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Molecular and Cellular Biology, March 2005, p. 1958-1970, Vol. 25, No. 5
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.5.1958-1970.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Functional Region of CENP-H Interacts with the Nuf2 Complex That Localizes to Centromere during Mitosis{dagger}

Yoshikazu Mikami,1 Tetsuya Hori,1 Hiroshi Kimura,2 and Tatsuo Fukagawa1*

PRESTO of JST, National Institute of Genetics and The Graduate University for Advanced Studies, Mishima, Shizuoka,1 Nuclear Function and Dynamics Unit, HMRO, Graduate School of Medicine, Kyoto University, Kyoto, Japan2

Received 2 July 2004/ Returned for modification 9 August 2004/ Accepted 23 November 2004

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.

* Corresponding author. Mailing address: National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. Phone: 81-55-981-6792. Fax: 81-55-981-6742. E-mail: tfukagaw{at}lab.nig.ac.jp.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, March 2005, p. 1958-1970, Vol. 25, No. 5
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.5.1958-1970.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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