CPI motifs are essential for the function of Aim21, Bsp1, and Twf1. (A) Top: Live-cell fluorescence microscopy showing reduced recruitment of Cap1-GFP to endocytic sites in cells lacking Aim21 or Bsp1 (SDY1292, SDY1350), or with the CPI motifs of Aim21 and Bsp1 mutated (SDY1474, SDY1512). In contrast, cells lacking Twf1 or with the CPI motif of Twf1 mutated display an increase in Cap1-GFP patch intensity (SDY1351 and SDY1513). Bottom: Live-cell fluorescence microscopy showing increased recruitment of Abp1-GFP to endocytic sites in cells lacking Aim21, Bsp1, or Twf1 (SDY1294, SDY1432, SDY1433), or with the CPI motifs of Aim21, Bsp1, or Twf1 mutated (SDY1434, SDY1514, SDY1515). Scale bars, 1 µm. (B) Quantification of Cap1-GFP peak fluorescence intensity at endocytic patches in WT, aim21∆, aim21CPI*, bsp1∆, bsp1CPI*, twf1∆, and twf1CPI* cells. From left to right, the mean peak patch/cytosol ratio = 32.08, 19.56, 18.73, 21.60, 23.13, 38.11, 38.49, and n = 50 for all groups. Error bars, mean with 95% CI. ns = not significant, **P ≤ 0.01, ****P ≤ 0.0001. (C) Quantification of Abp1-GFP peak fluorescence intensity at endocytic patches in WT, aim21∆, aim21CPI*, bsp1∆, bsp1CPI*, twf1∆, and twf1CPI* cells. From left to right, the mean peak patch/cytosol ratio = 49.63, 84.96, 84.36, 58.10, 51.25, 59.71, 61.68, and n = 50 for all groups. Error bars, mean with 95% CI. ns = not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. (D) Quantification of Abp1-GFP patch lifetime at endocytic patches in WT, aim21∆, aim21CPI*, bsp1∆, bsp1CPI*, twf1∆, and twf1CPI* cells. From left to right, mean patch lifetime = 18.06, 26.06, 24.12, 22.17, 20.14, 23.02 s, 22.61, and n = 50 for all groups. Error bars, mean with 95% CI. ns = not significant, *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001. (E) Cell extracts from Cap1-GFP strains used for fluorescence microscopy (Fig. 2 C, panel A, Fig. 3 A, Fig. 4 D, and Fig. 7 E) were analyzed by immunoblotting (IB) using antibodies to the GFP tag (anti-GFP). The parent strain, BY4741, is shown as a negative control. Bottom: Coomassie-stained gel of extracts show equal loading. The strains display comparable expression of Cap1-GFP except for the cap2∆ strain, which was expected. Source data are available for this figure: SourceData FS2.
Figure S2.

CPI motifs are essential for the function of Aim21, Bsp1, and Twf1. (A) Top: Live-cell fluorescence microscopy showing reduced recruitment of Cap1-GFP to endocytic sites in cells lacking Aim21 or Bsp1 (SDY1292, SDY1350), or with the CPI motifs of Aim21 and Bsp1 mutated (SDY1474, SDY1512). In contrast, cells lacking Twf1 or with the CPI motif of Twf1 mutated display an increase in Cap1-GFP patch intensity (SDY1351 and SDY1513). Bottom: Live-cell fluorescence microscopy showing increased recruitment of Abp1-GFP to endocytic sites in cells lacking Aim21, Bsp1, or Twf1 (SDY1294, SDY1432, SDY1433), or with the CPI motifs of Aim21, Bsp1, or Twf1 mutated (SDY1434, SDY1514, SDY1515). Scale bars, 1 µm. (B) Quantification of Cap1-GFP peak fluorescence intensity at endocytic patches in WT, aim21∆, aim21CPI*, bsp1∆, bsp1CPI*, twf1∆, and twf1CPI* cells. From left to right, the mean peak patch/cytosol ratio = 32.08, 19.56, 18.73, 21.60, 23.13, 38.11, 38.49, and n = 50 for all groups. Error bars, mean with 95% CI. ns = not significant, **P ≤ 0.01, ****P ≤ 0.0001. (C) Quantification of Abp1-GFP peak fluorescence intensity at endocytic patches in WT, aim21∆, aim21CPI*, bsp1∆, bsp1CPI*, twf1∆, and twf1CPI* cells. From left to right, the mean peak patch/cytosol ratio = 49.63, 84.96, 84.36, 58.10, 51.25, 59.71, 61.68, and n = 50 for all groups. Error bars, mean with 95% CI. ns = not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. (D) Quantification of Abp1-GFP patch lifetime at endocytic patches in WT, aim21∆, aim21CPI*, bsp1∆, bsp1CPI*, twf1∆, and twf1CPI* cells. From left to right, mean patch lifetime = 18.06, 26.06, 24.12, 22.17, 20.14, 23.02 s, 22.61, and n = 50 for all groups. Error bars, mean with 95% CI. ns = not significant, *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001. (E) Cell extracts from Cap1-GFP strains used for fluorescence microscopy (Fig. 2 C, panel A, Fig. 3 A, Fig. 4 D, and Fig. 7 E) were analyzed by immunoblotting (IB) using antibodies to the GFP tag (anti-GFP). The parent strain, BY4741, is shown as a negative control. Bottom: Coomassie-stained gel of extracts show equal loading. The strains display comparable expression of Cap1-GFP except for the cap2∆ strain, which was expected. Source data are available for this figure: SourceData FS2.

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