The spindle checkpoint of budding yeast depends on a tight complex betweenthe Mad1 and Mad2 proteins

The spindle checkpoint arrests the cell cycle at metaphase in the presence of defects in the mitotic spindle or in the attachment of chromosomes to th e spindle. When spindle assembly is disrupted, the budding yeast mad and bu b mutants fail to arrest and rapidly lose viability. We have cloned the MAD 2 gene, which encodes a protein of 196 amino acids that remains at a consta nt level during the cell cycle, Gel filtration and co-immunoprecipitation a nalyses reveal that Mad2p tightly associates with another spindle checkpoin t component, Mad1p. This association is independent of cell cycle stage and the presence or absence of other known checkpoint proteins. In addition, M ad2p binds to all of the different phosphorylated isoforms of Mad1p that ca n be resolved on SDS-PAGE. Deletion and mutational analysis of both protein s indicate that association of Mad2p with Mad1p is critical for checkpoint function and for hyperphosphorylation of Mad1p.