THE CHECKPOINT PROTEIN MAD2 AND THE MITOTIC REGULATOR CDC20 FORM A TERNARY COMPLEX WITH THE ANAPHASE-PROMOTING COMPLEX TO CONTROL ANAPHASE INITIATION

The spindle assembly checkpoint mechanism delays anaphase initiation u ntil all chromosomes are aligned at the metaphase plate. Activation of the anaphase-promoting complex (APC) by binding of CDC20 and CDH1 is required for exit from mitosis, and APC has been implicated as a targe t for the checkpoint intervention. We show that the human checkpoint p rotein hlMAD2 prevents activation of APC by forming a hMAD2-CDC20-APC complex. When injected into Xenopus embryos, hMAD2 arrests cells at mi tosis with an inactive APC. The recombinant hMAD2 protein exists in tw o-folded states: a tetramer and a monomer. Both the tetramer and the m onomer bind to CDC20, but only the tetramer inhibits activation of APC and blocks cell cycle progression. Thus, hMAD2 binding is not suffici ent for inhibition, and a change in hMAD2 structure may play a role in transducing the checkpoint signal. There are at least three different forms of mitotic APC that can be detected in vivo: an inactive hMAD2- CDC20-APC ternary complex present at metaphase, a CDC20-APC binary com plex active in degrading specific substrates at anaphase, and a CDH1-A PC complex active later in mitosis and in G(1). We conclude that the c heckpoint-mediated cell cycle arrest involves hMAD2 receiving an upstr eam signal to inhibit activation of APC.