A novel role for the GTPase-activating protein Bud2 in the spindle position checkpoint.

Abstract

The spindle position checkpoint (SPC) ensures correct mitotic spindle position before allowing mitotic exit in the budding yeast Saccharomyces cerevisiae. In a candidate screen for checkpoint genes, we identified bud2Δ as deficient for the SPC. Bud2 is a GTPase activating protein (GAP), and the only known substrate of Bud2 was Rsr1/Bud1, a Ras-like GTPase and a central component of the bud-site-selection pathway. Mutants lacking Rsr1/Bud1 had no checkpoint defect, as did strains lacking and overexpressing Bud5, a guanine-nucleotide exchange factor (GEF) for Rsr1/Bud1. Thus, the checkpoint function of Bud2 is distinct from its role in bud site selection. The catalytic activity of the Bud2 GAP domain was required for the checkpoint, based on the failure of the known catalytic point mutant Bud2(R682A) to function in the checkpoint. Based on assays of heterozygous diploids, bud2(R682A), was dominant for loss of checkpoint but recessive for bud-site-selection failure, further indicating a separation of function. Tem1 is a Ras-like protein and is the critical regulator of mitotic exit, sitting atop the mitotic exit network (MEN). Tem1 is a likely target for Bud2, supported by genetic analyses that exclude other Ras-like proteins.