Fig. 3. Vps4p directly binds Vps20p and Vta1p in vitro and binding is ATP-independent. Wild-type Vps4p, and both ATP hydrolysis mutant (E233Q) and ATP binding mutant (K179A) forms of Vps4p were tagged at the C-terminus with 6HIS and expressed in E. coli. Each protein was affinity purified using the 6HIS tag and incubated with beads bearing GST-Vps20p, GST-Vta1p or GST only in the presence or absence of added ATP. Unbound proteins (unbound) were recovered in the supernatants. After washing the beads, the specifically bound proteins (bound) were eluted by heating in Laemmli sample buffer. Proteins in both bound and unbound samples were resolved by SDS-PAGE, and transferred to PVDF membranes. Wild-type and mutant forms of Vps4p-6HIS were detected by immunoblotting with a pentaHIS-specific monoclonal antiserum. In each set of experiments the exposure times for the gels containing bound and unbound samples were identical.