Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats anddoes not require intact kinetochores

The kinetochore checkpoint pathway, involving the Mad1, Mad2, Mad3, Bub1, B ub3 and Mps1 proteins, prevents anaphase entry and mitotic exit by inhibiti ng the anaphase promoting complex activator Cdc20 in response to monopolar attachment of sister kinetochores to spindle fibres. We show here that Cdc2 0, which had previously been shown to interact physically with Mad2 and Mad 3, 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. For mation of the Bub3-Cdc20 complex requires all kinetochore checkpoint protei ns 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 chec kpoint response. We suggest that Bub3 could serve as a platform for interac tions between kinetochore checkpoint proteins, and its association with Mad 2, Mad3 and Cdc20 might be instrumental for checkpoint activation.