Mitotic Exit Control in Budding Yeast: Regulators and Dynamics

In budding yeast, the phosphatase Cdc14 is released from nucleolus to promote mitotic exit (ME). Cdc14 release and ME is controlled by mitotic cyclin-Cdk oscillation, the FEAR network including a non-proteolytic function of separase (Esp1), and the Mitotic Exit Network (MEN) indirectly activated by spindle elongation through cohesin cleavage by the proteolytic function of Esp1. The MEN contributes strongly to ME efficiency. Esp1 contributes to Cdc14 release and ME kinetics mainly through cohesin cleavage: the Esp1 requirement can be largely bypassed if cells are provided Esp1-independent means of separating sister chromatids. In the absence of Esp1 activity we observed only a minor ME delay consistent with a FEAR defect. Esp1 overexpression drives ME in Cdc20-depleted cells arrested in metaphase. We have found that this activity of overexpressed Esp1 depended on spindle integrity and the MEN. Quantitative measure of Cdc14 localization indicates efficient Cdc14 release upon MEN activation; release driven by Esp1 in the absence of microtubules was inefficient and incapable of driving ME. Reducing mitotic cyclin-Cdk activity is critical for ME, but Cdc14 release and resequestration is not blocked by endogenous undegradable mitotic cyclin Clb2. Using quantitative time-lapse microscopy, we demonstrate an intrinsic oscillatory module controlling Cdc14 localization. This autonomous Cdc14 release oscillator functions at constant cyclin-Cdk levels by titrated introduction of undegradable Clb2, and at cell-cycle-average Clb2 levels given a block of cell cycle progression by actin depolymerization. Using genetic manipulations, we demonstrate that this oscillator can operate in free-running cell cycles even without undegradable Clb2. The Cdc14-Cdh1-Cdc5 negative feedback is the primary mechanism driving this release oscillator. Its mechanism and regulation of its frequency by Clb2-Cdk, suggest the hypothesis that intrinsically autonomous Cdc14 release cycles are locked at once per cell cycle through entrainment by the cyclin-Cdk oscillator. This concept incorporates autonomous cell cycle oscillators previously reported into a coherent cell cycle control by cyclin-Cdk oscillation, therefore, may have broad implications for the structure and evolution of eukaryotic cell cycle