MAD1P, A PHOSPHOPROTEIN COMPONENT OF THE SPINDLE ASSEMBLY CHECKPOINT IN BUDDING YEAST

The spindle assembly checkpoint prevents cells from initiating anaphas e until the spindle has been fully assembled. We previously isolated m itotic arrest deficient (mad) mutants that inactivate this checkpoint and thus increase the sensitivity of cells to benomyl, a drug that int erferes with mitotic spindle assembly by depolymerizing microtubules. We have cloned the MAD1 gene and show that when it is disrupted yeast cells have the same phenotype as the previously isolated mad1 mutants: they fail to delay the metaphase to anaphase transition in response t o microtubule depolymerization. MAD1 is predicted to encode a 90-kD co iled-coil protein. Anti-Mad1p antibodies give a novel punctate nuclear staining pattern and cell fractionation reveals that the bulk of Mad1 p is soluble. Mad1p becomes by perphosphorylated when wild-type cells are arrested in mitosis by benomyl treatment, or by placing a cold sen sitive tubulin mutant at the restrictive temperature. This modificatio n does not occur in G1-arrested cells treated with benomyl or in cells arrested in mitosis by defects in the mitotic cyclin proteolysis mach inery, suggesting that Mad1p hyperphosphorylation is a step in the act ivation of the spindle assembly checkpoint. Analysis of Mad1p phosphor ylation in other spindle assembly checkpoint mutants reveals that this response to microtubule-disrupting agents is defective in some (mad2, bub1, and bub3) but not all (mad3, bub2) mutant strains. We discuss t he possible functions of Mad1p at this cell cycle checkpoint.