CPI motifs differentially affect the capping function of CP. (A) Left: Pyrene-labeled actin (1 µM, 20% labeled) was polymerized in the absence or presence of His-Cap1/2 (CP, 100 nM). In addition, reactions were performed with various concentrations of either unlabeled Aim21^CPI or Aim21^CPI* peptides. His-Tda2 was included to allow for the formation of the Tda2–Aim21 complex. While Aim21^CPI partially inhibited the capping function of CP at high concentrations, Aim21^CPI* had no effect. Right: Quantification of the relative polymerization rate from three independent pyrene actin polymerization assays. From left to right, mean relative polymerization rate = 1.00, 0.23, 0.98, 0.31, 0.45, 0.54, and 0.22. Error bars, mean ± SEM. *P ≤ 0.05. (B) Left: Pyrene actin polymerization assays were performed as in A with various concentrations of either unlabeled Bsp1^CPI or Bsp1^CPI* peptides. While Bsp1^CPI inhibited the capping function of CP at high concentrations, Bsp1^CPI* had no effect. Right: Quantification of the relative polymerization rate from three independent pyrene actin polymerization assays. From left to right, mean relative polymerization rate = 1.00, 0.23, 0.97, 0.37, 0.58, 0.77, and 0.23. Error bars, mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. (C) Left: Pyrene actin polymerization assays were performed as in A with various concentrations of either unlabeled Twf1^CPI or Twf1^CPI* peptides. Twf1^CPI had no effect on the capping function of CP, even at high concentrations. Right: Quantification of the relative polymerization rate from three independent pyrene actin polymerization assays. From left to right, mean relative polymerization rate = 1.00, 0.23, 0.94, 0.24, 0.21, 0.23, and 0.26. Error bars, mean ± SEM.