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J. Biol. Chem., Vol. 278, Issue 44, 43571-43579, October 31, 2003

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Calcium Regulates ATP-sensitive Microtubule Binding by Chlamydomonas Outer Arm Dynein^*

Miho Sakato and Stephen M. King

From the Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06030-3305

The Chlamydomonas outer dynein arm contains three distinct heavy chains (, , and ) that exhibit different motor properties. The LC4 protein, which binds 1–2 Ca²⁺ with K_Ca = 3 x 10^–5 M, is associated with the heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca²⁺ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the heavy chain exhibits a dominant Ca²⁺-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the and heavy chains does not occur at Ca²⁺ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the heavy chain or in dyneins that lack both the heavy chain and the motor domain of the heavy chain. These findings strongly suggest that Ca²⁺ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.

Received for publication, June 4, 2003 , and in revised form, August 1, 2003.

^* This study was supported by National Institutes of Health Grant GM51293. 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.

Investigator of the Patrick and Catherine Weldon Donaghue Medical Research Foundation. To whom correspondence should be addressed: Dept. of Biochemistry, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3305. Tel.: 860-679-3347; Fax: 860-679-3408; E-mail: steve{at}king2.uchc.edu.

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