Intrinsic membrane targeting of the flagellar export ATPase FliI: interaction with acidic phospholipids and FliH.
Intrinsic membrane targeting of the flagellar export ATPase FliI: interaction with acidic phospholipids and FliH.
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DOI：
10.1016/s0022-2836(02)00172-9
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发表时间：
2002-05-10
影响因子：
5.6
通讯作者：
Hughes, C
中科院分区：
文献类型：
--
作者：
Auvray, F;Ozin, AJ;Claret, L;Hughes, C
专门的 ATP 酶 FliI 对于鞭毛组装过程中鞭毛轴向蛋白亚基的输出至关重要。我们在运动型鼠伤寒沙门氏菌中建立了 FliI 及其调节辅助蛋白 FliH 的正常细胞位置，并确定了参与 FliH2/FliI 异源三聚化的区域。在存在和不存在构成鞭毛输出机器和基体的蛋白质的情况下，FliI 和 FliH 都定位于细胞质膜。膜结合紧密，FliI 和 FliH 在体外与大肠杆菌磷脂相互作用，无论是单独的还是作为预先形成的 FliH2/FliI 复合物，无论是否存在 ATP。酵母双杂交分析和 Pull-down 测定表明，FliH (H105-235) 的 C 末端一半指导 FliH 同二聚化，并与 FliI (I1-155) 的 N 末端区域相互作用，而 FliI (I1-155) 反过来又与含有 ATPase 结构域的蛋白质的其余部分 (I156-456) 发生分子内相互作用。 FliH105-235 与 FliI 的相互作用足以发挥 FliH 介导的 ATP 酶活性下调。分离的 FliI 的基础 ATP 酶活性被细菌（酸性）磷脂刺激十倍，因此活性比在没有磷脂的情况下与 FliH 结合时高 100 倍。结果表明 FliI 与充分表征的 SecA ATP 酶之间存在相似性，后者可促进一般蛋白质分泌。他们认为，FliI 和 FliH 在接触鞭毛分泌机制之前本质上是靶向内膜的，FliH105-235 和膜磷脂都与 FliI 相互作用，将 ATP 水解与鞭毛组装结合起来。
The specialised ATPase FliI is central to export of flagellar axial protein subunits during flagellum assembly. We establish the normal cellular location of FliI and its regulatory accessory protein FliH in motile Salmonella typhimurium, and ascertain the regions involved in FliH2/FliI heterotrimerisation. Both FliI and FliH localised to the cytoplasmic membrane in the presence and in the absence of proteins making up the flagellar export machinery and basal body. Membrane association was tight, and FliI and FliH interacted with Escherichia coli phospholipids in vitro, both separately and as the preformed FliH2/FliI complex, in the presence or in the absence of ATP. Yeast two-hybrid analysis and pull-down assays revealed that the C-terminal half of FliH (H105-235) directs FliH homodimerisation, and interacts with the N-terminal region of FliI (I1-155), which in turn has an intra-molecular interaction with the remainder of the protein (I156-456) containing the ATPase domain. The FliH105-235 interaction with FliI was sufficient to exert the FliH-mediated down-regulation of ATPase activity. The basal ATPase activity of isolated FliI was stimulated tenfold by bacterial (acidic) phospholipids, such that activity was 100-fold higher than when bound by FliH in the absence of phospholipids. The results indicate similarities between FliI and the well-characterised SecA ATPase that energises general protein secretion. They suggest that FliI and FliH are intrinsically targeted to the inner membrane before contacting the flagellar secretion machinery, with both FliH105-235 and membrane phospholipids interacting with FliI to couple ATP hydrolysis to flagellum assembly.