Pom1 phosphorylates the Cdr2 C-terminal basic domain to modulate Cdr2 association with membranes. (A) Localization of GFP–Cdr2-Cter in wild-type (wt) and pom1Δ cells or in cells expressing the Pom1-chimera and of GFP–Cdr2-CterΔbsc in wild-type cells. (B) GFP–Cdr2-Cter fluorescence intensity ratio between the cell tip and the medial cortex in strains shown in A: Cdr2-Cter (n = 31), Cdr2-Cter pom1Δ (n = 39), Cdr2-Cter Pom1-chimera (n = 37), and Cdr2-CterΔbsc (n = 34) from two experiments. T1, tip of lowest intensity; T2, tip of highest intensity. Horizontal bars are means. (C) Scheme highlighting the sequence of Cdr2 basic domain and residues found to be phosphorylated in vivo (red). (D) In vitro kinase assay of GST-Pom1 on MBP-Cdr2(518–620) with the indicated mutations. (top) Phosphorimager detection of 32P incorporation. (bottom) Silver-stained gel. The top band represents Pom1 autophosphorylation. Molecular masses are indicated. Black bars indicate that intervening lanes have been spliced out. (E) Localization of GFP–Cdr2-Cterbsc-3D (S604D, S607D, and S616D) or GFP–Cdr2-Cterbsc-3A (S604A, S607A, and S616A). (F) GFP–Cdr2-Cter fluorescence intensity ratio between the cell tip and the medial cortex in strains shown in E. Cdr2-Cter (n = 45), Cterbsc-3D (n = 39), and Cterbsc-3A (n = 44) from one experiment representative of two repeats. Horizontal bars are means. Gray bars represent t tests: *, P < 5 × 10−2; **, P < 5 × 10−3. Bars, 5 µm.
