Slow and continuous ESCRT-III recycling and ILV biogenesis in snf7*, vps2* double mutants. (A) vps4-ts mutants, vps4Δ mutants and snf7*, vps2*, vps4-ts mutants were shifted to the nonpermissive temperature (37°C) for 4 h. 15 min before cells were shifted back to 26°C, 50 µg/ml cycloheximide was added. Cells were incubated for 2 and 4 h at 26°C. Subcellular fractionation was performed at the indicated time points. Cytoplasmic fractions were analyzed by SDS-PAGE and Western blotting. (B) Cells were incubated with cycloheximide (CHX) and labeled for 5 min with [³⁵S]methionine. Autoradiogram and Coomassie staining of cell lysates is shown. (C) Electron tomography of cryofixed snf7*, vps2*, vps4-ts mutants at the indicated temperatures and times. 2D slices from tomographic reconstructions and models from 400-nm sections are shown. Arrowheads point to enlarged budding profiles. Limiting MVB membrane (yellow), ILVs (red), vacuolar membrane (brown), nuclear envelope (blue), and class E–like structures (green) are depicted. Bars, 150 nm.