HA-tagging of putative flagellar proteins in Chlamydomonas reinhardtii identifies a novel protein of intraflagellar transport complex B.
HA-tagging of putative flagellar proteins in Chlamydomonas reinhardtii identifies a novel protein of intraflagellar transport complex B.
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DOI：
10.1002/cm.20369
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发表时间：
2009-08
影响因子：
--
通讯作者：
Witman, George B.
中科院分区：
文献类型：
--
作者：
Lechtreck, Karl-Ferdinand;Luro, Scott;Awata, Junya;Witman, George B.
关键词：
对绿藻莱茵衣藻鞭毛的蛋白质组学分析已鉴定出 600 多种假定的鞭毛蛋白。克隆编码先前未表征的九个基因以及先前描述的PACRG蛋白的基因，插入到在基因产物的C末端添加三重HA标签的载体中，并转化到莱茵衣藻中。通过蛋白质印迹证实表达。间接免疫荧光定位了鞭毛中的所有十种融合蛋白； PACRG 定位于外部双联微管的一个子集。对于某些蛋白质，在细胞体内观察到额外的信号。 FAP232-HA就是后者之一，它沿着鞭毛呈点状分布，并在基体处聚集。这种模式是鞭毛内转运 (IFT) 蛋白的特征。 FAP232-HA 与 IFT 蛋白 IFT46 共定位，并与蔗糖梯度中的 IFT 颗粒共沉淀。此外，它与 IFT 复合物 B 蛋白 IFT46 发生共免疫沉淀，但不与 IFT 复合物 A 蛋白 IFT139 发生共免疫沉淀。我们得出结论，FAP232是IFT复合物B的新成分，并将其重命名为IFT25。 IFT25 的同源物编码在组装纤毛或鞭毛的生物体子集的基因组中；莱茵衣藻 IFT25 与相应的人类蛋白有 37% 的同一性。缺乏纤毛和鞭毛的生物体的基因组中不存在编码 IFT25 同源物的基因，有趣的是，黑腹果蝇和秀丽隐杆线虫的基因组中也不存在编码 IFT25 同源物的基因，这表明 IFT25 在 IFT 中具有特殊的作用，而在线虫和果蝇中的纤毛或鞭毛的组装并不需要 IFT25。
Proteomic analysis of flagella from the green alga Chlamydomonas reinhardtii has identified over 600 putative flagella 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 ten 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.
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影响因子：
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作者：
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通讯作者：
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