Regulated degradation of the APC coactivator Cdc20

Cdc20 is a highly conserved activator of the anaphase-promoting complex (APC), promoting cell-cycle-regulated ubiquitination and proteolysis of a number of critical cell-cycle-regulatory targets including securin and mitotic cyclins. APC-Cdc20 activity is tightly regulated, and this regulation is likely important for accurate cell cycle control. One significant component of Cdc20 regulation is thought to be Cdc20 proteolysis. However, published literature suggests different mechanisms and requirements for Cdc20 proteolysis. The degree to which Cdc20 proteolysis is cell-cycle regulated, the dependence of Cdc20 proteolysis on Cdc20 destruction boxes (recognition sequences for APC-mediated ubiqutination, either by Cdc20 or by the related Cdh1 APC activator), and the need for APC itself for Cdc20 proteolysis all have been disputed to varying extents. In animals, Cdc20 proteolysis is thought to be mediated by Cdh1, contributing an intrinsic order of APC activation by Cdc20 and then by Cdh1. One report suggests a Cdh1 requirement for Cdc20 proteolysis in budding yeast; this idea has not been tested further.

We characterized Cdc20 proteolysis using Cdc20 expressed from its endogenous locus; previous studies generally employed strongly overexpressed Cdc20, which can cause significant artifacts. We analyzed Cdc20 proteolysis with or without mutations in previously identified destruction box sequences, using varying methods of cell cycle synchronization, and in the presence or absence of Cdh1. Cdc20 instability is only partially dependent on destruction boxes. A much stronger dependence on Cdh1 for Cdc20 proteolysis was observed, but Cdh1-independent proteolysis was also clearly observed. Cdc20 proteolysis independent of both destruction boxes and Cdh1 was especially detectable around the G1/S transition; Cdh1-dependent proteolysis was most notable in late mitosis and G1.

Cdc20 proteolysis is under complex control, with different systems operating at different points in the cell cycle. This complexity is likely to explain apparent conflicts in previously published literature on this subject. A major mode of control of Cdc20 proteolysis occurs in late mitosis/early G1 and is Cdh1-dependent, as in animal cells; this mode may contribute to the known sequential activation of the APC by Cdc20 followed by Cdh1. An independent mode of Cdc20 proteolysis, independent of destruction boxes and Cdh1, occurs at G1/S; we do not know the mechanism or function of this mode of proteolysis, but speculate that it may contribute to sharpening and restricting activation of APC-Cdc20 to early mitosis.