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 structure, kinetochore formation and sister chromatid separation. However, the molecular architecture and the precise dynamic function of the centromere-kinetochore complex during mitosis remain poorly understood. Here we report the isolation and characterization of human CENP-H. Confocal microscopic analyses of HeLa cells with anti-human CENP-H-specific antibody demonstrated that CENP-H colocalizes with inner kinetochore plate proteins CENP-A and CENP-C in both interphase and metaphase. CENP-H was present outside centromeric heterochromatin, where CENP-B is localized, and inside the kinetochore corona, where CENP-E is localized during prometaphase. Furthermore, CENP-H was detected at neocentromeres, but not at inactive centromeres in stable dicentric 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 in which both FLAG-tagged CENP-H and hemagglutinin-tagged CENP-H were expressed. These results suggest that CENP-H multimers localize constitutively to the inner kinetochore plate and play an important fundamental role in organization and function of the active human centromere-kinetochore complex.
Sugata
N
N
Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
Li
S
S
Earnshaw
W C
WC
Yen
T J
TJ
Yoda
K
K
Masumoto
H
H
Munekata
E
E
Warburton
P E
PE
Todokoro
K
K
D000595:Amino Acid Sequence; D001324:Autoantigens; D002503:Centromere / Q000378:metabolism*; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D018386:Kinetochores / Q000378:metabolism*; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence; D008938:Mitosis; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment
D001324:Autoantigens; C000619700:CENPA protein, human; C121933:CENPH protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; C060905:centromere protein C