Ubiquitin-mediated proteolytic degradation is fundamental to eukaryotic cell cycle progression. From late mitosis through early G1, the Anaphase Promoting Complex (APC) is essential for cell-cycle relevant proteolytic degradation, and its activity is targeted to appropriate substrates by the evolutionarily conserved coactivators Cdc20 and Cdh1. After an initial wave of APC-Cdc20 activity, APC-Cdh1 degrades multiple mitotic proteins from mitotic exit through G1; inhibitory phosphorylation of Cdh1 by CDK and Polo kinase may allow accumulation of Cdh1 targets in the subsequent cell cycle. I demonstrate lethality of exact endogenous gene replacement of CDH1 with the CDK-unphosphorylatable CDH1-m11 allele; neither polo kinase sites nor polo interaction motifs are required for Cdh1 regulation. CDH1-m11 cells arrest in the first cycle with replicated DNA; ~30% of these cells have bipolar spindles. Construction of bipolar spindles in these cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1. CDH1-m11 cells with bipolar spindles fail to progress to anaphase, suggesting that Cdh1 inhibits multiple spindle-regulatory pathways. Expression of undegradable mitotic cyclin causes spindle pole body separation (a key step in bipolar spindle assembly) in CDH1-m11 cells; thus mitotic cyclins are a significant target for Cdh1 with respect to bipolar spindle assembly, and reciprocally, cyclin-Cdk activity is the most significant mechanism for Cdh1 inactivation. Cdc20 has been proposed to be a Cdh1 target, but regulation of Cdc20 proteolysis has been controversial. My experiments demonstrate that degradation of Cdc20 can be dependent on Cdh1 and Cdc20 destruction boxes, but Cdh1- and db-independent modes of Cdc20 proteolysis are also effective in limiting Cdc20 levels. To better understand the mechanisms by which multisite CDK phosphorylation inhibits Cdh1, I employed a novel recombination approach to create a series of partially unphosphorylatable CDH1 alleles ablating contiguous sites beginning at either the N or C terminus. Strains lacking N-terminal phosphorylation sites were strictly dependent upon ACM1 and S-phase cyclins for viability, and a fraction of cells displayed evidence of hyperactive APC-Cdh1, in contrast to a non-overlapping larger set of C-terminal site ablations.

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy