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Abstract:
The kinetochore checkpoint pathway, involving the Mad1, Mad2, Mad3,
Bub1, Bub3 and Mps1 proteins, prevents anaphase entry and mitotic exit
by inhibiting the anaphase promoting complex activator Cdc20 in response
to monopolar attachment of sister kinetochores to spindle fibres. We
show here that Cdc20, which had previously been shown to interact
physically with Mad2 and Mad3, associates also with Bub3 an association
is up-regulated upon checkpoint activation. Moreover, co-fractionation
experiments suggest that Mad2, Mad3 and Bub3 may be concomitantly
present in protein complexes with Cdc20. Formation of the Bub3-Cdc20
complex requires all kinetochore checkpoint proteins but, surprisingly,
not intact kinetochores. Conversely, point mutations altering the
conserved WD40 motifs of Bub3, which might be involved in the formation
of a beta -propeller fold devoted to protein-protein interactions,
disrupt its association with Mad2. Mad3 and Cdc20. as well as proper
checkpoint response. We suggest that Bub3 could serve as a platform for
interactions between kinetochore checkpoint proteins, and its
association with Mad2, Mad3 and Cdc20 might be instrumental for
checkpoint activation.
