Two functional thioredoxins containg redox-senesitive vicinal dithiols from the Chlamydomonas outer dynein arm.
We describe here the molecular cloning and analysis of the Mr 14,000 and 16,000 outer arm dynein light chains (DLCs) from Chlamydomonas flagella. Within the outer arm, the Mr 14,000 DLC apparently is associated with the intermediate chains at the base of the soluble dynein particle; the Mr 16,000 DLC interacts directly with the a dynein heavy chain. Sequence analysis indicates that both molecules are novel members of the thioredoxin superfamily and share approximately 30% sequence identity with thioredoxin from Penicillium. Both DLCs have a perfect copy of the thioredoxin active site (WCGPCK); the Mr 16,000 DLC also contains the canonical P-loop motif (AX4GKS). There is a single gene for both DLCs within Chlamydomonas and only single messages that were upregulated more than 10-fold upon deflagellation were observed on Northern blots. Both recombinant DLCs were specifically eluted from a phenylarsine oxide matrix with beta-mercaptoethanol indicating that they contain vicinal dithiols competent to undergo reversible oxidation/reduction. Furthermore, we demonstrate that outer (but not inner) arm dynein may he purified on the basis of its affinity for phenylarsine oxide suggesting that the predicted redox-sensitive vicinal dithiols exist within the native complex.
Patel-King
R S
RS
Department of Biochemistry, University of Connecticut Health Center, Farmington 06032-3305, USA.
Benashki
S E
SE
Harrison
A
A
King
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016825:Chlamydomonas reinhardtii / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004398:Dyneins / Q000737:chemistry* / Q000235:genetics; D017125:Genes, Protozoan; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D016054:DNA, Protozoan; D004220:Disulfides; D013879:Thioredoxins; D004398:Dyneins