The FliP and FliR proteins of Salmonella typhimurium, putative components of the type III flagellar export apparatus, are located in the flagellar basal body
The FliP and FliR proteins of Salmonella typhimurium, putative components of the type III flagellar export apparatus, are located in the flagellar basal body
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DOI：
10.1046/j.1365-2958.1997.6412010.x
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发表时间：
1997-12-01
影响因子：
3.6
通讯作者：
Macnab, RM
中科院分区：
文献类型：
--
作者：
Fan, F;Ohnishi, K;Macnab, RM
鼠伤寒沙门氏菌鞭毛的大部分结构组分通过鞭毛特异性途径输出，这是III型分泌途径家族的成员。人们对这一过程的输出装置知之甚少。先前的研究表明，两种蛋白质，约23和26 kDa的大小和未知的遗传来源，被纳入鞭毛基体在一个非常早期的阶段鞭毛组装。在本研究中，我们证明，这些基体蛋白是Flip（在其成熟形式的信号肽裂解后）和FliR分别。这两种蛋白质在其他III型分泌系统中都有同源物。通过在FliR和MS环蛋白FliF上放置FLAG表位标签，并用抗FLAG单克隆抗体免疫印迹分离的钩基体复合物，我们估计（使用FLAG标记的FliF作为内部参考）FliR的化学计量小于每个基体三个拷贝。使用来自早期定量放射性标记分析的数据进行了化学计量的独立估计，对于FliP，每个基体产生约四个或五个亚基的值，对于FliR，每个基体产生约一个亚基的值。使用抗FLAG抗体和金蛋白A的免疫电镜表明，FliR位于MS环附近。我们建议，鞭毛出口装置包含Flip和FliR，并且该装置嵌入在MS环的中心孔中的膜片中。
Most of the structural components of the flagellum of Salmonella typhimurium are exported through a flagellum-specific pathway, which is a member of the family of type III secretory pathways. The export apparatus for this process is poorly understood. A previous study has shown that two proteins, about 23 and 26 kDa in size and of unknown genetic origin, are incorporated into the flagellar basal body at a very early stage of flagellar assembly. In the present study, we demonstrate that these basal body proteins are Flip (in its mature form after signal peptide cleavage) and FliR respectively. Both of these proteins have homologues in other type III secretion systems. By placing a FLAG epitope tag on FliR and the MS-ring protein FliF and immunoblotting isolated hook basal body complexes with anti-FLAG monoclonal antibody, we estimate (using the FLAG-tagged FliF as an internal reference) that the stoichiometry of FliR is fewer than three copies per basal body. An independent estimate of stoichiometry was made using data from an earlier quantitative radiolabelling analysis, yielding values of around four or five subunits per basal body for FliP and around one subunit per basal body for FliR. Immunoelectron microscopy using anti-FLAG antibody and gold-protein A suggests that FliR is located near the MS ring. We propose that the flagellar export apparatus contains Flip and FliR and that this apparatus is embedded in a patch of membrane in the central pore of the MS ring.