ABSTRACT: The centromere is a specialized domain that facilitates chromosome segregation during mitosis and serves as the site for kinetochore formation. KINETOCHORE NULL2 (αKNL2) is essential for the recognition and loading of the centromeric histone H3 variant, CENH3, to 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. Bioinformatic and functional analysis identified three SUMOylation and two SUMO-interacting motif (SIM) sites in the C-terminal region of αKNL2, which are critical for growth, fertility, and chromosome alignment. Of the three SUMOylation sites, Lys474 was 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 MS analysis revealed that SUMO1 modified several lysine residues, including K424, K445, K511, K540, K572, K583, K585, and K598, indicating extensive SUMOylation across the C-terminal region. In contrast, SUMO3-dependent modification was detected only at K424, K511, and K598. These findings are consistent with our in vitro assay results, which showed stronger SUMOylation by the SUMO1 isoform than SUMO3.
David Potesil 
