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

Our results suggest that a full length MAD1 molecule may employ NTD and CTD to at least transiently interact with the substrates (O-MAD2) and the products (C-MAD2) even the intermediate states (I-MAD2, represented by MAD2 N10 in Fig. 3C ) of the MAD2 O-C conversion reaction (50) . The interaction with O-MAD2 might increase local concentration of substrates, driving the conversion reaction. Similarly, enrichment of I-MAD2 by MAD1-NTD or CTD could facilitate its interaction with CDC20 (29, 30) , although conversion to C-MAD2 can certainly be accomplished in the absence of CDC20 (12, 13) . Following the law of mass action, the NTD or CTD-retained C-MAD2 could also drive its interaction with BUBR1 as a step of MCC assembly (36) . These possible scenarios may underlie the compromised mitotic checkpoint responses as seen when cells were transfected with mCherry-Mis12-MAD1 NTD or -MAD1 CTD (Fig. 1 ). In consistence with this idea, our data indicated that MAD1-NTD and MAD1-CTD bind to a new interface on MAD2. Such a binding mode is postulated to be advantageous as the I-MAD2 or C-MAD2 products anchored on MAD1 then have two other protein-protein interaction interfaces readily available: the safety belt for CDC20 and the dimerization domain for BUBR1 to assemble into the MCC, the anaphase onset inhibitor (1, 4) . In addition, the MAD2 interactions with NTD or CTD must be weak or transient in cells as mCherry-Mis12-MAD1 NTD and MAD1 CTD did not show significant reduction in co-immunoprecipitated MAD2 and FLAG-MAD1 NTD only shows faint MAD2 binding (Fig. 1) . Such transient interactions are likely beneficial for stepwise relay mechanisms to facilitate MAD2 O-C conversion.