Human CENP-H multimers colocalize with CENP-A and CENP-C at active centromere-kinetochore complexes

Centromere and kinetochore proteins have a pivotal role in centromere struc ture, kinetochore formation;Ind sister chromatid separation. However, the m olecular architecture and the precise dynamic function of the centromere-ki netochore complex during mitosis remain poorly understood. Here we report t he isolation and characterization of human CENP-H, Confocal microscopic ana lyses of HeLa cells with anti-human CENP-H-specific antibody demonstrated t hat CENP-H colocalizes with inner kinetochore plate proteins CENP-A and CEN P-C in both interphase and metaphase. CENP-H was present outside centromeri c heterochromatin, where CENP-B is localized, and inside the kinetochore co rona, where CENP-E is localized during prometaphase. Furthermore, CENP-H wa s detected at neocentromeres, but not at inactive centromeres in stable dic entric chromosomes. In vitro binding assays of human CENP-H with centromere -kinetochore proteins suggest that the CENP-H binds to itself and MCAK, but not to CENP-A, CENP-B or CENP-C, CENP-H multimers were observed in cells i n which both FLAG-tagged CENP-H and hemagglutinin-tagged CENP-H were expres sed, These results suggest that CENP-H multimers localize constitutively to the inner kinetochore plate and play an important fundamental role in orga nization and function of the active human centromere-kinetochore complex.