Interaction between FliJ and FlhA, Components of the Bacterial Flagellar Type III Export Apparatus
Interaction between FliJ and FlhA, Components of the Bacterial Flagellar Type III Export Apparatus
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DOI：
10.1128/jb.01711-12
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发表时间：
2013-02-01
影响因子：
3.2
通讯作者：
Minamino, Tohru
中科院分区：
文献类型：
--
作者：
Ibuki, Tatsuya;Uchida, Yumiko;Minamino, Tohru
可溶性蛋白FliJ与膜蛋白FlhA沿着在细菌鞭毛蛋白输出的能量偶联机制中发挥作用。水溶性FliH（X）-FliI（6）ATP酶环复合物允许FliJ有效地与FlhA相互作用。然而，FliJ的FlhA结合位点仍然未知。在此，我们对FliJ的高度保守的残基Gln 38、Leu 42、Tyr 45、Tyr 49、Phe 72、Leu 76、Ala 79和His 83形成的区域进行了遗传分析。FliI（6）-FliJ环复合物的结构模型表明它们延伸出FliI（6）环。谷胱甘肽S-转移酶（GST）-FliJ抑制野生型细胞和fliH-fliI flhB（P28 T）旁路突变体的运动和鞭毛蛋白输出。下拉测定揭示，输出装置的输出活性降低是由于GST-FliJ与FlhA的结合。FliJ的F72 A和L76 A突变显著降低了FliJ对FlhA的结合亲和力，从而抑制了GST-FliJ对蛋白输出的抑制作用。F72 A和L76 A突变在存在FliH和FliI的情况下是耐受的，但在它们不存在的情况下显著降低了运动性。这两个突变既不影响与FliI的相互作用，也不影响FliI ATP酶活性。这些结果表明，FliJ（F72 A）和FliJ（L76 A）需要FliH和FliI的支持才能发挥其输出功能。因此，我们认为FliJ的保守表面参与了与FlhA的相互作用。
A soluble protein, FliJ, along with a membrane protein, FlhA, plays a role in the energy coupling mechanism for bacterial flagellar protein export. The water-soluble FliH(X)-FliI(6) ATPase ring complex allows FliJ to efficiently interact with FlhA. However, the FlhA binding site of FliJ remains unknown. Here, we carried out genetic analysis of a region formed by well-conserved residues-Gln38, Leu42, Tyr45, Tyr49, Phe72, Leu76, Ala79, and His83-of FliJ. A structural model of the FliI(6)-FliJ ring complex suggests that they extend out of the FliI(6) ring. Glutathione S-transferase (GST)-FliJ inhibited the motility of and flagellar protein export by both wild-type cells and a fliH-fliI flhB(P28T) bypass mutant. Pulldown assays revealed that the reduced export activity of the export apparatus results from the binding of GST-FliJ to FlhA. The F72A and L76A mutations of FliJ significantly reduced the binding affinity of FliJ for FlhA, thereby suppressing the inhibitory effect of GST-FliJ on the protein export. The F72A and L76A mutations were tolerated in the presence of FliH and FliI but considerably reduced motility in their absence. These two mutations affected neither the interaction with FliI nor the FliI ATPase activity. These results suggest that FliJ(F72A) and FliJ(L76A) require the support of FliH and FliI to exert their export function. Therefore, we propose that the well-conserved surface of FliJ is involved in the interaction with FlhA.