Bim1p is phosphorylated by the Ipl1–Sli15 complex. (A) Silver-stained gel of purified Bim1–S-Tag-TEV-ZZ from yeast extracts. TAP, tandem affinity purification. (B) Schematic representation of phosphorylation sites found after purification of Bim1p from yeast extracts and after in vitro phosphorylation of recombinant Bim1p with purified Ipl1–Sli15 complex. The phosphorylation sites cluster in the flexible linker region connecting the conserved CH and EB1 domains. (C) Validation of the mapped Bim1p phosphorylation sites. Recombinant Bim1p^WT and mutants either preventing (6A) or mimicking (6D) Ipl1 phosphorylation were phosphorylated with the Ipl1–Sli15 complex in vitro. Note the elimination of phosphorylation upon changing the mapped phosphorylation sites. (D) Mapping the Bim1 interaction with the Ipl1–Sli15 complex. Different N- and C-terminal truncations of Bim1p were expressed as GST fusion proteins in E. coli and used in pull-down assays with His6-tagged recombinant Ipl1–Sli15 complex in vitro. Binding to Ipl1–Sli15 depends on the C-terminal domain of Bim1p. (E) Analytical SEC of Bim1p and Ipl1p alone or in combination indicates the formation of a stable complex between the two proteins.