Fig. 2. Vps4p in yeast lysates associates with Vps20p and Vta1p and association is ATP-independent. Vps20p and Vta1p were expressed as GST fusions in E. coli and purified on glutathione-agarose beads. Vps4p, either tagged with GFP or without tag, was expressed from a centromeric plasmid in vps4 (RH2906). Lysates were prepared from both strains and a 100,000 g supernatant (S3) was supplemented with 20 mM MgCl₂ and incubated with beads bearing GST only or GST-Vps20p (A), or with beads bearing GST only or GST-Vta1p (B) with or without pretreatment of the lysates with apyrase to deplete endogenous 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. Vps4p-GFP was detected by immunoblotting with a GFP-specific polyclonal antiserum. In each set of experiments the exposure times for the gels containing bound and unbound samples were identical.