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Abstract:
The spindle assembly checkpoint (SAC) coordinates mitotic progression
with sister chromatid alignment. In mitosis, the checkpoint machinery
accumulates at kinetochores, which are scaffolds devoted to microtubule
capture. The checkpoint protein Mad2 (mitotic arrest defficient 2)
adopts two conformations: open (O- Mad2) and closed (C- Mad2). C-Mad2
forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore
receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In
HeLa cells, an essential interaction between C- and O-Mad2 conformers
allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In
this study, we show that the interaction of the O and C conformers of
Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles
impaired in this interaction fail to restore the SAC in a mad2 deletion
strain. The corresponding mutant proteins bind Mad1 normally, but their
ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical
and genetic evidence shows that the interaction of O- and C- Mad2 is
essential for the SAC and is conserved in evolution.
