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Originally published In Press as doi:10.1074/jbc.M604817200 on July 7, 2006
J. Biol. Chem., Vol. 281, Issue 36, 26004-26013, September 8, 2006
Homotetrameric Form of Cin8p, a Saccharomyces cerevisiae Kinesin-5 Motor, Is Essential for Its in Vivo Function*
Emily R. Hildebrandt 1,
Larisa Gheber ,
Tami Kingsbury , and
M. Andrew Hoyt 2
From the
Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218 and the Departments of Clinical Biochemistry and Chemistry, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
Kinesin-5 motor proteins are evolutionarily conserved and perform essential roles in mitotic spindle assembly and spindle elongation during anaphase. Previous studies demonstrated a specialized homotetrameric structure with two pairs of catalytic domains, one at each end of a dumbbell-shaped molecule. This suggests that they perform their spindle roles by cross-linking and sliding antiparallel spindle microtubules. However, the exact kinesin-5 sequence elements that are important for formation of the tetrameric complexes have not yet been identified. In addition, it has not been demonstrated that the homotetrameric form of these proteins is essential for their biological functions. Thus, we investigated a series of Saccharomyces cerevisiae Cin8p truncations and internal deletions, in order to identify structural elements in the Cin8p sequence that are required for Cin8p functionality, spindle localization, and multimerization. We found that all variants of Cin8p that are functional in vivo form tetrameric complexes. The first coiled-coil domain in the stalk of Cin8p, a feature that is shared by all kinesin-5 homologues, is required for its dimerization, and sequences in the last part of the stalk, specifically those likely involved in coiled-coil formation, are required for Cin8p tetramerization. We also found that dimeric forms of Cin8p that are nonfunctional in vivo can nonetheless bind to microtubules. These findings suggest that binding of microtubules is not sufficient for the functionality of Cin8p and that microtubule cross-linking by the tetrameric complex is essential for Cin8p mitotic functions.
Received for publication, May 18, 2006
* This work was supported in part by Israel-United States Bi-National Grant 2003141 (to L. G. and M. A. H.), Israeli Science Foundation Grant 822/04 (to L. G.), and National Institutes of Health Grant GM40714 (to M. A. H.). 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.
The on-line version of this article (available at http://www.jbc.org) contains Tables S1 and S2 and a supplemental figure.
1 Supported in Part by NRSA Grant GM18745 from the National Institutes of Health.
2 To whom correspondence should be addressed: Dept. of Biology, The Johns Hopkins University, 3400 North Charles St., Baltimore, MD 21218-2685. Tel.: 410-516-7299; Fax: 410-516-5213; E-mail: hoyt{at}jhu.edu.

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Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
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