The CPI motifs of Aim21 and Bsp1 are required for proper recruitment of CP to CME sites. (A) Live-cell fluorescence microscopy showing reduced recruitment of Cap1-GFP to endocytic sites in cells with the CPI motifs of Aim21 or Bsp1 mutated (SDY1474 and SDY1512), or in cells with the CPI mutations combined (SDY1604). (B) Quantification of Cap1-GFP peak fluorescence intensity at endocytic patches in WT, aim21^CPI*, bsp1^CPI*, and aim21^CPI*/bsp1^CPI* cells. From left to right, mean peak patch/cytosol ratio = 32.08, 18.73, 23.13, 5.75, and n = 50 for all groups. Error bars, mean with 95% CI. ****P ≤ 0.0001. (C) Top: Live-cell fluorescence microscopy showing strong colocalization of Cap1-GFP with Abp1-mCherry at endocytic patches (SDY1698). Bottom: Live-cell fluorescence microscopy showing reduced recruitment of Cap1-GFP and increased recruitment of Abp1-mCherry to endocytic patches in cells with the CPI motifs of Aim21 and Bsp1 mutated (SDY1702). Endocytic patches used for construction of kymographs to the right are indicated by arrowheads. Scale bars, 1 µm.