Figure 4.

p31^comet is a competitive inhibitor of O-Mad2:C-Mad2 assembly. (A) Mad2^ΔC and p31^comet compete for C-Mad2^wt bound to GST-Cdc20. Mad2^ΔC (3 μM) and p31^comet (0.3–3 μM) can separately bind to ∼ 1 μM Cdc20:C-Mad2^wt on beads (lanes 2 and 6, respectively). Relative stoichiometries of Mad2^ΔC and p31^comet are indicated. When mixed together (lane 5), Mad2^ΔC and p31^comet are unable to enter a single complex. Already at equimolar concentrations, p31^comet prevents O-Mad2^ΔC from binding C-Mad2 (lane 5). (B) GST-Cdc20 pull-down assay with double C-Mad2 point mutants additionally harboring the R133A mutation. Experiments were repeated three times with identical results.