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J. Biol. Chem., Vol. 279, Issue 49, 51354-51361, December 3, 2004

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Mechanistic Analysis of the Saccharomyces cerevisiae Kinesin Kar3^*

Andrew T. Mackey, Lisa R. Sproul, Christopher A. Sontag¶, Lisa L. Satterwhite||, John J. Correia||, and Susan P. Gilbert**

From the Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, the ^¶Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216, and the ^||Department of Medicine-Cardiology, Duke University Medical Center, Durham, North Carolina 27710

Kar3 is a minus-end-directed microtubule motor that is implicated in meiotic and mitotic spindle function in Saccharomyces cerevisiae. To date, the only truncated protein of Kar3 that has been reported to promote unidirectional movement in vitro is GSTKar3. This motor contains an NH₂-terminal glutathione S-transferase (GST) tag followed by the Kar3 sequence that is predicted to form an extended -helical coiled-coil. The -helical domain leads into the neck linker and COOH-terminal motor domain. Kar3 does not homodimerize with itself but forms a heterodimer with either Cik1 or Vik1, both of which are non-motor polypeptides. We evaluated the microtubule-GSTKar3 complex in comparison to the microtubule-Kar3 motor domain complex to determine the distinctive mechanistic features required for GSTKar3 motility. Our results indicate that ATP binding was significantly faster for GSTKar3 than that observed previously for the Kar3 motor domain. In addition, microtubule-activated ADP release resulted in an intermediate that bound ADP weakly in contrast to the Kar3 motor domain, suggesting that after ADP release, the microtubule-GSTKar3 motor binds ATP in preference to ADP. The kinetics also showed that GST-Kar3 readily detached from the microtubule rather than remaining bound for multiple ATP turnovers. These results indicate that the extended -helical domain NH₂-terminal to the catalytic core provides the structural transitions in response to the ATPase cycle that are critical for motility and that dimerization is not specifically required. This study provides the foundation to define the mechanistic contributions of Cik1 and Vik1 for Kar3 force generation and function in vivo.

Received for publication, June 4, 2004 , and in revised form, August 30, 2004.

^* This work was supported in part by NIGMS National Institutes of Health Grant GM54141 and NIAMSD National Institute of Health, Career Development Award K02-AR47841 from the Department of Health and Human Services (to S. P. G.). 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 Figs. S1–S2.

Recipient of an Andrew Mellon predoctoral fellowship. Present address: Dept. of Molecular, Cellular, and Developmental Biology, Yale University, P.O. Box 208103, New Haven, CT 06520-8103.

^** To whom correspondence should be addressed: Dept. of Biological Sciences, 518 Langley Hall, University of Pittsburgh, Pittsburgh, PA 15260. Tel.: 412-624-5842; Fax: 412-624-4759; E-mail: spg1{at}pitt.edu.

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