Figure 1.
Aim21, Bsp1, and Twf1 contain CPI motifs. (A) Top: Crystal structure of the chicken CP complex 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 displayed in red. The N-terminus of the CPI motif is denoted with N’. Bottom: The amino acid sequences of the S. cerevisiae proteins containing potential CPI motifs are shown. For Twf1, * denotes the stop codon. For comparison, the previously described consensus CPI motif sequence (Hernandez-Valladares et al., 2010) is shown above. (B) A GST pulldown assay was performed with GST fused to fragments of Aim21 (491–545), Bsp1 (552–576), Twf1 (308–332), or Inp51 (874–900). 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 fragments of Aim21, Bsp1, and Twf1 demonstrated interaction with His-Cap1/2, while the fragment of Inp51 and GST alone did not. (C) A fluorescence polarization assay was performed using 10 nM FITC-labeled Aim21 peptide spanning amino acids 493–540 (FITC-Aim21CPI) and various concentrations of His-Cap1/2 (CP). Reactions included 25 µM His-Tda2 to form the Tda2–Aim21 complex. Data points represent the average and standard deviation from a single experiment performed in three technical replicates fit to a one-site binding isotherm. Note that the error is too small for many data points to produce an error bar larger than the symbol and is therefore not visible. (D) A fluorescence polarization assay was performed as in C using 2.5 nM FITC-labeled Bsp1 peptide spanning amino acids 552–576 (FITC-Bsp1CPI) and various concentrations of His-Cap1/2 (CP). (E) A fluorescence polarization assay was performed as in C using 10 nM FITC-Labeled Twf1 peptide spanning amino acids 308–332 (FITC-Twf1CPI) and various concentrations of His-Cap1/2 (CP). (F) Dissociation constants of CP and Aim21CPI, Bsp1CPI, or Twf1CPI, respectively. Dissociation constants represent the mean ± SEM from three independent experiments each fit to a one-site binding isotherm. (G) Live-cell fluorescence microscopy showing strong colocalization of (top) Aim21-GFP, (middle) Bsp1-GFP, and (bottom) Twf1-GFP with Cap1-mCherry at endocytic patches, respectively (SDY1518, SDY1520, and SDY1522). Endocytic patches used for construction of kymographs to the right are indicated by arrowheads. Scale bars, 1 µm. Source data are available for this figure: SourceData F1.

Aim21, Bsp1, and Twf1 contain CPI motifs. (A) Top: Crystal structure of the chicken CP complex 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 displayed in red. The N-terminus of the CPI motif is denoted with N’. Bottom: The amino acid sequences of the S. cerevisiae proteins containing potential CPI motifs are shown. For Twf1, * denotes the stop codon. For comparison, the previously described consensus CPI motif sequence (Hernandez-Valladares et al., 2010) is shown above. (B) A GST pulldown assay was performed with GST fused to fragments of Aim21 (491–545), Bsp1 (552–576), Twf1 (308–332), or Inp51 (874–900). 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 fragments of Aim21, Bsp1, and Twf1 demonstrated interaction with His-Cap1/2, while the fragment of Inp51 and GST alone did not. (C) A fluorescence polarization assay was performed using 10 nM FITC-labeled Aim21 peptide spanning amino acids 493–540 (FITC-Aim21CPI) and various concentrations of His-Cap1/2 (CP). Reactions included 25 µM His-Tda2 to form the Tda2–Aim21 complex. Data points represent the average and standard deviation from a single experiment performed in three technical replicates fit to a one-site binding isotherm. Note that the error is too small for many data points to produce an error bar larger than the symbol and is therefore not visible. (D) A fluorescence polarization assay was performed as in C using 2.5 nM FITC-labeled Bsp1 peptide spanning amino acids 552–576 (FITC-Bsp1CPI) and various concentrations of His-Cap1/2 (CP). (E) A fluorescence polarization assay was performed as in C using 10 nM FITC-Labeled Twf1 peptide spanning amino acids 308–332 (FITC-Twf1CPI) and various concentrations of His-Cap1/2 (CP). (F) Dissociation constants of CP and Aim21CPI, Bsp1CPI, or Twf1CPI, respectively. Dissociation constants represent the mean ± SEM from three independent experiments each fit to a one-site binding isotherm. (G) Live-cell fluorescence microscopy showing strong colocalization of (top) Aim21-GFP, (middle) Bsp1-GFP, and (bottom) Twf1-GFP with Cap1-mCherry at endocytic patches, respectively (SDY1518, SDY1520, and SDY1522). Endocytic patches used for construction of kymographs to the right are indicated by arrowheads. Scale bars, 1 µm. Source data are available for this figure: SourceData F1.

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