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Originally published In Press as doi:10.1074/jbc.M001124200 on April 14, 2000

J. Biol. Chem., Vol. 275, Issue 29, 22187-22195, July 21, 2000
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A Kinesin Mutation That Uncouples Motor Domains and Desensitizes the gamma -Phosphate Sensor*

Katherine M. BrendzaDagger §, Christopher A. Sontag, William M. SaxtonDagger ||, and Susan P. Gilbert**Dagger Dagger

From the Dagger  Department of Biology, Indiana University, Bloomington, Indiana 47405, the  Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, and the ** Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr291 of Drosophila kinesin heavy chain is identical in all superfamily members and is located in alpha -helix 5 on the microtubule-binding surface of the catalytic motor domain. Substitution of methionine at Thr291 results in complete loss of function in vivo. In vitro, the T291M mutation disrupts the ATPase cross-bridge cycle of a kinesin motor/neck construct, K401-4 (Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514). The pre-steady-state kinetic analysis presented here shows that ATP binding is weakened significantly, and the rate of ATP hydrolysis is increased. The mutant motor also fails to distinguish ATP from ADP, suggesting that the contacts important for sensing the gamma -phosphate have been altered. The results indicate that there is a signaling defect between the motor domains of the T291M dimer. The ATPase cycles of the two motor domains appear to become kinetically uncoupled, causing them to work more independently rather than in the strict, coordinated fashion that is typical of kinesin.

* This work was supported by National Institutes of Health Grants GM-54141 (to S. P. G.) and GM-46295 (to W. M. S.), American Cancer Society (ACS) Grant IRG-58-35 (to S. P. G.), and March of Dimes Birth Defects Foundation Grant 5-FY95-1136 (to S. P. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a Predoctoral Fellowship from the American Heart Association (AHA), Indiana Affiliate, Inc. Present address: Dept. of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110.

|| Supported by an AHA Established Investigatorship with funds contributed in part by the AHA, Indiana Affiliate, Inc.

Dagger Dagger Supported in part by an ACS Junior Faculty Research Award (JFRA-618). To whom correspondence should be addressed: Dept. of Biological Sciences, University of Pittsburgh, 518 Langley Hall, Fifth and Ruskin, Pittsburgh, PA 15260. Tel.: 412-624-5842; Fax: 412-624-9311; E-mail: spg1+@pitt.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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