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

The spindle assembly checkpoint (SAC) monitors and promotes kinetochoremicrotubule attachment during mitosis. Bubl and BubRl, SAC components, originated from duplication of an ancestor gene. Subsequent subfunctionalization established subordination: Bubl, recruited first to kinetochores, promotes successive BubRl recruitment. Because both Bubl and BubRl hetero- dimerize with Bub3, a targeting adaptor for phosphorylated kinetochores, the molecular basis for such sub-functionalization is unclear. We demonstrate that Bubl, but not BubRl, enhances binding of Bub3 to phosphorylated kinetochores. Grafting a short motif of Bubl onto BubRl promotes Bubl-independent kinetochore recruitment of BubRl. Such gain-of-function BubRl mutant cannot sustain a functional checkpoint. We demonstrate that kinetochore localization of BubRl relies on direct hetero-dimerization with Bubl at a pseudo-symmetric interface. Such 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.