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 th...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 forαKNL2 interactors identified components of the SUMOylation pathway.However,the role ofαKNL2 SUMOylation in Arabidopsis has not yet been determined.In this study,we demonstrated that the C-terminal region ofαKNL2(designatedαKNL2-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 ofαKNL2-C identified three SUMOylation sites and two SUMOinteracting 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 ofαKNL2,underscoring the importance ofαKNL2 SUMOylation for its function.Additionally,both in vitro and in vivo assays showed thatαKNL2-C undergoes SUMOylation by SUMO1 or SUMO3.The Arabidopsis SUMO protease mutant ulp1d-2 exhibits a mild accumulation of SUMOylatedαKNL2.We further showed that SUMOylation ofαKNL2 promotes its binding to CENH3 and controls protein stability.Our findings demonstrate that C-terminal SUMOylation ofαKNL2 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.展开更多
基金supported by Deutsche Forschungsgemeinschaft(DFG)grant DFG LE 2299/5-1J.V.acknowledges support from ProteoCure COST(European Cooperation in Science and Technology)Action CA20113+1 种基金which funded the research stay during which the methodology applied in this publication was acquired.J.R.C.was supported by DFG grant DFG LE 2299/8-1.J.J.P.was supported by the Czech Science Foundation(GA23-05284S)The CEITEC Proteomics Core Facility of CIISB,Instruct-CZ Centre,was supported by MEYS CR(LM2023042,CZ.02.01.01/00/23_015/0008175,and e-INFRA CZ[ID:90254]).
文摘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 forαKNL2 interactors identified components of the SUMOylation pathway.However,the role ofαKNL2 SUMOylation in Arabidopsis has not yet been determined.In this study,we demonstrated that the C-terminal region ofαKNL2(designatedαKNL2-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 ofαKNL2-C identified three SUMOylation sites and two SUMOinteracting 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 ofαKNL2,underscoring the importance ofαKNL2 SUMOylation for its function.Additionally,both in vitro and in vivo assays showed thatαKNL2-C undergoes SUMOylation by SUMO1 or SUMO3.The Arabidopsis SUMO protease mutant ulp1d-2 exhibits a mild accumulation of SUMOylatedαKNL2.We further showed that SUMOylation ofαKNL2 promotes its binding to CENH3 and controls protein stability.Our findings demonstrate that C-terminal SUMOylation ofαKNL2 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.
