Aim21 and Bsp1 localize to CME sites through interactions with SH3 domain–containing proteins. (A) Cell extracts from Aim21-GFP strains used for fluorescence microscopy (Fig. 5 B and panel 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 shows equal loading. The strains display comparable expression of Aim21-GFP. (B) Cell extracts from Bsp1-GFP strains used for fluorescence microscopy (Fig. 5 B and panel 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 shows equal loading. The strains display comparable expression of Bsp1-GFP. (C) Cell extracts from Twf1-GFP strains used for fluorescence microscopy (Fig. 5, B and F) 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 shows equal loading. The strains display comparable expression of Twf1-GFP. (D) Yeast two-hybrid analysis of cells co-transformed with plasmids expressing the GAL4 activation (pGAD424) and binding (pGBT9) domains fused to the SH3 domain of various endocytic factors and polyproline motif (PxxP)–rich regions of Aim21 or Bsp1, respectively. Cells were spotted onto plates containing histidine (+HIS, control) or selective medium lacking histidine (−HIS). Cell growth, indicative of an interaction between proteins, was detected for Aim21 with Abp1 and Bbc1, and for Bsp1 with Abp1, Lsb3, Rvs167, Sla1, and Ysc84. (E) Top: Live-cell fluorescence microscopy showing decreased recruitment of Aim21-GFP to endocytic sites in cells lacking Abp1, Bbc1, or both Abp1 and Bbc1 (SDY1011, SDY1658, SDY1663). Bottom: Live-cell fluorescence microscopy showing decreased recruitment of Bsp1-GFP to endocytic sites in cells lacking Abp1 (SDY1659). In contrast, cells lacking Lsb3, Rvs167, or Ysc84 display an increase in Bsp1-GFP patch intensity (SDY1660, SDY1700, and SDY1661). (F) Quantification of Aim21-GFP peak fluorescence intensity at endocytic patches in WT, abp1∆, bbc1∆, and abp1∆/bbc1∆ cells. From left to right, mean peak patch/cytosol ratio = 17.61, 11.82, 9.70, 4.56, and n = 50 for all groups. Error bars, mean with 95% CI. ****P ≤ 0.0001. (G) Quantification of Bsp1-GFP peak fluorescence intensity at endocytic patches in WT, abp1∆, lsb3∆, rvs167∆, and ysc84∆ cells. From left to right, the mean peak patch/cytosol ratio = 17.50, 7.29, 20.26, 19.24, 21.49, and n = 50 for all groups. Error bars, mean with 95% CI. ns = not significant, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Source data are available for this figure: SourceData FS4.
Figure S4.

Aim21 and Bsp1 localize to CME sites through interactions with SH3 domain–containing proteins. (A) Cell extracts from Aim21-GFP strains used for fluorescence microscopy (Fig. 5 B and panel 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 shows equal loading. The strains display comparable expression of Aim21-GFP. (B) Cell extracts from Bsp1-GFP strains used for fluorescence microscopy (Fig. 5 B and panel 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 shows equal loading. The strains display comparable expression of Bsp1-GFP. (C) Cell extracts from Twf1-GFP strains used for fluorescence microscopy (Fig. 5, B and F) 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 shows equal loading. The strains display comparable expression of Twf1-GFP. (D) Yeast two-hybrid analysis of cells co-transformed with plasmids expressing the GAL4 activation (pGAD424) and binding (pGBT9) domains fused to the SH3 domain of various endocytic factors and polyproline motif (PxxP)–rich regions of Aim21 or Bsp1, respectively. Cells were spotted onto plates containing histidine (+HIS, control) or selective medium lacking histidine (−HIS). Cell growth, indicative of an interaction between proteins, was detected for Aim21 with Abp1 and Bbc1, and for Bsp1 with Abp1, Lsb3, Rvs167, Sla1, and Ysc84. (E) Top: Live-cell fluorescence microscopy showing decreased recruitment of Aim21-GFP to endocytic sites in cells lacking Abp1, Bbc1, or both Abp1 and Bbc1 (SDY1011, SDY1658, SDY1663). Bottom: Live-cell fluorescence microscopy showing decreased recruitment of Bsp1-GFP to endocytic sites in cells lacking Abp1 (SDY1659). In contrast, cells lacking Lsb3, Rvs167, or Ysc84 display an increase in Bsp1-GFP patch intensity (SDY1660, SDY1700, and SDY1661). (F) Quantification of Aim21-GFP peak fluorescence intensity at endocytic patches in WT, abp1∆, bbc1∆, and abp1∆/bbc1∆ cells. From left to right, mean peak patch/cytosol ratio = 17.61, 11.82, 9.70, 4.56, and n = 50 for all groups. Error bars, mean with 95% CI. ****P ≤ 0.0001. (G) Quantification of Bsp1-GFP peak fluorescence intensity at endocytic patches in WT, abp1∆, lsb3∆, rvs167∆, and ysc84∆ cells. From left to right, the mean peak patch/cytosol ratio = 17.50, 7.29, 20.26, 19.24, 21.49, and n = 50 for all groups. Error bars, mean with 95% CI. ns = not significant, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Source data are available for this figure: SourceData FS4.

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