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J. Biol. Chem., Vol. 278, Issue 43, 42652-42659, October 24, 2003

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DC3, the Smallest Subunit of the Chlamydomonas Flagellar Outer Dynein Arm-docking Complex, Is a Redox-sensitive Calcium-binding Protein^*

Diane M. Casey, Toshiki Yagi, Ritsu Kamiya, and George B. Witman¶

From the Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655 and the Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113, Japan

The outer dynein arm-docking complex (ODA-DC) targets the outer dynein arm to its correct binding site on the flagellar axoneme. The Chlamydomonas ODA-DC contains three proteins; loss of any one prevents normal assembly of the outer arm, leading to a slow, jerky swimming phenotype. We showed previously that the smallest ODA-DC subunit, DC3, has four EF-hands (Casey, D. M., Inaba, K., Pazour, G. J., Takada, S., Wakabayashi, K., Wilkerson, C. G., Kamiya, R., and Witman, G. B. (2003) Mol. Biol. Cell 14, 3650-3663). Two of the EF-hands fit the consensus pattern for calcium binding, and one of these contains two cysteine residues within its binding loop. To determine whether the predicted EF-hands are functional, we purified bacterially expressed wild-type DC3 and analyzed its calcium-binding potential in the presence and absence of dithiothreitol and Mg²⁺. The protein bound one calcium ion with an affinity (K_d) of 1 x 10^-5 M. Calcium binding was observed only in the presence of dithiothreitol and thus is redox-sensitive. DC3 also bound Mg²⁺ at physiological concentrations but with a much lower affinity. Changing the essential glutamate to glutamine in both EF-hands eliminated the calcium binding activity of the bacterially expressed protein. To investigate the role of the EF-hands in vivo, we transformed the modified DC3 gene into a Chlamydomonas insertional mutant lacking DC3. The transformed strain swam normally, assembled a normal number of outer arms, and had a normal photoshock response, indicating that the Glu to Gln mutations did not affect ODA-DC assembly, outer arm assembly, or Ca²⁺-mediated outer arm activity. Thus, DC3 is a true calcium-binding protein, but the function of this activity remains unknown.

Received for publication, March 25, 2003 , and in revised form, August 13, 2003.

^* This work was supported by National Institutes of Health Grant GM30626 (to G. B. W.), by the Robert W. Booth Fund at the Greater Worcester Community Foundation (to G. B. W.), by a National Institutes of Health Individual National Research Service Award (predoctoral) (to D. M. C.), by a Summer Program in Japan fellowship from the National Science Foundation and the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to D. M. C.), and by a grant from MEXT (to T. Y. and R. K.). 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: Dept. of Cell Biology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655. Tel.: 508-856-4038; Fax: 508-856-1033; E-mail: george.witman{at}umassmed.edu.

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