APC15 mediates CDC20 autoubiquitylation by APC/CMCC and disassembly of the mitotic checkpoint complex

Abstract

The anaphase-promoting complex/cyclosome bound to CDC20 (APC/CCDC20) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) which inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which has been proposed to require CDC20 auto-ubiquitylation. Here we characterized APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C’s MCC binding site, is required for APC/CMCC-dependent CDC20 auto-ubiquitylation and degradation, and for timely anaphase initiation, but is dispensable for substrate ubiquitylation by APC/CCDC20 and APC/CCDH1. Our results support the view that MCC is continuously assembled and disassembled to enable rapid activation of APC/CCDC20 and that CDC20 auto-ubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators, and report the first generation of recombinant human APC/C.

biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes

“…12); CDH1 is an APC/C coactivator required to recruit substrates to the APC/C during mitotic exit and the G1 and G0 phase (13); and EMI1 and SKP1 form a heterodimeric inhibitor of APC/C-CDH1 (14). Earlier studies relied on the coexpression of human recombinant APC/C subunits from several baculoviruses (14)(15)(16). Using biGBac, we were able to assemble all 17 cDNAs previously cloned into pLIB vectors in a defined order into five different pBIG1 constructs and assembled the PGCs from these into one pBIG2 construct (FW_II-1; all pBIG1 and pBIG2 expression constructs used in this study are listed in Tables S3 and S4).…”

WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity

“…3 A and B) (12,19,20). In these structures the small subunit Apc15, which is required for Cdc20 autoubiquitination (21)(22)(23), adopts an extended conformation anchored to Apc5 by its N terminus and bridging Apc5 and Apc8A through its adjacent N-terminal helix (Apc15 NTH ) ( Fig. 3 A and B).…”

“…Apc10 and the coactivators are responsible for APC/C substrate recruitment. Apc15 is required for Cdc20 autoubiquitination in the context of the mitotic checkpoint complex (MCC), thereby regulating APC/C-MCC disassembly (21)(22)(23).…”

The Biochemistry of Mitosis

“…It is still not understood how inactivation of the SAC and activation of the APC/C are so closely coupled; in particular, this implies that the MCC must have a very short half-life, which is difficult to reconcile with the potency of the SAC that can inhibit the APC/C when only a few kinetochores are unattached (Rieder et al 1995;Collin et al 2013;Dick and Gerlich 2013). But recent attention has focused on mutual inhibition between the MCC and the APC/C (Reddy et al 2007), which requires a particular APC/C subunit, APC15 (Mansfeld et al 2011;Foster and Morgan 2012;Uzunova et al 2012). In animal cells, a second important player for MCC disassembly is the p31 comet protein (Habu et al 2002;Teichner et al 2011;Westhorpe et al 2011), which has recently been implicated in targeting an AAA ATPase to the MCC to initiate its disassembly (Eytan et al 2014;Wang et al 2014).…”