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

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/212704 doi: bioRxiv preprint MAD1 domains in the mitotic checkpoint (d) and (e) Immunoprecipitation using anti-6×His antibody to detect His-tagged MAD1-CTD interaction with untagged MAD2 L13A (d) or MAD2 ∆d) (e) in the presence/absence of GST-MPS1 kinase, ATP or MPS1 inhibitor reversine. In the classical model, a MAD1 dimer tightly binds two C-MAD2 molecules at its MIM region to become the catalyst for MAD2 O-C conversion. Our work showed that MAD1-NTD and CTD have additional, probably weaker, binding sites for both O-MAD2 and C-MAD2. Cell imaging results suggested that MAD1-NTD and CTD positively contribute to the MAD2 O-C conversion. Furthermore, the possible functions of several residues in the CTD domain were revealed. T716 phosphorylation by MPS1 and S610 are required for full MAD1 activity. Y634 might be a residue whose phosphorylation negatively impacts the O-C conversion. Similarly, the NTD:CTD interaction (not shown) may restrain the catalytic efficiency of MAD1, but the interaction can be disrupted by action of MPS1 kinase. It remains unclear whether both C-MAD2 molecules bound to the MIM regions are equally engaged in MAD2 O-C conversion.