The group used the mass spectrometry (MS) expertise of Scott Anderson and John Yates (Scripps Research Institute, La Jolla, CA) to identify 28 yeast kinetochore proteins (including 5 previously unidentified proteins) in 4 subcomplexes. The same analysis identified 18 phosphorylation sites, 10 of which appear to be Ipl1p targets.
Mutation of individual sites for Ipl1p phosphorylation did not yield any phenotype. But mutation of four sites in the microtubule-binding Dam1p was lethal, and mutant combinations focused on Dam1p led to either constitutive attachment of kinetochores to microtubules (when sites were converted to alanines) or weakened attachment (when sites were converted to phosphorylation-mimicking aspartate residues).
The identification of specific sites for kinetochore–attachment regulation is a victory for the MS approach. “It's one thing to find kinase targets,” says coauthor David Drubin, “but it's another to find the important target.” ▪