Aim21, Bsp1, and Twf1 contain CPI motifs. (A) Three independent fluorescence polarization assays were performed using 10 nM FITC-labeled Aim21 peptide spanning amino acids 493–540 (FITC-Aim21^CPI) and various concentrations of His-Cap1/2 (CP). Reactions included 25 µM His-Tda2 to form the Tda2–Aim21 complex. Each data point represents the average and SD 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 CP concentration data points to produce an error bar larger than the symbol. The dissociation constant determined for individual experiments is listed at the lower right of each graph. (B) Three independent fluorescence polarization assays were performed as in A using 2.5 nM FITC-labeled Bsp1 peptide spanning amino acids 552–576 (FITC-Bsp1^CPI) and various concentrations of His-Cap1/2 (CP). (C) Three independent fluorescence polarization assays were performed as in A using 10 nM FITC-labeled Twf1 peptide spanning amino acids 308–332 (FITC-Twf1^CPI) and various concentrations of His-Cap1/2 (CP). (D) Fluorescence microscopy images taken with 1-s exposure times of cells expressing Cap1-mCherry along with either Aim21-GFP, Bsp1-GFP, or Twf1-GFP (SDY1518, SDY1520, SDY1522). Aim21-GFP and Twf1-GFP display higher overall fluorescence intensity levels than Bsp1-GFP, likely reflecting higher expression levels. Scale bars, 5 μm.