The functional region of CENP-H interacts with the Nuf2 complex that localizes to centromere during mitosis.
CENP-H is a constitutive centromere component that localizes to the centromere throughout the cell cycle. Because CENP-H is colocalized with CENP-A and CENP-C, it is thought to be an inner centromere protein. We previously generated a conditional loss-of-function mutant of CENP-H and showed that CENP-H is required for targeting of CENP-C to the centromere in chicken DT40 cells. In the present study, we used this mutant to identify the functional region of chicken CENP-H necessary for centromere targeting and cell viability. This region was found by yeast two-hybrid analysis to interact with Hec1, which is a member of the Nuf2 complex that transiently localizes to the centromere during mitosis. Coimmunoprecipitation experiments revealed that CENP-H interacts with the Nuf2 complex in chicken DT40 cells. Photobleaching experiments showed that both Hec1 and CENP-H form stable associations with the centromeres during mitosis, suggesting that Hec1 acts as a structural component of centromeres during mitosis. On the basis of these results and previously published data, we propose that the Nuf2 complex functions as a connector between the inner and outer kinetochores.
Mikami
Yoshikazu
Y
PRESTO of JST, National Institute of Genetics and The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan.
Hori
Tetsuya
T
Kimura
Hiroshi
H
Fukagawa
Tatsuo
T
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002503:Centromere / Q000737:chemistry / Q000378:metabolism*; D002645:Chickens / Q000235:genetics; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D005816:Genetic Complementation Test; D047468:Immunoprecipitation; D008938:Mitosis / Q000502:physiology*; D008954:Models, Biological; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism*; D038761:Photobleaching; D025941:Protein Interaction Mapping; D017384:Sequence Deletion / Q000235:genetics; D020798:Two-Hybrid System Techniques
D002868:Chromosomal Proteins, Non-Histone; D009687:Nuclear Proteins