Keith R.Willison1, J.Wade Harper2 and

The anaphase-promoting complex (APC) is a multi-subunit E3 ubiquitin ligase that targets speci®c cell cycle-related proteins for degradation, regulating pro-gression from metaphase to anaphase and exit from mitosis. The APC is regulated by binding of the co-activator proteins Cdc20p and Cdh1p, and by phos-phorylation. We have developed a puri®cation strategy that allowed us to purify the budding yeast APC to near homogeneity and identify two novel APC-associated proteins, Swm1p and Mnd2p. Using an in vitro ubiquitylation system and a native gel bind-ing assay, we have characterized the properties of wild-type and mutant APC. We show that both the D and KEN boxes contribute to substrate recognition and that coactivator is required for substrate binding. APC lacking Apc9p or Doc1p/Apc10 have impaired E3 ligase activities. However, whereas Apc9p is required for structural stability and the incorporation of Cdc27p into the APC complex, Doc1p/Apc10 plays a speci®c role in substrate recognition by APC±coactivator complexes. These results imply that Doc1p/Apc10 may play a role to regulate the binding of speci®c substrates, similar to that of the coactivators