KI Motifs of Human Knl1 Enhance Assembly of Comprehensive Spindle Checkpoint Complexes around MELT Repeats

Abstract

The KMN network, a ten-subunit protein complex, mediates the interaction of kinetochores with spindle microtubules and recruits spindle assembly checkpoint (SAC) constituents to halt cells in mitosis until attainment of sister chromatid biorientation. Two types of motifs in the KMN subunit Knl1 interact with SAC proteins. Lys-Ile (KI) motifs, found in vertebrates, interact with the TPR motifs of Bub1 and BubR1. Met-Glu-Leu-Thr (MELT) repeats, ubiquitous in evolution, recruit the Bub3/Bub1 complex in a phosphorylation-dependent manner. The exact contributions of KI and MELT motifs to SAC signaling and chromosome alignment are unclear. We report here that KI motifs cooperate strongly with the neighboring single MELT motif in the N-terminal 250 residues (Knl1(1-250)) of human Knl1 to seed a comprehensive assembly of SAC proteins. In cells depleted of endogenous Knl1, kinetochore-targeted Knl1(1-250) suffices to restore SAC and chromosome alignment. Individual MELT repeats outside of Knl1(1-250), which lack flanking KI motifs, establish qualitatively similar sets of interactions, but less efficiently. MELT sequences on Knl1 emerge from our analysis as the platforms on which SAC complexes become assembled. Our results show that KI motifs are enhancers of MELT function in assembling SAC signaling complexes, and that they might have evolved to limit the expansion of MELT motifs by providing a more robust mechanism of SAC signaling around a single MELT. We shed light on the mechanism of Bub1 and BubR1 recruitment and identify crucial questions for future studies.