BuGZ facilitates loading of spindle assembly checkpoint proteins to kinetochores in early mitosis

BuGZ is a kinetochore component that binds to and stabilizes Bub3, a key player in mitotic spindle assembly checkpoint signaling. Bub3 is required for kinetochore recruitment of Bub1 and BubR1, two proteins that have essential and distinct roles in the checkpoint. Both Bub1 and BubR1 localize to kinetochores through interactions with Bub3, which are mediated through conserved GLEBS domains in both Bub1 and BubR1. BuGZ also has a GLEBS domain, which is required for its kinetochore localization as well, presumably mediated through Bub3 binding. Although much is understood about the requirements for Bub1 and BubR1 interaction with Bub3 and kinetochores, much less is known regarding BuGZ's requirements. Here, we used a series of mutants to demonstrate that BuGZ kinetochore localization requires only its core GLEBS domain, which is distinct from the requirements for both Bub1 and BubR1. Furthermore, we found that the kinetics of Bub1, BubR1, and BuGZ loading to kinetochores differ, with BuGZ localizing prior to BubR1 and Bub1. To better understand how complexes containing Bub3 and its binding partners are loaded to kinetochores, we carried out size-exclusion chromatography and analyzed Bub3-containing complexes from cells under different spindle assembly checkpoint signaling conditions. We found that prior to kinetochore formation, Bub3 is complexed with BuGZ but not Bub1 or BubR1. Our results point to a model in which BuGZ stabilizes Bub3 and promotes Bub3 loading onto kinetochores in early mitosis, which, in turn, facilitates Bub1 and BubR1 kinetochore recruitment and spindle assembly checkpoint signaling.