p31comet-mediated extraction of Mad2 from the MCC promotes efficient mitotic exit

Abstract

Accurate chromosome segregation requires the spindle assembly checkpoint to be active at the onset of mitosis, before being silenced following chromosome alignment. p31comet is a checkpoint antagonist in that its inhibition delays mitotic exit, whereas its overexpression overrides the checkpoint. How exactly p31comet antagonises the checkpoint is unclear. A prevalent model is that p31comet acts as a ‘cap’ by inhibiting recruitment of the open conformation form of Mad2 (O-Mad2) to the kinetochore-bound complex of Mad1–C-Mad2 (closed conformation Mad2), an essential step that is required for checkpoint activation. Here, we show that although p31comet localises to kinetochores in mitosis, modulation of its activity has no effect on recruitment of O-Mad2 to kinetochores. Rather, our observations support a checkpoint-silencing role for p31comet downstream of kinetochores. We show that p31comet binds Mad2 when it is bound to the mitotic checkpoint complex (MCC) components BubR1 and Cdc20. Furthermore, RNAi-mediated inhibition of p31comet results in more Mad2 bound to BubR1–Cdc20, and conversely, overexpression of p31comet results in less Mad2 bound to BubR1–Cdc20. Addition of recombinant p31comet to checkpoint-arrested extracts removes Mad2 from the MCC, whereas a p31comet mutant that cannot bind Mad2 has no effect. Significantly, expression of a Mad2 mutant that cannot bind p31comet prolongs the metaphase to anaphase transition. Taken together, our data support the notion that p31comet negatively regulates the spindle assembly checkpoint by extracting Mad2 from the MCC.