INSIGHTS INTO THE MECHANISM OF RECRUITMENT OF CHECKPOINT PROTEINS BUB1 AND BUBR1 TO KINETOCHORE SITES

Bub1 and BubR1 are essential components of the spindle assembly checkpoint (SAC), a ubiquitous safety mechanism required for accurate segregation of chromosomes during mitosis. Recruitment to mitotic kinetochores, protein assemblies built on the centromeric DNA, might be essential for the functions of Bub1 and BubR1, but the exact recruitment mechanism has been unknown. During my doctoral work, I have tried to investigate this issue at a molecular level. Previously, kinetochore recruitment of Bub1 and BubR1 had been proposed to rely on the interaction of their tetratricopeptide repeats (TPR repeats) with two motifs, named the KI motifs, in the outer kinetochore protein Knl1. In the first part of my doctoral work, I demonstrate that point mutations on the TPR repeats that impair the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo have essentially no macroscopic effect on the localization of Bub1 and BubR1 at kinetochores. Indeed, we have been able to define a 62-residue segment of Bub1, comprising a motif that mediates the interaction with another checkpoint protein, Bub3, as the minimal kinetochore-binding domain of Bub1. Subsequent studies in other laboratories have identified multiple Met-Glu-Leu-Thr (MELT) motifs in the kinetochore protein Knl1 as crucial docking sites, when phosphorylated by the Mps1 kinase, for the recruitment of Bub1 and BubR1. In the second part of my work, I therefore began to test the hypothesis that Bub1 recognizes MELT repeats on Knl1, through the minimal kinetochore targeting domain and its partner Bub3. Within the context of this new model, I have re-investigated the question whether the KI1 and KI2 motifs have any role in the interaction of Bub1 and BubR1 with Knl1. I provide evidence that Knl11-250 (N-terminal 250 residues of Knl1), with a single MELT motif, drive the assembly of complexes that included all SAC proteins and are sufficient to respond robustly to spindle poisons. Interestingly, I have found that the KI motifs, which flank the MELT motif in Knl11-250, strongly enhance the interaction with SAC components. Conversely, MELT motifs outside of Knl11-250, which lack flanking KI motifs, establish qualitatively similar sets of interactions, but less efficiently. Thus, my analyses indicate that MELT motifs act as independent docking sites for Bub1/Bub3 and assembly stations for SAC signaling complexes, and that KI motifs are MELT enhancers. Collectively, my work has contributed to elucidating important aspects of the molecular mechanism of kinetochore recruitment of two fundamental components of the spindle assembly checkpoint.