Proper localization and function of CP are required to maintain normal levels of actin at endocytic sites. (A) Live-cell fluorescence microscopy showing increased recruitment of Abp1-GFP to endocytic sites in cells lacking Cap1 (SDY1480), with the α-tentacle of Cap1 mutated (SDY1580), with the CPI motifs of Aim21 and Bsp1 mutated (SDY1610), or with the mutations combined (SDY1634). Scale bar, 1 µm. (B) Quantification of Abp1-GFP peak fluorescence intensity at endocytic patches in WT, cap1∆, cap1*, aim21^CPI*/bsp1^CPI*, and aim21^CPI*/bsp1^CPI*/cap1* cells. From left to right, mean peak patch/cytosol ratio = 49.63, 79.03, 80.27, 85.68, 82.70, and n = 50 for all groups. Error bars, mean with 95% CI. ns = not significant, ****P ≤ 0.0001. (C) Quantification of Abp1-GFP patch lifetime at endocytic patches in WT, cap1∆, cap1*, aim21^CPI*/bsp1^CPI*, and aim21^CPI*/bsp1^CPI*/cap1* cells. From left to right, mean patch lifetime = 18.06, 33.52, 30.82, 26.02, 30.24 s, and n = 50 for all groups. Error bars, mean with 95% CI. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001. (D) A GST pulldown assay was performed with GST fused to the CPI motifs of Aim21, Bsp1, and Twf1 or a fragment of Inp51. Each GST fusion protein was incubated with either WT CP (His-Cap1/2) or a CP with a mutated His-Cap1 subunit α-tentacle (His-Cap1*/2), as indicated in Fig. 1 B. Aim21 was additionally incubated with His-Tda2 to form the Tda2/Aim21 complex. Bound proteins were analyzed by SDS-PAGE and Coomassie staining. The mutations to His-Cap1* do not affect the interaction between the CPI motifs and His-Cap1*/2. Source data are available for this figure: SourceData FS3.