Rewiring of the phosphoproteome executes two meiotic divisions in budding yeast.

The cell cycle is ordered by a controlled network of kinases and phosphatases. To generate gametes via meiosis, two distinct and sequential chromosome segregation events occur without an intervening S phase. How canonical cell cycle controls are modified for meiosis is not well understood. Here, using highly synchronous budding yeast populations, we reveal how the global proteome and phosphoproteome change during the meiotic divisions. While protein abundance changes are limited to key cell cycle regulators, dynamic phosphorylation changes are pervasive. Our data indicate that two waves of cyclin-dependent kinase (Cdc28Cdk1) and Polo (Cdc5Polo) kinase activity drive successive meiotic divisions. These two distinct phases of phosphorylation are ensured by the meiosis-specific Spo13 protein, which rewires the phosphoproteome. Spo13 binds to Cdc5Polo to promote phosphorylation in meiosis I, particularly of substrates containing a variant of the canonical Cdc5Polo motif. Overall, our findings reveal that a master regulator of meiosis directs the activity of a kinase to change the phosphorylation landscape and elicit a developmental cascade.

Autophosphorylation is sufficient to release Mps1 kinase from native kinetochores.

Accurate mitosis depends on a surveillance system called the spindle assembly checkpoint. This checkpoint acts at kinetochores, which attach chromosomes to the dynamic tips of spindle microtubules. When a kinetochore is unattached or improperly attached, the protein kinase Mps1 phosphorylates kinetochore components, catalyzing the generation of a diffusible "wait" signal that delays anaphase and gives the cell time to correct the error. When a kinetochore becomes properly attached, its checkpoint signal is silenced to allow progression into anaphase. Recently, microtubules were found to compete directly against recombinant human Mps1 fragments for binding to the major microtubule-binding kinetochore element Ndc80c, suggesting a direct competition model for silencing the checkpoint signal at properly attached kinetochores. Here, by developing single-particle fluorescence-based assays, we tested whether such direct competition occurs in the context of native kinetochores isolated from yeast. Mps1 levels were not reduced on kinetochore particles bound laterally to the sides of microtubules or on particles tracking processively with disassembling tips. Instead, we found that Mps1 kinase activity was sufficient to promote its release from the isolated kinetochores. Mps1 autophosphorylation, rather than phosphorylation of other kinetochore components, was responsible for this dissociation. Our findings suggest that checkpoint silencing in yeast does not arise from a direct competition between Mps1 and microtubules, and that phosphoregulation of Mps1 may be a critical aspect of the silencing mechanism.

Kinetochore-bound Mps1 regulates kinetochore-microtubule attachments via Ndc80 phosphorylation.

Dividing cells detect and correct erroneous kinetochore-microtubule attachments during mitosis, thereby avoiding chromosome missegregation. The Aurora B kinase phosphorylates microtubule-binding elements specifically at incorrectly attached kinetochores, promoting their release and providing another chance for proper attachments to form. However, growing evidence suggests that the Mps1 kinase is also required for error correction. Here we directly examine how Mps1 activity affects kinetochore-microtubule attachments using a reconstitution-based approach that allows us to separate its effects from Aurora B activity. When endogenous Mps1 that copurifies with kinetochores is activated in vitro, it weakens their attachments to microtubules via phosphorylation of Ndc80, a major microtubule-binding protein. This phosphorylation contributes to error correction because phospho-deficient Ndc80 mutants exhibit genetic interactions and segregation defects when combined with mutants in other error correction pathways. In addition, Mps1 phosphorylation of Ndc80 is stimulated on kinetochores lacking tension. These data suggest that Mps1 provides an additional mechanism for correcting erroneous kinetochore-microtubule attachments, complementing the well-known activity of Aurora B.

Purification of kinetochores from the budding yeast Saccharomyces cerevisiae.

Chromosome segregation relies on forces generated by spindle microtubules that are translated into chromosome movement through interactions with kinetochores, highly conserved macromolecular machines that assemble on a specialized centromeric chromatin structure. Kinetochores not only have to stably attach to growing and shrinking microtubules, but they also need to recruit spindle assembly checkpoint proteins to halt cell cycle progression when there are attachment defects. Even the simplest kinetochore in budding yeast contains more than 50 unique components that are present in multiple copies, totaling more than 250 proteins in a single kinetochore. The complex nature of kinetochores makes it challenging to elucidate the contributions of individual components to its various functions. In addition, it is difficult to manipulate forces in vivo to understand how they regulate kinetochore-microtubule attachments and the checkpoint. To address these issues, we developed a technique to purify kinetochores from budding yeast that can be used to analyze kinetochore functions and composition as well as to reconstitute kinetochore-microtubule attachments in vitro.