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MBC in Press, published online ahead of print December 19, 2007
Mol. Biol. Cell 10.1091/mbc.E07-06-0556

A more recent version of this article appeared on March 1, 2008
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Submitted on June 12, 2007
Revised on November 21, 2007
Accepted on December 10, 2007

CENP-O-Class Proteins Form a Stable Complex and Are Required for Proper Kinetochore Function

Tetsuya Hori,* Masahiro Okada,* Katsumi Maenaka,{dagger} and Tatsuo Fukagawa*

*Department of Molecular Genetics, National Institute of Genetics and The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan; {dagger}Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan

Monitoring Editor: Ted Salmon

We previously identified a multi-subunit complex (CENP-H/I complex) in kinetochores from human and chicken cells. We showed that the CENP-H/I complex is divided into three functional classes. In the present study, we investigated CENP-O-class proteins, which include CENP-O, -P, -Q, -R, and -50 (U). We created chicken DT40 cell knockouts of each of these proteins and found that all knockout lines were viable but showed slow proliferation and mitotic defects. Kinetochore localization of CENP-O, -P, -Q, and -50 was interdependent, but kinetochore localization of these proteins was observed in CENP-R-deficient cells. A coexpression assay in bacteria showed that CENP-O, -P, -Q, and -50 proteins form a stable complex that can associate with CENP-R. Phenotype analysis of knockout cells showed that all proteins except for CENP-R were required for recovery from spindle damage, and phosphorylation of CENP-50 was essential for recovery from spindle damage. We also found that treatment with the proteasome inhibitor MG132 partially rescued the severe mitotic phenotype observed in response to release from nocodazole block in CENP-50-deficient cells. This suggests that CENP-O-class proteins are involved in the prevention of premature sister chromatid separation during recovery from spindle damage.

Address correspondence to: Tatsuo Fukagawa (tfukagaw{at}lab.nig.ac.jp)






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