Regulation of Bim1p MT binding by multisite linker phosphorylation. (A) Equal amounts (1 µM) of Bim1^WT, Bim1^WT phosphorylated with Ipl1–Sli15, and the phosphomimicking Bim1^6D mutant were incubated with increasing amounts of taxol-stabilized MTs (0–10 µM). Bim1p bound to MTs was separated from the unbound fraction by ultracentrifugation, and the amount of Bim1p in the supernatant and pellet was quantified. The binding affinity curves represent averaged data from three independent experiments. (B) Bim1^WT was bound to 2.5 µM MTs in the presence or absence of ATP. Increasing concentrations of the Ipl1–Sli15 complex were added, the binding reaction was centrifuged, and the supernatant and pellet fractions were separated and analyzed by SDS-PAGE. The top panel displays the quantification from two independent experiments. (A and B) Error bars denote standard error of the mean. (C) Bim1^WT, Bim1^6A, and Bim1^6D variants were fused to 3× GFP, expressed as the sole source of Bim1p in yeast, and visualized by live cell microscopy. Examples of large-budded yeast cells are shown. Note the faint spindle staining of the 6D mutant. (D) Bim1^WT, Bim1^6A, and Bim1^6D mutants are expressed at similar levels, as indicated by anti-GFP Western blotting. Bar, 6 µm.