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,George B Witman

doi:10.1074/jbc.m303064200

Abstract

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 (Kd) of approximately 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.

Highlights

The outer dynein arm-docking complex (ODA-DC) targets the outer dynein arm to its correct binding site on the flagellar axoneme
Expressed DC3 Is Biologically Functional—DC3 was expressed in E. coli as a C-terminal fusion with glutathione S-transferase (GST)
To determine whether bacterially expressed DC3 was biologically functional, the GST tag was removed by digesting the fusion protein with PreScissionTM protease (Amersham Biosciences), and the free DC3 was purified on a glutathione-Sepharose 4B column (Amersham Biosciences) (Fig. 3B)

Summary

Introduction

The outer dynein arm-docking complex (ODA-DC) targets the outer dynein arm to its correct binding site on the flagellar axoneme. To determine the in vivo function of the EF-hands of DC3, we used site-directed mutagenesis to inactivate them and transformed the mutated gene into a DC3-null strain that assembles only some of the outer dynein arms, swims slowly, and does not generate symmetrical bends during photoshock.

Results

Conclusion