Cdr2 hydrophobic β4–β5 loop regulates clustering. (A) Localization of GFP-tagged Cdr2-Cter, Cdr2-Cter mutated in the F residues of the β4–β5 loop (Cdr2-Cter^FF*: F704D and F705D), or Cdr2-Cter^Δbsc. Bar, 5 µm. (bottom) 1.75× magnification of the boxed regions. Bar, 1 µm. (B) Fluorescence intensity along the cortex in regions boxed in A, representative of the cell population. (C) Pull-down assay between differentially tagged Cdr2-Cter, Cdr2-Cter^FF*, or Cdr2-Cter^Δbsc. Overexpressed GST–Cdr2-Cter, GST–Cdr2-Cter^FF*, or GST–Cdr2-Cter^Δbsc were coupled to glutathione beads and mixed with extracts of cells expressing Cdr2-Cter–myc₁₂, Cdr2-Cter^FF*–myc₁₂, or Cdr2-Cter^Δbsc*–myc₁₂, respectively. Cdr2-Cter was revealed in input and pull-down fractions with anti-GST or antimyc antibodies. (right) Normalized Cter-myc signals in GST pull-down measured in three independent experiments. Molecular masses are indicated. Error bars show SDs. (D) Mean FRAP of mEGFP-tagged Cdr2 and Cdr2^FF* mutant on the medial cortex as indicated. Error bars show SDs. Cdr2 t_1/2 = ∼3 min; Cdr2^FF* t_1/2 < 1 min. (E) Localization of mEGFP-tagged Cdr2 or Cdr2^FF*. Arrows show cell tip localization of Cdr2^FF*. Inverted black and white pictures are depicted. Bar, 5 µm. (right) Percentage of cells with Cdr2 or Cdr2^FF* detected at cell tips. n > 80 cells. A.U., arbitrary unit; B, bleach; WB, Western blot.