HA-tagging of putative flagellar proteins in Chlamydomonas reinhardtii identifies a novel protein of intraflagellar transport complex B

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dc.contributor.authorLechtreck, Karl-Ferdinand
dc.contributor.authorLuro, Scott
dc.contributor.authorAwata, Junya
dc.contributor.authorWitman, George B.
dc.contributor.departmentDepartment of Cell Biology
dc.date2022-08-11T08:11:05.000
dc.date.accessioned2022-08-23T17:33:04Z
dc.date.available2022-08-23T17:33:04Z
dc.date.issued2009-08-22
dc.date.submitted2011-01-21
dc.description.abstractProteomic 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.
dc.identifier.citationCell Motil Cytoskeleton. 2009 Aug;66(8):469-82. <a href="http://dx.doi.org/10.1002/cm.20369">Link to article on publisher's site</a>
dc.identifier.contextkey1739061
dc.identifier.doi10.1002/cm.20369
dc.identifier.issn0886-1544 (Linking)
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/witman/3
dc.identifier.submissionpathwitman/3
dc.identifier.urihttps://hdl.handle.net/20.500.14038/51088
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=19382199&dopt=Abstract">Link to Article in PubMed</a>
dc.relation.urlhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2922027/pdf/nihms139411.pdf
dc.source.issue8
dc.source.journaltitleCell motility and the cytoskeleton
dc.source.pages469-82
dc.source.volume66
dc.subjectAmino Acid Sequence
dc.subjectAnimals
dc.subjectBlotting, Western
dc.subjectChlamydomonas reinhardtii
dc.subjectFlagella
dc.subjectFluorescent Antibody Technique, Indirect
dc.subjectImmunoprecipitation
dc.subjectModels, Genetic
dc.subjectMolecular Sequence Data
dc.subjectPolymerase Chain Reaction
dc.subjectProtein Binding
dc.subjectProtozoan Proteins
dc.subjectRecombinant Fusion Proteins
dc.subjectSequence Homology, Amino Acid
dc.subjectCell Biology
dc.titleHA-tagging of putative flagellar proteins in Chlamydomonas reinhardtii identifies a novel protein of intraflagellar transport complex B
dc.typeJournal Article
dspace.entity.typePublication
html.description.abstract<p>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.</p>
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