KNL2 degradation is regulated by APC/C ubiquitin ligase

ABSTRACT: Centromeres are specialized chromosomal regions where kinetochore proteins form a protein complex that regulates the cell cycle. KNL2 is an essential protein for kinetochore assembly and plays a significant role in the loading of CENH3. Therefore, the level of KNL2 in plants and other organisms needed to be tightly regulated. Overexpressed AtKNL2 is shown to be degraded via the proteasome degradation pathway by applying an MG115 inhibitor. However, the molecular mechanisms behind the degradation pathway of KNL2 is unknown. Therefore, here we show that the APC/C-dependent degradation of KNL2 is required for maintaining the level and localization of KNL2. We confirmed that the ubiquitin-proteasome system polyubiquitinates and degrades KNL2. Further, IP-MS and Y2H library screening assays performed with KNL2 identified 32 proteins from the ubiquitination pathway. The direct interaction of KNL2 with APC2 was identified using BiFC interaction analysis. Further, the APC10 and Cdc20, core subunit of APC/C, interacts directly with KNL2 as shown by BiFC and Y2H, which is required for substrate recruitment. Moreover, Apcin inhibitor treatment suppresses the degradation of KNL2 in Arabidopsis, validating the involvement of APC/C in KNL2 degradation. Furthermore, our findings highlight the significance of the conserved D-box1 region as a degron for KNL2 in Arabidopsis, facilitating its interaction with APC/C. Further, the interaction of ubiquitin E2 conjugating enzymes (UBC19 and UBC20) with KNL2 has been demonstrated and transfer ubiquitin protein to the lysine sites 336K and 339K of KNL2. Thus, our findings advance our knowledge of cell cycle dynamics by providing insightful information about the molecular mechanisms governing KNL2 regulation.

INSTRUMENT(S):

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell

SUBMITTER: Astrid Bruckmann  

LAB HEAD: Dr. Astrid Bruckmann

PROVIDER: PXD051741 | Pride | 2025-07-14

REPOSITORIES: Pride