BubR1 recruitment to the kinetochore via Bub1 enhances Spindle Assembly Checkpoint signaling

During mitosis, unattached kinetochores in a dividing cell activate the Spindle Assembly Checkpoint (SAC) and delay anaphase onset by generating the anaphase-inhibitory Mitotic Checkpoint Complex (MCC). These kinetochores generate the MCC by recruiting MCC constituent proteins, including BubR1. In principle, BubR1 recruitment to signaling kinetochores should increase its local concentration and promote MCC formation. However, in human cells BubR1 is mainly thought to sensitize the SAC to silencing; whether BubR1 localization to signaling kinetochores per se enhances SAC signaling activity remains unknown. Therefore, we used ectopic SAC activation systems (eSAC) to isolate two molecules that recruit BubR1 to the kinetochore: the checkpoint protein Bub1 and the KI and MELT motifs in the kinetochore protein KNL1, and observed their contribution to eSAC signaling. Our quantitative analyses and mathematical modeling show that the Bub1-mediated BubR1 recruitment to the human kinetochore promotes SAC signaling and highlight BubR1s dual role of directly strengthening the SAC and indirectly silencing it.