A molecular basis for the differential roles of Bub1 and BubR1 in the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) monitors and promotes kinetochore-microtubule attachment during Mitosis. BUB1 and BUBR1, SAC components, originated from duplication of an ancestor gene. Subsequent sub-functionalization established subordination: BUB1, recruited first to kinetochores, promotes successive BUBR1 recruitment. Because both BUB1 and BUBR1 hetero-dimerize with Bub3, a targeting adaptor for phosphorylated kinetochores, the molecular basis for such sub-functionalization is unclear. We demonstrate that BUB1, but not BUBR1, enhances binding of Bub3 to phosphorylated kinetochores. Grafting a short motif of BUB1 onto BUBR1 promotes Bub1-independent kinetochore recruitment of BUBR1. This gain-of-function BUBR1 mutant cannot sustain a functional checkpoint. We demonstrate that kinetochore localization of BUBR1 relies on direct hetero-dimerization with BUB1 at a pseudo-symmetric interface. This pseudo-symmetric interaction underpins a template-copy relationship crucial for kinetochore-microtubule attachment and SAC signaling. Our results illustrate how gene duplication and sub-functionalization shape the workings of an essential molecular network.