The C-terminal SUMOylation-dependent regulation of αKNL2 governs its centromere targeting and interaction with CENH3.

The centromere is a specialized domain that facilitates chromosome segregation during mitosis and serves as the site of kinetochore formation. KINETOCHORE NULL2 (αKNL2) is essential for the recognition and loading of the centromeric histone H3 variant CENH3 at centromeres. A yeast two-hybrid screen for αKNL2 interactors identified components of the SUMOylation pathway. However, the role of αKNL2 SUMOylation in Arabidopsis has not yet been determined. In this study, we demonstrated that the C-terminal region of αKNL2 (designated αKNL2-C) interacts with small ubiquitin-like modifier 3 (SUMO3) and ULP1d, as shown by bimolecular fluorescence complementation and co-immunoprecipitation assays. Bioinformatic and functional analyses of αKNL2-C identified three SUMOylation sites and two SUMO-interacting motifs, which were shown to be critical for growth, fertility, and chromosome alignment. Of the three SUMOylation sites, Lys474 and Lys511 are the most critical for the centromeric localization of αKNL2, underscoring the importance of αKNL2 SUMOylation for its function. Additionally, both in vitro and in vivo assays showed that αKNL2-C undergoes SUMOylation by SUMO1 or SUMO3. The Arabidopsis SUMO protease mutant ulp1d-2 exhibits a mild accumulation of SUMOylated αKNL2. We further showed that SUMOylation of αKNL2 promotes its binding to CENH3 and controls protein stability. Our findings demonstrate that C-terminal SUMOylation of αKNL2 is crucial for its centromeric localization, interaction with CENH3, and kinetochore assembly, emphasizing the significance of post-translational modifications in chromosome segregation and cell division in plants.