Coordination between discrete Mitotic Arrest Deficient 1 (MAD1) domains is required for efficient mitotic checkpoint signaling

Wenbin Ji; Yibo Luo; Ejaz Ahmad; Song-Tao Liu

Both phospho-resistant and phosphomimic mutants at individual sites were then prepared. The mutants at S598 or S624 did not show obvious defects in the mitotic checkpoint when fused with mCherry-Mis12. However, the mutants at S610 and T716 showed significant differences in mitotic arrest durations as compared to the MAD1 WT fusion (Fig. 5c) . Furthermore, the T716E mutant maintained the checkpoint longer than the T716A mutant (441±38 vs 242±48 min, P<0.05, students' t-test), indicating that T716 is likely the residue activated by MPS1 for mitotic checkpoint signaling. However, the mCherry-Mis12 fusion of MAD1 T716E mutant could not maintain mitotic arrest when transfected cells were challenged by reversine (Fig. S4c) , suggesting that either the phosphomimic mutant is imperfect or MPS1 has additional key substrates required for a functional mitotic checkpoint.