Arp2/3 ATP hydrolysis mutants nucleate actin at levels near that of wild-type Arp2/3 complex. (A) Arp2Q137A does not hydrolyze ATP, in contrast to wild-type Arp2. We cross-linked azido-γ-[³²P]ATP to WT and Arp2Q137A yeast Arp2/3. The VCA domain of the yeast NPF, Las17, was added to stimulate ATP hydrolysis by Arp2/3. Samples were removed from the reaction tubes at the indicated times, methanol precipitated, resuspended in sample buffer, and resolved by SDS-PAGE. Wild-type yeast Arp2/3 shows an appreciable loss of ³²P signal over time, signifying hydrolysis and subsequent dissociation of the γ³²-phosphate of cross-linked nucleotide from the ATP-binding pocket. (B) Fluorimetry of pyrene-labeled actin demonstrating Arp2/3-accelerated actin polymerization with WT and ATP hydrolysis mutant human Arp2/3 variants. Reactions were performed in 1× KMEI (50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, and 10 mM imidazole, pH 7.0) with 4 µM actin (5% pyrene labeled), 41 nM human Arp2/3, and 100 nM nWASP WWCA. Each pyrene reaction was performed two times. The data shown are from a single representative experiment.