Effects of CPI motifs on the capping function of CP using yeast actin. (A) Actin was purified from budding yeast using the DNaseI affinity purification method (Goode, 2002). Samples of the high-speed supernantant (HSS), column washes, and column elution were analyzed by SDS-PAGE and Coomassie staining. Yeast actin (∼41 kD) was successfully purified from yeast with a high degree of purity. (B) Actin pelleting experiments were performed using either rabbit skeletal muscle actin or actin purified from yeast (2 µM). Yeast actin was more abundant in the pellet sample (P) than the supernatant sample (S) when mixed with polymerization buffer (+Poly), demonstrating the actin was assembly competent following purification. (C) Yeast actin was combined with pyrene-labeled actin (1 µM total, 20% pyrene-labeled) and polymerized in the absence or presence of His-Cap1/2 (CP, 100 nM). In addition, reactions were performed with 500 nM of unlabeled Aim21^CPI, Bsp1^CPI, or Twf1^CPI peptides. His-Tda2 was included in the Aim21^CPI reaction to allow for formation of the Tda2–Aim21 complex. While Aim21^CPI and Bsp1^CPI partially inhibited the capping function of CP, Twf1^CPI had no effect. These results are consistent with the results seen using rabbit skeletal muscle actin. (D) Quantification of the relative polymerization rate from three independent pyrene actin polymerization assays. From left to right, mean relative polymerization rate = 1.00, 0.19, 0.45, 0.69, and 0.18. Error bars, mean ± SEM. ns = not significant, *P ≤ 0.05, **P ≤ 0.01. Source data are available for this figure: SourceData FS5.