Genomics and proteomics studies in Chlamydomonas have revealed that an axoneme is composed of 200-600 types of proteins, including uncharacterized proteins collectively named flagellar-associated proteins (FAPs). Nine FAPs contain the EF-hand motif; however, they have not yet been well characterized. To find components responsible for Chlamydomonas-specific waveform changes coupled with intracellular Ca2+ concentrations, we focused on FAP85, an EF-hand motif-containing FAP specific to Chlamydomonas and its relatives. We cloned the cDNA encoding FAP85, expressed it in Escherichia coli cells, and generated a polyclonal antibody against the expressed protein. Immunoblotting showed that FAP85 was present in every axoneme of several flagellar mutants lacking major axonemal components. Immuno-electron microscopy revealed that anti-FAP85 antibodies were found only on the inner wall of A-tubules of the doublets exposed by N-lauroylsarcosine (Sarkosyl) treatment. The zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) applied to 0.6 M KCl-extracted axonemes generated a 75-kDa complex containing β-tubulin and FAP85. Further characterization of FAP85 and its effects on microtubule dynamics showed that FAP85 binds to tubulin and stabilized microtubules. According to these results, we conclude that FAP85 is a novel member of microtubule-binding proteins, localizing on the inner wall of the A-tubule and stabilizing microtubules.Key words: Chlamydomonas, flagella, doublet microtubule, microtubule inner proteins.

中文翻译：

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鞭毛相关蛋白FAP85是稳定微管的微管内部蛋白。

衣藻的基因组学和蛋白质组学研究表明，轴蛋白由200-600种蛋白质组成，包括未表征的蛋白质（统称为鞭毛相关蛋白质（FAP））。九个FAP包含EF手基序；但是，它们尚未被很好地表征。为了找到负责衣原体特定波形变化以及细胞内Ca2 +浓度的成分，我们集中于FAP85，FAP85是衣原体及其亲属特有的含有EF手基序的FAP。我们克隆了编码FAP85的cDNA，在大肠杆菌细胞中表达，并产生了针对表达蛋白的多克隆抗体。免疫印迹表明，FAP85存在于几个缺乏主要轴突成分的鞭毛突变体的每个轴突中。免疫电子显微镜显示，仅在通过N-月桂酰肌氨酸（Sarkosyl）处理暴露的双峰的A管内壁上发现抗FAP85抗体。将零长度交联剂1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）应用于0.6 M KCl提取的轴酮生成75-kDa的复合物，其中包含β-微管蛋白和FAP85。FAP85的进一步表征及其对微管动力学的影响表明，FAP85与微管蛋白和稳定的微管结合。根据这些结果，我们得出结论，FAP85是一种微管结合蛋白的新型成员，位于A管的内壁并稳定微管。