T584 phosphorylation switches Sid4 affinity for Ppc89 to affinity for CK1δ^Hhp1/CK1δ^Hhp2. (A) A scheme showing the configuration of strains expressing fusions between the carboxyl terminus of Sid4 and GFP to monitor affinity for the SPB in B. (B) Maximum projections of z stacks of GFP fluorescence that span the diameter of the cell. BF, brightfield. (C) Qualitative assessment of the impact of the indicated mutations upon the ability of the Sid4-GFP fusion protein to be recruited to the SPB. (D) GFP-Trap precipitation of the Sid4.GFP fusions used in A–C reveal association of wild-type and phospho-blocking T584V fusions with Ppc89.3Pk (detected with the 336 monoclonal antibody that recognizes the Pk epitope). IP, immunoprecipitation. (E) Yeast two-hybrid assays using the indicated baits and prey suggest that phosphorylation switches the affinity of unphosphorylated Sid4 for Ppc89 to an affinity for CK1δ^Hhp1/CK1δ^Hhp2. n = 3. For full dilution series, see Fig. S1 C. SD-L-T, SD-Leu-Trp; SD-L-T-H, SD-Leu-Trp-His.