UCSF

The critical event of mitosis is the equal segregation of duplicated chromosomes to opposite poles of the cell. As would be expected for such a significant event in the life of a cell, this process is highly regulated. We explored how the regulation of the protein Cdc20 through ubiquitination contributes to proper mitotic progression in budding yeast. Cdc20 is an activating subunit of the Anaphase Promoting Complex (APC/C), the E3 ubiquitin ligase that promotes anaphase by ubiquitinating, and thereby targeting for destruction, specific mitotic inhibitors. We found that Cdc20 is also a substrate of the APC/C, which occurs through an autoubiquitination mechanism while Cdc20 is bound to the APC/C in its activator binding orientation. This activity is cell cycle regulated, and peaks in late mitosis. Using a Cdc20 mutant that is poorly ubiquitinated throughout the cell cycle, we found that this turnover is required to reset Cdc20 levels to allow cells to establish a Spindle Assembly Checkpoint (SAC) in the subsequent cell cycle. Cdc20 has also been observed to be an unstable protein during an SAC arrest, although the mechanism has largely been unexplored. Using purified components, we found that two essential SAC components, Mad2 and Mad3 (which is found in a complex with Bub3), have opposite effects on Cdc20 autoubiquitination. Mad2 inhibits full binding of Cdc20 to the APC/C, thereby inhibiting Cdc20 autoubiquitination. The Mad3-Bub3 complex increases Cdc20 binding to the APC/C, an effect that is enhanced by the presence of Mad2, effectively stimulating Cdc20 autoubiquitination. Specific inhibition of this mechanism, by deletion of the APC/C subunit Mnd2/Apc15, allowed cells to establish and SAC arrest, but delay release from the arrest. Together these results show that Cdc20 autoubiquitination has two opposing functions on mitotic progression: Cdc20 autoubiquitination is required in late mitosis to allow cells to establish an SAC arrest in the subsequent cell cycle, and Cdc20 autoubiquitination in the arrest is required for SAC inactivation.