(A–D) Actin assembly assays of the wild-type WRC230VCA (A), Sra1 Y967A (B), Sra1 S969F (C), and Sra1 C179R (D), with corresponding reaction conditions shown on the right. Reactions contain 4 μM actin (5% pyrene labeled), 10 nM Arp2/3 complex, 100 nM WRC230VCA or VCA, and indicated amount of Rac1 Q61L loaded with GMPPNP. (E) Maximum actin assembly rates at different Rac1 concentrations derived from data in (A–D) (Doolittle et al., 2013). (F) Maximum actin assembly rates at different concentrations of distinct WRC-Rac1 species, obtained from the data shown in Figure 1—figure supplement 1C for WRC activation by Rac1 Q61L/P29S. Concentrations of the indicated species of WRC-Rac1 complexes (shown as cartoons) formed at different Rac1 concentrations in the actin assembly assay were calculated using DynaFit (Kuzmic, 1996) by using a two-site cyclic model shown in Figure 5—figure supplement 2A, using KDD of 0.265 μM and KDA of 11.52 μM (derived from Figure 5, assuming no cooperativity between the A and D Site, since the actin assembly assays were performed in solution). Black data plot the summed concentration of all WRC-Rac1 species, while red data plot only WRC-(Rac1)2. A linear fit of the latter data yields r2 of 0.98.