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

(NTD and CTD) are required for efficient mitotic checkpoint signaling. In studying the MAD2 O-C conversion, earlier work has detailed the conformational changes of MAD2 (6, 32) . We reasoned that better characterization of the MAD1:C-MAD2 catalyst would provide further mechanistic insights into the conversion reaction and hence the signal amplification step of the mitotic checkpoint. We noted that even though MAD1 is commonly depicted as a rigid coiled coil protein, parts of its NTD and CTD have been shown or were predicted to remain disordered or adopt other structures (Fig. 1a) (Fig. S1) (11, 33, 34) . We first investigated possible contribution of MAD1 NTD and MAD1 CTD to the mitotic checkpoint using a "separation-of-function" system developed by Maldonado and Kapoor (26) . In this system, a mCherry-Mis12-MAD1 fusion construct was exploited to examine "catalytic efficiency" of the MAD1:C-MAD2 catalyst without concerns over the "kinetochore targeting" aspect of its regulation (26) (Fig. 1b) . Although endogenous MAD1 and MAD2 disappear from metaphase kinetochores which presumably are occupied by spindle microtubules, expression of wild type MAD1 (MAD1 WT ) fused with "constitutive" kinetochore protein Mis12 retained MAD1 at metaphase plate and recruited GFP-MAD2 L13A to these metaphase kinetochores (26, 28) (Fig. 1c, Fig. S2 ). MAD2 L13A is a MAD2 mutant locked in C-conformation (13, 32) . The persistence of MAD1 and MAD2 at metaphase attached kinetochores was sufficient to trigger a >12 hr mitotic arrest in HeLa cells (26, 28) (Fig. 1d) . The arrest is dependent on C-MAD2 binding to MAD1, as cells expressing the fusion with MAD2-binding deficient MAD1 AA mutant (K541A, L543A in MIM) finished mitosis within ~60 min on average (Fig. 1d, Fig. S2 ) (26) . Note no GFP-MAD2 L13A is localized at metaphase kinetochores containing mCherry-Mis12-MAD1 AA , although GFP-MAD2 L13A does appear at the last few unattached kinetochores most likely due to presence of endogenous MAD1 there (Fig. S2 , compare the second and third columns). Furthermore, co-expression of MAD2 C10 , an Oconformer locked mutant of MAD2 (6,7), abolished the mitotic arrest in MAD1 WT transfected cells (data not shown), corroborating that the arrest is due to O-C conversion dependent checkpoint responses (28, 35, 36) . In consistence with previous reports (14) (15) (16) (17) , MAD1 missing (597-718) residues (MAD1 CTD ), even as a fusion with Mis12, could not maintain mitotic arrest (98±7 vs 749±22 min for MAD1 WT , mean±SD, P<0.0001, student's t-test, there might be an underestimation for MAD1 WT transfected cells as the movies last 13 hrs). Moreover, a specific MAD1 Y634E mutant also abolished the mitotic arrest, while a MAD1 Y634F mutant did not significantly impact mitotic duration (Fig. 1d) . Y634 is situated close to the junction between the coiled coil subdomain (597-637 residues) and the globular subdomain (638-718 residues) of the MAD1 CTD (33) . We noticed this site during screening potential MAD1 phospho-mutants as Y634 was reported to be phosphorylated in vivo (37) . Interestingly, MAD1 missing (1-485) residues (MAD1 NTD ) cannot maintain prolonged mitosis either (average duration: 101±22 min, Fig 1d) .