Rearranging the binding of Vps4 to the ESCRT-III complex. (A) Schematic representation of the ESCRT-III complex. (B) In vitro pull-down assay with ESCRT-III chimeras. Their domain organization is shown. GST alone or the indicated GST–ESCRT-III chimeras were immobilized on beads and incubated for 10 min at RT with Vps4-E233Q and 1 mM ATP. Bound fractions were analyzed by SDS-PAGE, Western blotting (WB), or Coomassie staining. (C and D) Vps4-HA (C) or Vps4-E233Q-HA (D) were immunoprecipitated (IP) from cell lysates of the indicated mutants and analyzed by SDS-PAGE and Western blotting. IN, input.