During mitosis, faithful chromosome segregation depends on the formation of proper kinetochore-microtubule attachment. This process is tightly controlled by the Spindle Assembly Checkpoint (SAC), which requires kinetochore recruitment of several SAC proteins to assemble the Mitotic Checkpoint Complex (MCC). The MCC assembly rate is limited by a structural rearrangement of the MAD2 protein, required for its binding to CDC20. While the spontaneous MAD2 conversion requires hours in vitro, it requires only few seconds in cells, indicating that the conversion is catalyzed. However, the conversion of MAD2 occurs spontaneously in the presence of a catalytic scaffold (MAD1:MAD2, BUB1:BUB3, MPS1), despite being a kinetically disfavored reaction. Understanding how the kinetic barrier is lowered at the kinetochore and not favored in the cytoplasm is of key interest and remains largely obscure. Here, the main aim of my study was to understand how kinetochores stimulate the generation of the checkpoint effector, MCC during SAC signaling. 

In this study, I reconstituted a synthetic SAC functional kinetochore from individually purified proteins to directly address how kinetochores accomplish significantly higher acceleration of MCC formation in cells. The study provides a detailed analysis of the role of the outer kinetochore in MCC formation, utilizing a previously established FRET sensor to measure MCC in real-time in vitro. The results of this study demonstrate the importance of BUB1:BUB3 recruitment to the kinetochore via KNL1 in bringing together catalytic components at the kinetochore, thus enabling the spatial positioning of CDC20 MIM and MAD2 to catalyze their interaction. Additionally, this study highlights the role of MPS1-dependent phosphorylation in the stepwise assembly of BUB1:CDC20:MAD1. 

Overall, these findings shed light on the mechanisms underlying MCC formation at the kinetochore and provide insight into the role of specific proteins in facilitating this process. Altogether, we demonstrate that KMN accelerates the MCC assembly by increasing the local concentration of required substrates and catalysts.