Fig. 4. Actin and profilin do not interfere with each other's binding to Srv2. (A) The presence of profilin does not alter the affinity of Srv2 for G-actin. Addition of 1 µM Srv2 or Srv2-201 to 0.2 µM NBD-labeled ADP-G-actin resulted in a 30% increase in the fluorescence. Addition of profilin (0-40 µM) did not significantly reduce the NBD fluorescence signal, suggesting that profilin does not affect the binding of Srv2 or Srv2-201 to ADP-G-actin. Standard deviations are indicated by error bars. (B) Actin-binding does not change Srv2 affinity for profilin. Supernatant depletion pull-down assays were carried out with reactions containing 2 µM profilin; the average of five independent assays is shown. Lane 1, profilin alone; lanes 2-6, 20 µM GST-Srv2 on beads; lanes 3-6, variable concentrations of ADP-G-actin (1, 2, 4, 10 µM). Addition of ADP-G-actin does not change the amount of profilin in the supernatant, demonstrating that actin monomers do not interfere with Srv2-profilin interaction. Results using Srv2-201 show that profilin does not bind indirectly to Srv2 through interaction with G-actin in this assay. Standard deviations are indicated by error bars.