Structure of human Mad1 C-terminal domain reveals its involvement in kinetochore targeting

Abstract

The spindle checkpoint prevents aneuploidy by delaying anaphase onset until all sister chromatids achieve proper microtubule attachment. The kinetochore-bound checkpoint protein complex Mad1-Mad2 promotes the conformational activation of Mad2 and serves as a catalytic engine of checkpoint signaling. How Mad1 is targeted to kinetochores is not understood. Here, we report the crystal structure of the conserved C-terminal domain (CTD) of human Mad1. Mad1 CTD forms a homodimer and, unexpectedly, has a fold similar to those of the kinetochore-binding domains of Spc25 and Csm1. Nonoverlapping Mad1 fragments retain detectable kinetochore targeting. Deletion of the CTD diminishes, does not abolish, Mad1 kinetochore localization. Mutagenesis studies further map the functional interface of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Our results indicate that CTD is a part of an extensive kinetochore-binding interface of Mad1, and rationalize graded kinetochore targeting of Mad1 during checkpoint signaling.dimerization | mitosis | X-ray crystallography | multivalency | chromosome

Dissecting the roles of human BUB1 in the spindle assembly checkpoint

“…Although our observed lack of correlation between BUB1 and MAD1 at kinetochores agrees with some reports (Hewitt et al, 2010;Vleugel et al, 2013), it contrasts with others (Kim et al, 2012;Klebig et al, 2009). We noted that although we assessed the levels of MAD1 at kinetochores in cells that had been treated with nocodazole for several hours, others did so in unperturbed cells, which are likely to have spent less time in mitosis (Klebig et al, 2009).…”

Endopolyploidization and flowering time are antagonistically regulated by checkpoint component MAD1 and immunity modulator MOS1

“…1f). In addition, DAB (3,3 0 -Diaminobenzidine) staining was more pronouncedly reduced in the bon1 mos1-6 mutant (Fig. 1g), indicating that elevation of H 2 O 2 production in bon1 was more suppressed by the mos1-6 mutation than the mos1-4 mutation.…”

“…The closed form of MAD2 is bound to MAD1 and serves as a template for the conversion of the open form MAD2 into the closed form 2 . Closed MAD2 binds to CDC20 and the formation of MAD2-CDC20 complex promotes the binding of BUB3 and MAD3/BUB1-related 1 (BUBR1) to form mitotic checkpoint complex 3,4 . Functional mitotic checkpoint complex prevents aberrant chromosome segregation at mitosis until all chromosomes are properly attached to spindles.…”

Graded requirement for the spliceosome in cell cycle progression

“…Antibodies against Apc2, Mad2, Mad1, and BubR1 have been previously described. [44][45][46] The following antibodies were purchased from commercial sources: anti-SNRPB (Sigma), anti-g-tubulin (Sigma), anti-actin (Millipore) and anti-g-H2AX (Millipore).…”