| The anaphase promoting complex/cyclosome (APC) is a highly conserved ubiquitin ligase that controls passage through the cell cycle by targeting many proteins for proteolysis. The complex is composed of at least thirteen core subunits, eight of which are essential, and two activating subunits, Cdc20 (essential) and Cdh1/Hct1 (non-essential). Previously, it was not known which APC targets are sufficient to explain the essential nature of the complex. Here, we show that each of the 8 normally essential APC subunits is rendered nonessential (is "bypass-suppressed") by the simultaneous removal/inhibition of the APC substrates securin (Pds1) and B-type cyclin/CDK (Clb/CDK). In strains lacking the APC, levels of Clb2 and Clb3 remain constant, yet Clb/CDK activity oscillates as cells cycle. We have used an updated version of a mathematical model of the yeast cell cycle to model strains that lack the APC. With a few modifications, the model accurately simulates the viability of Apc - strains, as well as the phenotypes of 27 other previously characterized strains. We used these new strains to analyze the architecture of the APC. We found that the largest subunit, Apc1, serves as a scaffold that associates independently with two separable subcomplexes, one that contains Apc2 (Cullin), Apc11 (RING), and Doc1/Apc10, and another that contains the three TPR subunits (Cdc27, Cdc16, and Cdc23). We find that the three TPR subunits display a sequential binding dependency, with Cdc27 the most peripheral, Cdc23 the most internal and Cdc16 between. Apc4, Apc5, Cdc23 and Apc1 associate interdependently, such that loss of any one subunit greatly reduces binding between the remaining three. Intriguingly, the cullin and TPR subunits both contribute to the binding of Cdh1 to the APC. We have also found that the IR domain of Cdc20 is dispensable for viability, suggesting that Cdc20 can activate the APC through another domain. We have provided an updated model for the subunit architecture of the APC. |
