Prior to anaphase, a protein known as securin binds to and inhibits separase (see Figure 19-25). Once all kinetochores have attached to spindle microtubules in the correct bi-oriented manner, the APC/C ubiquitin-protein ligase, directed by specificity factor Cdc20 (together, the complex is known as APC/C^Cdc20), ubiquitinylates securin (note that this specificity factor is distinct from Cdh1, which targets APC/C substrates for degradation later during mitosis and G₁). Polyubiquitinylated securin is rapidly degraded by proteasomes, thereby releasing separase.

902

APC/C^Cdc20 is phosphorylated in prophase by mitotic CDK phosphorylation of several APC/C subunits. However, this phosphorylated APC/C^Cdc20 is not active until all chromosomes have become bi-oriented on the mitotic spindle. As we will see in Section 19.7, APC/C^Cdc20 is inhibited by a checkpoint pathway that ensures that anaphase is not initiated until all chromosomes have achieved proper attachment to the mitotic apparatus. Cdc20 is inhibited until every kinetochore has attached to microtubules and tension has been applied to the kinetochores of all sister chromatids, pulling them toward opposite spindle poles. In vertebrate cells, separase is also regulated by phosphorylation. Mitotic CDK activity inhibits separase during prophase and metaphase. Only when mitotic CDK activity begins to decline at the metaphase-anaphase transition through APC/C^Cdc20-mediated protein degradation can separase become active and trigger chromosome segregation.

Once cohesins are cleaved, anaphase chromosome movement ensues. As discussed in Chapter 18, chromosome segregation is mediated by microtubule depolymerization and motor proteins as the spindle poles move away from each other. Decline in mitotic CDK activity is important for these anaphase chromosome movements. When mitotic CDK inactivation is inhibited, anaphase does occur, but it is abnormal. Dephosphorylation of a number of microtubule-associated proteins that affect microtubule dynamics appears important for this process. In budding yeast, this dephosphorylation is brought about by the protein phosphatase Cdc14, which, as we will see, plays an essential role in the final cell cycle stage, exit from mitosis.