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HA-tagging of putative flagellar proteins in Chlamydomonas reinhardtii identifies a novel protein of intraflagellar transport complex B.
- Source :
-
Cell motility and the cytoskeleton [Cell Motil Cytoskeleton] 2009 Aug; Vol. 66 (8), pp. 469-82. - Publication Year :
- 2009
-
Abstract
- Proteomic analysis of flagella from the green alga Chlamydomonas reinhardtii has identified over 600 putative flagellar proteins. The genes encoding nine of these not previously characterized plus the previously described PACRG protein were cloned, inserted into a vector adding a triple-HA tag to the C-terminus of the gene product, and transformed into C. reinhardtii. Expression was confirmed by western blotting. Indirect immunofluorescence located all 10 fusion proteins in the flagellum; PACRG was localized to a subset of outer doublet microtubules. For some proteins, additional signal was observed in the cell body. Among the latter was FAP232-HA, which showed a spotted distribution along the flagella and an accumulation at the basal bodies. This pattern is characteristic for intraflagellar transport (IFT) proteins. FAP232-HA co-localized with the IFT protein IFT46 and co-sedimented with IFT particles in sucrose gradients. Furthermore, it co-immunoprecipitated with IFT complex B protein IFT46, but not with IFT complex A protein IFT139. We conclude that FAP232 is a novel component of IFT complex B and rename it IFT25. Homologues of IFT25 are encoded in the genomes of a subset of organisms that assemble cilia or flagella; C. reinhardtii IFT25 is 37% identical to the corresponding human protein. Genes encoding IFT25 homologues are absent from the genomes of organisms that lack cilia and flagella and, interestingly, also from those of Drosophila melanogaster and Caenorhabditis elegans, suggesting that IFT25 has a specialized role in IFT that is not required for the assembly of cilia or flagella in the worm and fly. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.
- Subjects :
- Amino Acid Sequence
Animals
Blotting, Western
Chlamydomonas reinhardtii genetics
Fluorescent Antibody Technique, Indirect
Immunoprecipitation
Models, Genetic
Molecular Sequence Data
Polymerase Chain Reaction
Protein Binding
Protozoan Proteins chemistry
Protozoan Proteins genetics
Recombinant Fusion Proteins chemistry
Recombinant Fusion Proteins genetics
Sequence Homology, Amino Acid
Chlamydomonas reinhardtii metabolism
Flagella metabolism
Protozoan Proteins metabolism
Recombinant Fusion Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1097-0169
- Volume :
- 66
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Cell motility and the cytoskeleton
- Publication Type :
- Academic Journal
- Accession number :
- 19382199
- Full Text :
- https://doi.org/10.1002/cm.20369