CP:CPI interactions occur through an overlapping binding site. (A) Top: Crystal structure of chicken CP bound to the CPI motif of human CD2AP (PDB entry 3AA6). The α-subunit (Cap1) is displayed in dark blue, the β-subunit (Cap2) in light blue, and the CPI motif of CD2AP is shown in red. Bottom: The CP complex is rotated 90⁰ for a view of the stalk region of CP. The N-terminus and C-terminus of the CPI motif are labeled N’ and C’, respectively. The CP:CPI interaction occurs largely through a binding interface on the β-subunit. (B) A GST pulldown assay was performed with GST fused to the CPI motifs of Aim21 (top), Bsp1 (middle), or Twf1 (bottom). Each GST fusion protein was incubated with either His-Cap1/2 or with a CP containing a mutated version of the Cap2 subunit potentially affecting interaction with the CPI motifs (His-Cap1/2D44A, His-Cap1/2D64A, His-Cap1/2D88A, and a mutant combining the three mutations, His-Cap1/244,64,88A). 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 Cap2 affect the interaction with the GST-CPI motif fusions in varying ways and abolish the interaction when combined. (C) Pyrene-labeled actin (1 µM, 20% labeled) was polymerized in the absence or presence of His-Cap1/2 (CP, 100 nM) or His-Cap1/244,64,88A (CP2*, 100 nM). In addition, reactions were performed with either 500 nM of unlabeled Aim21CPI, Bsp1CPI, or Twf1CPI peptides. His-Tda2 was included in the Aim21CPI reaction to allow for formation of the Tda2–Aim21 complex. CP2* displayed an equivalent capping ability to CP, and the capping function of CP2* was not affected by any of the CPI motif peptides. (D) Quantification of the relative polymerization rate from three independent pyrene actin polymerization assays in C. From left to right, mean relative polymerization rate = 1.00, 0.25, 0.32, 0.30, 0.31, and 0.31. Error bars, mean ± SD. ns = not significant. (E) Top: Live-cell fluorescence microscopy showing reduced recruitment of Cap1-GFP to endocytic sites in cells with the CPI motif binding site of Cap2 mutated (SDY1596). Bottom: Live-cell fluorescence microscopy showing increased recruitment of Abp1-GFP to endocytic sites in cells with the CPI motif binding site of Cap2 mutated (SDY1599). Scale bars, 1 µm. (F) Quantification of Cap1-GFP peak fluorescence intensity at endocytic patches in WT and cap2* cells. From left to right, mean peak patch/cytosol ratio = 32.08, 8.44, and n = 50 for all groups. Error bars, mean with 95% CI. ****P ≤ 0.0001. (G) Quantification of Abp1-GFP peak fluorescence intensity at endocytic patches in WT and cap2* cells. From left to right, mean peak patch/cytosol ratio = 49.63, 70.40, and n = 50 for all groups. Error bars, mean with 95% CI. ****P ≤ 0.0001. (H) Quantification of Abp1-GFP patch lifetime at endocytic patches in WT and cap2* cells. From left to right, mean patch lifetime = 18.06, 22.96 s, and n = 50 for all groups. Error bars, mean with 95% CI. ****P ≤ 0.0001. Source data are available for this figure: SourceData F7.
Figure 7.

CP:CPI interactions occur through an overlapping binding site. (A) Top: Crystal structure of chicken CP bound to the CPI motif of human CD2AP (PDB entry 3AA6). The α-subunit (Cap1) is displayed in dark blue, the β-subunit (Cap2) in light blue, and the CPI motif of CD2AP is shown in red. Bottom: The CP complex is rotated 90⁰ for a view of the stalk region of CP. The N-terminus and C-terminus of the CPI motif are labeled N’ and C’, respectively. The CP:CPI interaction occurs largely through a binding interface on the β-subunit. (B) A GST pulldown assay was performed with GST fused to the CPI motifs of Aim21 (top), Bsp1 (middle), or Twf1 (bottom). Each GST fusion protein was incubated with either His-Cap1/2 or with a CP containing a mutated version of the Cap2 subunit potentially affecting interaction with the CPI motifs (His-Cap1/2D44A, His-Cap1/2D64A, His-Cap1/2D88A, and a mutant combining the three mutations, His-Cap1/244,64,88A). 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 Cap2 affect the interaction with the GST-CPI motif fusions in varying ways and abolish the interaction when combined. (C) Pyrene-labeled actin (1 µM, 20% labeled) was polymerized in the absence or presence of His-Cap1/2 (CP, 100 nM) or His-Cap1/244,64,88A (CP2*, 100 nM). In addition, reactions were performed with either 500 nM of unlabeled Aim21CPI, Bsp1CPI, or Twf1CPI peptides. His-Tda2 was included in the Aim21CPI reaction to allow for formation of the Tda2–Aim21 complex. CP2* displayed an equivalent capping ability to CP, and the capping function of CP2* was not affected by any of the CPI motif peptides. (D) Quantification of the relative polymerization rate from three independent pyrene actin polymerization assays in C. From left to right, mean relative polymerization rate = 1.00, 0.25, 0.32, 0.30, 0.31, and 0.31. Error bars, mean ± SD. ns = not significant. (E) Top: Live-cell fluorescence microscopy showing reduced recruitment of Cap1-GFP to endocytic sites in cells with the CPI motif binding site of Cap2 mutated (SDY1596). Bottom: Live-cell fluorescence microscopy showing increased recruitment of Abp1-GFP to endocytic sites in cells with the CPI motif binding site of Cap2 mutated (SDY1599). Scale bars, 1 µm. (F) Quantification of Cap1-GFP peak fluorescence intensity at endocytic patches in WT and cap2* cells. From left to right, mean peak patch/cytosol ratio = 32.08, 8.44, and n = 50 for all groups. Error bars, mean with 95% CI. ****P ≤ 0.0001. (G) Quantification of Abp1-GFP peak fluorescence intensity at endocytic patches in WT and cap2* cells. From left to right, mean peak patch/cytosol ratio = 49.63, 70.40, and n = 50 for all groups. Error bars, mean with 95% CI. ****P ≤ 0.0001. (H) Quantification of Abp1-GFP patch lifetime at endocytic patches in WT and cap2* cells. From left to right, mean patch lifetime = 18.06, 22.96 s, and n = 50 for all groups. Error bars, mean with 95% CI. ****P ≤ 0.0001. Source data are available for this figure: SourceData F7.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal