Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1

The anaphase promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell‐cycle regulators by the 26S proteasome. Cdc20 and Cdh1 are WD40‐containing APC co‐activators that bind destruction boxes (DB) and KEN boxes within substrates to recruit them to the APC for ubiquitination. Acm1 is an APC Cdh1 inhibitor that utilizes a DB and a KEN box to bind Cdh1 and prevent substrate binding, although Acm1 itself is not a substrate. We investigated what differentiates an APC substrate from an inhibitor. We identified the Acm1 A‐motif that interacts with Cdh1 and together with the DB and KEN box is required for APC Cdh1 inhibition. A genetic screen identified Cdh1 WD40 domain residues important for Acm1 A‐motif interaction and inhibition that appears to reside near Cdh1 residues important for DB recognition. Specific lysine insertion mutations within Acm1 promoted its ubiquitination by APC Cdh1 whereas lysine removal from the APC substrate Hsl1 converted it into a potent APC Cdh1 inhibitor. These findings suggest that tight Cdh1 binding combined with the inaccessibility of ubiquitinatable lysines contributes to pseudosubstrate inhibition of APC Cdh1 .