A kinetochore protein two-hybrid library screen identifies a MAD-1–BUB-1 interaction. (A, left) Schematic of checkpoint signaling mediated by kinetochore-anchored Mad1–Mad2 complexes. The inactive (O, open) and active (C, closed) conformers of Mad2 are depicted. (right) Summary of requirements for MAD-1–MAD-2 targeting to unattached kinetochores in C. elegans. (B) Summary of kinetochore protein two-hybrid screen. Strong indicates growth in low and high stringency conditions; weak indicates growth only under low stringency conditions. (C) Mapping of the region of MAD-1 that interacts with BUB-1 (FL). (D) Biochemical analysis of the MAD-1–BUB-1 interaction. Experimental schematic is on the left. Bead eluates were analyzed by anti–BUB-1 immunoblotting (top right) and Coomassie staining (bottom right). The data shown are representative of three experiments.