Microtubule-binding mutants of KNL-1 do not affect the interaction with PP1 and vice versa. (A) The conserved PP1 (PP1c)-docking motif SILK-RRVSF is highlighted, and mutations generated in the docking motif are indicated. (B) KNL-1 interacts with the C. elegans PP1 homologs GSP-1 and -2 but not GSP-3 and -4 in yeast two-hybrid assays (see also Fig. S3, A and B). (C) KNL-1 interaction with GSP-1/2 requires the RRVSF motif (see also Figs. 6 D and S3 C). (D) The 4A and Δ9 mutants do not perturb interaction of KNL-1 with GSP-1 and -2 in yeast two-hybrid analysis. KBP-5, a KNL-1–binding protein that interacts with the C-terminal half of KNL-1, serves as a positive control. (E) Biochemical analysis of KNL-1 interaction with GSP-2. Indicated variants of KNL-1^1–505–6xHis immobilized on nickel agarose beads were incubated with GST–GSP-2, and bead-bound proteins were analyzed by SDS-PAGE. Molecular mass is indicated in kilodaltons. (F) PP1-docking mutant of KNL-1 interacts normally with microtubules. Assays using purified KNL-1^1–505 variants were performed as described in Fig. 1 (C and E). The WT data are reproduced from Fig. 1 E. Error bars represent the SEM with a 95% confidence interval. Bar, 10 µm.