Fig. 1.

The “wait anaphase” signal is generated at improperly attached kinetochores. (A) Mad2 exists in two major conformations, open (O-Mad2, light red indented circles), which is mainly a free, cytoplasmic form, and closed (C-Mad2, bright red circles), which is either Mad1 or Cdc20 bound. Kinetochore-bound Mad1 dimers associate with Mad2 in the closed form at unattached kinetochores. Dimerization between O-Mad2 and C-Mad2 results in the release of a C-Mad2 molecule capable of binding to and inhibiting Cdc20; (inhibited Cdc20 is shown in green). The pathway for APC/C^Cdc20 inhibition is kinetochore dependent. (B) Mad3/BubR1-Bub3 can bind to and inhibit active Cdc20 (shown in blue) independent of Mad2 and kinetochores. Through its N-terminal KEN motif, BubR1 can bind directly to and inhibit Cdc20. It is not clear if Cdc20 changes structural con- formation upon Mad2 or Mad3/BubR1 binding. (C) Both Mad2 and Mad3/BubR1 are required for checkpoint function in vivo and may function cooperatively to mediate APC/C^Cdc20 inhibition. Prior binding to Mad2 may prime Cdc20 for the interaction with the Mad3/BubR1-Bub3 complex. Mad3/BubR1 inhibits APC/C^Cdc20 activity by acting as a pseudosubstrate and/or by mediating Cdc20 ubiquitination and degradation, as denoted by the dashed arrow. Mad2 may dissociate from the inhibited APC/C^Cdc20 complex once it is formed, being released into the cytoplasm again as free O-Mad2.