DC3, the smallest subunit of the Chlamydomonas flagellar outer dynein arm-docking complex, is a redox-sensitive calcium-binding protein.
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.
Casey
Diane M
DM
Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA.
Yagi
Toshiki
T
Kamiya
Ritsu
R
Witman
George B
GB
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002696:Chlamydomonas / Q000737:chemistry* / Q000502:physiology; D004398:Dyneins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D005407:Flagella / Q000737:chemistry / Q000502:physiology; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D013550:Swimming
D002135:Calcium-Binding Proteins; D021122:Protein Subunits; D015800:Protozoan Proteins; D004398:Dyneins; D008274:Magnesium; D002118:Calcium