CPI motifs are essential for the function of Aim21, Bsp1, and Twf1. (A) Organization of Aim21, Bsp1, and Twf1 domains. The sequences of the CPI motifs for each of Aim21, Bsp1, and Twf1 are displayed, with the three basic-to-acidic point mutations to the CPI motifs that are used throughout this work shown in red. Aim21CPI* = Aim21504,507,509E. Bsp1CPI* = Bsp1564,566,569E. Twf1CPI* = Twf1322,324,328E. (B) A GST pulldown assay was performed with GST fused to the CPI motifs of Aim21, Bsp1, or Twf1. The GST-CPI fusions either contained no mutations (CPI) or three-point mutations to the CPI motif (CPI*), as indicated in A. Each GST fusion protein was incubated with His-Cap1/2, with Aim21 additionally incubated with His-Tda2 to form the Tda2/Aim21 complex. Bound proteins were analyzed by SDS-PAGE and Coomassie staining. The mutations to each CPI motif abolished the interaction with His-Cap1/2. (C) Top: 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, SDY1512). In contrast, cells with the CPI motif of Twf1 mutated display an increase in Cap1-GFP patch intensity (SDY1513). Bottom: Live-cell fluorescence microscopy showing increased recruitment of Abp1-GFP to endocytic sites in cells with the CPI motif of Aim21, Bsp1, or Twf1 mutated (SDY1434, SDY1514, SDY1515). Scale bars, 1 µm. (D) Quantification of Cap1-GFP peak fluorescence intensity at endocytic patches in WT, aim21CPI*, bsp1CPI*, and twf1CPI* cells. From left to right, mean peak patch/cytosol ratio = 32.08, 18.73, 23.13, 38.49, and n = 50 for all groups. Error bars, mean with 95% confidence interval (CI). **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. (E) Quantification of Abp1-GFP peak fluorescence intensity at endocytic patches in WT, aim21CPI*, bsp1CPI*, and twf1CPI* cells. From left to right, the mean peak patch/cytosol ratio = 49.63, 84.36, 51.25, 61.68, and n = 50 for all groups. Error bars, mean with 95% CI. ns = not significant, ***P ≤ 0.001, ****P ≤ 0.0001. (F) Quantification of Abp1-GFP patch lifetime at endocytic patches in WT, aim21CPI*, bsp1CPI*, and twf1CPI* cells. From left to right, mean patch lifetime = 18.06, 24.12, 20.14, 23.02 s, and n = 50 for all groups. Error bars, mean with 95% CI. ***P ≤ 0.001, ****P ≤ 0.0001. (G) Live-cell fluorescence microscopy showing decreased endocytic uptake of Mup1-GFP after 20 min incubation with methionine-rich media in cells with the CPI motif of Aim21, Bsp1, or Twf1 mutated (SDY1471, SDY1749, and SDY1759). Scale bars, 5 µm. (H) Quantification of Mup1-GFP relative plasma membrane fluorescence intensity in WT, aim21CPI*, bsp1CPI*, and twf1CPI* cells. From left to right, mean relative fluorescence intensity = 1.00, 1.00, 1.00, 1.00, 0.35, 0.81, 0.79, 0.66, and n = 52, 55, 73, 62, 53, 55, 64, 65. Error bars, mean with 95% CI. ****P ≤ 0.0001. Source data are available for this figure: SourceData F2.
Figure 2.

CPI motifs are essential for the function of Aim21, Bsp1, and Twf1. (A) Organization of Aim21, Bsp1, and Twf1 domains. The sequences of the CPI motifs for each of Aim21, Bsp1, and Twf1 are displayed, with the three basic-to-acidic point mutations to the CPI motifs that are used throughout this work shown in red. Aim21CPI* = Aim21504,507,509E. Bsp1CPI* = Bsp1564,566,569E. Twf1CPI* = Twf1322,324,328E. (B) A GST pulldown assay was performed with GST fused to the CPI motifs of Aim21, Bsp1, or Twf1. The GST-CPI fusions either contained no mutations (CPI) or three-point mutations to the CPI motif (CPI*), as indicated in A. Each GST fusion protein was incubated with His-Cap1/2, with Aim21 additionally incubated with His-Tda2 to form the Tda2/Aim21 complex. Bound proteins were analyzed by SDS-PAGE and Coomassie staining. The mutations to each CPI motif abolished the interaction with His-Cap1/2. (C) Top: 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, SDY1512). In contrast, cells with the CPI motif of Twf1 mutated display an increase in Cap1-GFP patch intensity (SDY1513). Bottom: Live-cell fluorescence microscopy showing increased recruitment of Abp1-GFP to endocytic sites in cells with the CPI motif of Aim21, Bsp1, or Twf1 mutated (SDY1434, SDY1514, SDY1515). Scale bars, 1 µm. (D) Quantification of Cap1-GFP peak fluorescence intensity at endocytic patches in WT, aim21CPI*, bsp1CPI*, and twf1CPI* cells. From left to right, mean peak patch/cytosol ratio = 32.08, 18.73, 23.13, 38.49, and n = 50 for all groups. Error bars, mean with 95% confidence interval (CI). **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. (E) Quantification of Abp1-GFP peak fluorescence intensity at endocytic patches in WT, aim21CPI*, bsp1CPI*, and twf1CPI* cells. From left to right, the mean peak patch/cytosol ratio = 49.63, 84.36, 51.25, 61.68, and n = 50 for all groups. Error bars, mean with 95% CI. ns = not significant, ***P ≤ 0.001, ****P ≤ 0.0001. (F) Quantification of Abp1-GFP patch lifetime at endocytic patches in WT, aim21CPI*, bsp1CPI*, and twf1CPI* cells. From left to right, mean patch lifetime = 18.06, 24.12, 20.14, 23.02 s, and n = 50 for all groups. Error bars, mean with 95% CI. ***P ≤ 0.001, ****P ≤ 0.0001. (G) Live-cell fluorescence microscopy showing decreased endocytic uptake of Mup1-GFP after 20 min incubation with methionine-rich media in cells with the CPI motif of Aim21, Bsp1, or Twf1 mutated (SDY1471, SDY1749, and SDY1759). Scale bars, 5 µm. (H) Quantification of Mup1-GFP relative plasma membrane fluorescence intensity in WT, aim21CPI*, bsp1CPI*, and twf1CPI* cells. From left to right, mean relative fluorescence intensity = 1.00, 1.00, 1.00, 1.00, 0.35, 0.81, 0.79, 0.66, and n = 52, 55, 73, 62, 53, 55, 64, 65. Error bars, mean with 95% CI. ****P ≤ 0.0001. Source data are available for this figure: SourceData F2.

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