鞭毛相关蛋白FAP85是一种稳定微管的微管内部蛋白。

IF 2 4区 生物学 Q4 CELL BIOLOGY
Cell structure and function Pub Date : 2018-02-16 Epub Date: 2017-12-28 DOI:10.1247/csf.17023
Junya Kirima, Kazuhiro Oiwa
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引用次数: 16

摘要

衣藻的基因组学和蛋白质组学研究表明,轴突素由200-600种蛋白质组成,其中包括统称为鞭毛相关蛋白(FAPs)的未鉴定蛋白质。9个fap包含EF-hand基序;然而,它们还没有被很好地描述。为了找到衣藻特异性波形变化与细胞内Ca2+浓度耦合的成分,我们重点研究了FAP85,这是一种含有EF-hand基元的衣藻及其亲缘体特异性FAP。我们克隆了编码FAP85的cDNA,在大肠杆菌细胞中表达,并制备了针对该表达蛋白的多克隆抗体。免疫印迹显示,FAP85存在于缺乏主要轴突成分的鞭毛突变体的每个轴突中。免疫电镜显示,抗fap85抗体仅在n -lauroylsarcos (sarkozy)处理的双细胞的a小管内壁上发现。零长度交联剂1-乙基-3-(3-二甲氨基丙基)碳二酰亚胺(EDC)作用于0.6 M kcl提取的轴突上,生成了含有β-微管蛋白和FAP85的75 kda复合物。进一步表征FAP85及其对微管动力学的影响表明,FAP85与微管蛋白结合并稳定微管。根据这些结果,我们得出结论,FAP85是微管结合蛋白的新成员,定位于a管内壁并稳定微管。关键词:衣藻,鞭毛,双微管,微管内蛋白。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Flagellar-associated Protein FAP85 Is a Microtubule Inner Protein That Stabilizes Microtubules.

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.

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来源期刊
Cell structure and function
Cell structure and function 生物-细胞生物学
CiteScore
2.50
自引率
0.00%
发文量
6
审稿时长
>12 weeks
期刊介绍: Cell Structure and Function is a fully peer-reviewed, fully Open Access journal. As the official English-language journal of the Japan Society for Cell Biology, it is published continuously online and biannually in print. Cell Structure and Function publishes important, original contributions in all areas of molecular and cell biology. The journal welcomes the submission of manuscripts on research areas such as the cell nucleus, chromosomes, and gene expression; the cytoskeleton and cell motility; cell adhesion and the extracellular matrix; cell growth, differentiation and death; signal transduction; the protein life cycle; membrane traffic; and organelles.
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