Abstract

During mitosis, the proper segregation of duplicated chromosomes is coordinated by a spindle checkpoint. The bifurcated spindle checkpoint blocks cell cycle progression at metaphase by monitoring unattached kinetochores and inhibits mitotic exit in response to the misorientation of the mitotic spindle. Ibd1p/Bfa1p is a spindle checkpoint regulator of budding yeast in the Bub2p checkpoint pathway for mitotic exit and its disruption abolishes mitotic arrest when proper organization of the mitotic spindle is inhibited. Ibd1p/Bfa1p localizes to the spindle pole body, a microtubule-organizing center in yeast, and its overexpression arrests the cell cycle in 80% of cells with an enlarged bud at mitosis and in 20% of cells with multiple buds. In this study, we found that the C-terminus of Ibd1p/Bfa1p physically interacts with Skp1p, a key component of SCF (Skp1/cullin/F-box) complex for ubiquitin-mediated proteolysis of cell cycle regulators as well as an evolutionally conserved kinetochore protein for cell cycle progression. A putative F-box motif was found in the C-terminus of Ibd1p/Bfa1p and its function was investigated by making mutants of conserved residues in the motif. These Ibd1p/Bfa1p mutants of a putative F-box interacted with Skp1p in vitro by two-hybrid assays as wild type Ibd1p/Bfa1p. Also, these Ibd1p/Bfa1p mutants displayed the overexpression phenotypes of wild type Ibd1p, when over-expressed under inducible promoters. These results suggest that a putative F-box motif of Ibd1p/Bfa1p is not essential for the interaction with Skp1p and its function in mitotic exit and cytokinesis.

• Keywords: mitosis; spindle checkpoint; IBD1/BFA1; SKP1; F-box motif; SCF (Skp1/cullin/F-box protein) complex;