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J. Biol. Chem., Vol. 280, Issue 32, 29088-29095, August 12, 2005

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Architecture of the Human Ndc80-Hec1 Complex, a Critical Constituent of the Outer Kinetochore^*

Claudio Ciferri, Jennifer De Luca¶, Silvia Monzani, Karin J. Ferrari, Dejan Ristic||, Claire Wyman||**, Holger Stark, John Kilmartin, Edward D. Salmon¶, and Andrea Musacchio¶¶

From the Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy, the Italian Foundation for Cancer Research (FIRC) Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy, the ^¶Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599, the ^||Department of Radiation Oncology, Daniel Den Hoed Cancer Center and the ^**Department of Cell Biology and Genetics, Erasmus Medical Center, P. O. Box 1738, 3000 DR, Rotterdam, The Netherlands, the Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Goettingen, Germany, and the Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom

The Ndc80 complex is a constituent of the outer plate of the kinetochore and plays a critical role in establishing the stable kinetochore-microtubule interactions required for chromosome segregation in mitosis. The Ndc80 complex is evolutionarily conserved and contains the four subunits Spc24, Spc25, Nuf2, and Ndc80 (whose human homologue is called Hec1). All four subunits are predicted to contain globular domains and extensive coiled coil regions. To gain an insight into the organization of the human Ndc80 complex, we reconstituted it using recombinant methods. The hydrodynamic properties of the recombinant Ndc80 complex are identical to those of the endogenous HeLa cell complex and are consistent with a 1:1:1:1 stoichiometry of the four subunits and a very elongated shape. Two tight Hec1-Nuf2 and Spc24-Spc25 subcomplexes, each stabilized by a parallel heterodimeric coiled coil, maintain this organization. These subcomplexes tetramerize via an interaction of the C- and N-terminal portions of the Hec1-Nuf2 and Spc24-Spc25 coiled coils, respectively. The recombinant complex displays normal kinetochore localization upon injection in HeLa cells and is therefore a faithful copy of the endogenous Ndc80 complex.

Received for publication, April 14, 2005 , and in revised form, May 31, 2005.

^* This work was supported by grants from the Italian Association for Cancer Research (AIRC), the Italian Ministry of Health, and the Human Frontier Science Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶¶ To whom correspondence should be addressed. Tel.: 39-02-57489829; Fax: 39-02-57489851; E-mail: andrea.musacchio{at}ifomieo-campus.it.

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