Roles of the extreme N-terminal region of FliH for efficient localization of the FliH-FliI complex to the bacterial flagellar type III export apparatus
Roles of the extreme N-terminal region of FliH for efficient localization of the FliH-FliI complex to the bacterial flagellar type III export apparatus
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DOI：
10.1111/j.1365-2958.2009.06946.x
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发表时间：
2009-12-01
影响因子：
3.6
通讯作者：
Namba, Keiichi
中科院分区：
文献类型：
--
作者：
Minamino, Tohru;Yoshimura, Shinsuke D. J.;Namba, Keiichi
大多数细菌鞭毛蛋白通过鞭毛III型蛋白输出装置进行自组装。FliI atp酶与其调节因子FliH形成复合物，促进输出底物初始进入由6个完整膜蛋白组成的输出门。FliH-FliI复合物还通过FliH-FliN相互作用与基底体的C环结合，从而有效地输出。然而，目前尚不清楚这些反应是如何在细胞内进行的。在这里，我们用荧光显微镜分析了FliI-YFP的亚细胞定位。FliI-YFP定位于鞭毛基部，其定位需要FliH和C环。FliI的定位不需要atp酶活性。FliI-YFP与FliH Delta 1形成复合物（缺失残基2-10），但复合物未显示出任何定位。FliH Delta 1不与FliN相互作用，通过丙氨酸扫描诱变发现，FliH中只有Trp-7和Trp-10是与FliN相互作用所必需的。FliH-FliI复合体的过量生产提高了fliN突变体的输出活性，而FliH(W7A)-FliI和FliH(W10A)-FliI复合体都没有提高输出活性，这表明FliH的Trp-7和Trp-10也是FliH-FliI复合体有效定位到输出门所必需的。
P>Most bacterial flagellar proteins are exported by the flagellar type III protein export apparatus for their self-assembly. FliI ATPase forms a complex with its regulator FliH and facilitates initial entry of export substrates to the export gate composed of six integral membrane proteins. The FliH-FliI complex also binds to the C ring of the basal body through a FliH-FliN interaction for efficient export. However, it remains unclear how these reactions proceed within the cell. Here, we analysed subcellular localization of FliI-YFP by fluorescence microscopy. FliI-YFP was localized to the flagellar base, and its localization required both FliH and the C ring. The ATPase activity of FliI was not required for its localization. FliI-YFP formed a complex with FliH Delta 1 (missing residues 2-10) but the complex did not show any localization. FliH Delta 1 did not interact with FliN, and alanine-scanning mutagenesis revealed that only Trp-7 and Trp-10 of FliH are essential for the interaction with FliN. Overproduction of the FliH-FliI complex improved the export activity of the fliN mutant whereas neither of the FliH(W7A)-FliI nor FliH(W10A)-FliI complexes did, suggesting that Trp-7 and Trp-10 of FliH are also required for efficient localization of the FliH-FliI complex to the export gate.