Role of the C-Terminal Cytoplasmic Domain of FlhA in Bacterial Flagellar Type III Protein Export
Role of the C-Terminal Cytoplasmic Domain of FlhA in Bacterial Flagellar Type III Protein Export
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DOI：
10.1128/jb.01328-09
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发表时间：
2010-04-01
影响因子：
3.2
通讯作者：
Namba, Keiichi
中科院分区：
文献类型：
--
作者：
Minamino, Tohru;Shimada, Masafumi;Namba, Keiichi
为了构建细菌鞭毛，许多鞭毛蛋白通过鞭毛III型蛋白输出装置输出到鞭毛结构的中央通道中。FlhA和FlhB是输出装置的整合膜蛋白，形成输出装置的可溶性组分FliH、FliI和FliJ的对接平台。FlhA的C-末端胞质结构域（FlhA（C））是蛋白质输出所必需的，但尚不清楚其如何起作用。在这里，我们分析了一种温度敏感性肠道沙门氏菌突变体，flhA（G368 C）突变体，其在编码FlhA（C）的序列中具有突变。G368 C突变没有消除与FliH、FliI、FliJ和FlhB的C-末端胞质结构域的相互作用，表明在FliH-FliI-FliJ-输出底物复合物结合到FlhA-FlhB平台后，突变阻断了输出过程。有限的蛋白水解显示FlhA（C）由至少三个亚结构域组成，柔性接头、FlhA（CN）和FlhA（CC），并且FlhA（CN）通过G368 C突变变得对蛋白水解敏感。基因内抑制突变被确定在这些子域和恢复鞭毛蛋白出口到相当大的程度。然而，这些抑制突变抑制蛋白酶的敏感性。我们认为，FlhA（C）不仅形成了FliH-FliI-FliJ-输出底物复合物的对接平台的一部分，而且还直接参与了输出底物转运到生长鞭毛结构的中央通道中。
For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhA(C)) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, which has a mutation in the sequence encoding FlhA(C). The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhA(C) consists of at least three subdomains, a flexible linker, FlhA(CN), and FlhA(CC), and that FlhA(CN) becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhA(C) not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure.