Oda5p, a novel axonemal protein required for assembly of the outer dynein arm and an associated adenylate kinase.
Of the uncloned ODA genes required for outer dynein arm assembly in Chlamydomonas, ODA5 and ODA10 are of particular interest because they do not encode known subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC), and because genetic studies suggest their products interact. Beginning with a tagged oda5 allele, we isolated genomic and cDNA clones of the wild-type gene. ODA5 predicts a novel, 66-kDa coiled-coil protein. Immunoblotting indicates Oda5p is an axonemal component that assembles onto the axoneme independently of the outer arm and ODA-DC and is uniquely missing in oda5 and oda10 axonemes. Oda5p is released from the axoneme by extraction with 0.6 M KCl, but the soluble Oda5p does not cosediment with the outer dynein arm/ODA-DC in sucrose gradients. Quantitative mass spectrometry by using isotope coded affinity tagging revealed that a previously unidentified adenylate kinase is reduced 35-50% in oda5 flagella. Direct enzymatic assays demonstrated a comparable reduction in adenylate kinase activity in oda5 flagella, and also in oda10 flagella, but not in flagella of other oda mutants. We propose that Oda5p is part of a novel axonemal complex that is required for outer arm assembly and anchors adenylate kinase in proximity to the arm.
Wirschell
Maureen
M
Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Pazour
Gregory
G
Yoda
Akinori
A
Hirono
Masafumi
M
Kamiya
Ritsu
R
Witman
George B
GB
D000263:Adenylate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004398:Dyneins / Q000378:metabolism*; D005407:Flagella / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D017125:Genes, Protozoan / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D015800:Protozoan Proteins; D012333:RNA, Messenger; D000263:Adenylate Kinase; D004398:Dyneins