane neck diameters measured in the tomograms. Error bars indicate the SD of the membrane neck diameters measured in the tomograms. (D) Size distribution of individual membrane neck diameters of the WT and the indicated mutants. (E) Membrane fraction (M) and cytoplasmic fraction (C) of the indicated mutants were analyzed by SDS-PAGE and Western blotting. (F) 2D slices and 3D models from 400-nm sections of the indicated mutants overexpressing Vps21 (TDH3-VPS21). Limiting MVB membrane (yellow), ILVs (red), vacuolar membrane (brown), and class E compartments (green) are shown. Bars, 150 nm. ***, P < 0.001.