Ubiquitin-dependent proteolysis of KNL2 driven by APC/CCDC20 is critical for centromere integrity and mitotic fidelity.

Kinetochores are large protein complexes that serve as attachment sites for spindle microtubules, ensuring proper chromosome segregation during cell division. KINETOCHORE NULL2 (αKNL2) is a key kinetochore protein required for the incorporation of the centromeric histone variant CENH3. The precise regulation of αKNL2 levels is crucial, but the molecular mechanisms controlling this process remain largely unexplored. In this study, we demonstrated that the Anaphase-Promoting Complex/Cyclosome (APC/C) mediates the ubiquitin-dependent proteolysis of αKNL2 during mitosis. Our findings revealed that αKNL2 accumulates in the presence of 26S proteasome inhibitors, and our yeast two-hybrid and proteomic screens showed that proteins from the ubiquitin-proteasome pathway interact with KNL2 in Arabidopsis (Arabidopsis thaliana) and nematode (Caenorhabditis elegans). Arabidopsis αKNL2 directly interacts with Anaphase-Promoting Complex subunit 10 (APC10) and Cell Division Cycle 20.1 (CDC20.1), two substrate recognition components of the APC/C. RNAi-mediated depletion of APC/C resulted in the accumulation and mislocalization of endogenous αKNL2. Additionally, mutation or deletion of the D-box1 region, or substitution of residues K336 and K339, impaired αKNL2 degradation. The expression of a proteasome-resistant αKNL2 variant in planta caused severe defects in growth, fertility, and mitotic division. These findings show that APC/CCDC20-mediated degradation of αKNL2 is critical for proper kinetochore function and centromere integrity.