Mad3 and its interaction with Cdc20. A. Schematic drawing of Saccharomyces cerevisiae Mad3 (homolog of human BubR1). Mad3 contains several APC/C degradation motifs: a destruction box (D box) and two KEN boxes. B. Three possible mechanisms of Mad3 binding to Cdc20. The monovalency model proposes the cooperative binding of multiple motifs into a single docking site in Cdc20. The multivalency model proposes that the multiple degradation motifs of Mad3 bind to different docking sites in Cdc20. In the shared-binding model, the degradation motifs of Mad3 bind to different sites in both Cdc20 and APC/C.

A dynamic model for MCC-mediated inhibition of APC/C. A. Mad3 uses its APC/C degradation motifs to bind to Cdc20 and blocks substrate binding of APC/C^Cdc20. Meanwhile, Mad3 binding to Cdc20 induces APC/C-dependent ubiquitination of Cdc20, which is antagonized by USP44. Ubiquitination of Cdc20 promotes the disassembly of MCC. Ubiquitinated Cdc20 is either degraded by the proteasome to reduce the cellular levels of Cdc20 or deubiquitinated by USP44. The deubiquitinated Cdc20 can be re-incorporated into MCC and associate with APC/C. Thus, upon checkpoint activation, a dynamic equilibrium of MCC formation and disassembly is achieved by the continuous cycles of ubiquitination and deubiquitination of Cdc20. This process directs the activity of APC/C towards Cdc20 and reduces its activity towards cyclin B and securin. B. Schematic drawing of the rates of MCC formation and disassembly during mitosis. Upon checkpoint activation, Mad3 binds to Cdc20 and inhibits APC/C, but Mad3 binding also induces Cdc20 ubiquitination and the disassembly of MCC. Thus, the rates of MCC formation and disassembly may both be enhanced during active spindle checkpoint signaling. An equilibrium is reached to keep the steady-state levels of MCC constant, analogous to a runner on a treadmill. This model is also consistent with the finding that, at any given time, only small pools of the Mad2, Cdc20, Bub3 and Mad3 molecules in a cell associate with APC/C. Once all sister chromatids achieve bi-orientation, the rate of MCC formation falls below that of MCC disassembly. The existing MCC complexes are rapidly disassembled, allowing the activation of APC/C^Cdc20 and checkpoint inactivation.