Closed MAD2 (C-MAD2) is selectively incorporated into the mitotic checkpoint complex (MCC)

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• The mitotic checkpoint is crucial for preventing aneuploidy by monitoring kinetochore-microtubule attachments.
• MAD2, a key protein, exists in open (O) and closed (C) conformations, with C-MAD2 levels increasing during mitosis.
• The role of MAD2 in the Mitotic Checkpoint Complex (MCC), the checkpoint's effector, remains debated.

Purpose of the Study:

• To investigate the role of C-MAD2 in the assembly and function of the MCC.
• To elucidate the protein-protein interactions involved in MCC formation.
• To establish a direct link between C-MAD2 and MCC effector function.

Main Methods:

• Analysis of endogenous human MCC assembly in different cell cycle phases.
• Expression of a C-conformation locked MAD2 mutant to study MCC induction.
• Biochemical assays using recombinant MCC and APC/C from mitotic HeLa cells.

Main Results:

• Endogenous MCC assembly involves initial formation of a BUBR1:BUB3:CDC20 complex, followed by C-MAD2 incorporation during mitosis.
• Intracellular C-MAD2 levels are a major limiting factor for MCC assembly, as demonstrated by MCC induction with a C-MAD2 mutant.
• Recombinant MCC containing C-MAD2 effectively inhibits APC/C, unlike a ternary complex lacking C-MAD2.

Conclusions:

• This study establishes a direct connection between the C-MAD2 signal transducer and the MCC effector.
• C-MAD2 is a critical component for MCC assembly and function in the mitotic checkpoint.
• Novel insights into the protein-protein interactions governing MCC assembly and mitotic checkpoint regulation were provided.