Mitotic chromosome segregation is initiated by the anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20. APC/CCDC20 is inhibited by the spindle assembly checkpoint (SAC) when chromosomes have not attached to spindle microtubules. Unattached kinetochores catalyze the formation of a diffusible APC/CCDC20 inhibitor that is composed of BUBR1, BUB3, MAD2 and a second molecule of CDC20. Kinetochore recruitment of these proteins as well as SAC activation rely on the mitotic kinase BUB1, but the molecular mechanism by which BUB1 accomplishes this in human cells is unknown. We show that BUBR1 and BUB3 kinetochore recruitment by BUB1 is dispensable for SAC activation. Unlike its yeast and nematode orthologs, human BUB1 does not associate stably with the MAD2 activator MAD1 and, although required for accelerating loading of MAD1 onto kinetochores, is dispensable for its steady-state levels there. Instead, we identify a 50 amino acid segment harboring the recently reported ABBA motif close to a KEN box as critical for BUB1's role in SAC signaling. The presence of this segment correlates with SAC activity and efficient binding of CDC20 but not MAD1 to kinetochores.
- Received February 5, 2015.
- Accepted July 2, 2015.
- © 2015. Published by The Company of Biologists Ltd