Auvray Frédéric, Ozin Amanda J, Claret Laurent, Hughes Colin
Department of Pathology, Cambridge University, Tennis Court Road, CB2 1QP, UK.
J Mol Biol. 2002 May 10;318(4):941-50. doi: 10.1016/S0022-2836(02)00172-9.
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 FliH(2)/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 FliH(2)/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.
在鞭毛组装过程中，特殊的ATP酶FliI对于鞭毛轴向蛋白亚基的输出至关重要。我们确定了FliI及其调节辅助蛋白FliH在运动型鼠伤寒沙门氏菌中的正常细胞定位，并确定了参与FliH(2)/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，并且FliI和FliH在体外与大肠杆菌磷脂相互作用，无论是单独还是作为预先形成的FliH(2)/FliI复合物，无论有无ATP。酵母双杂交分析和下拉试验表明，FliH的C端一半（H105 - 235）指导FliH同二聚化，并与FliI的N端区域（I1 - 155）相互作用，而该区域又与包含ATP酶结构域的蛋白质其余部分（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酶活性下调作用。分离的FliI的基础ATP酶活性被细菌（酸性）磷脂刺激了10倍，使得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与为一般蛋白质分泌提供能量的特征明确的SecA ATP酶之间存在相似性。它们表明FliI和FliH在接触鞭毛分泌机器之前内在地靶向内膜，FliH105 - 235和膜磷脂都与FliI相互作用，将ATP水解与鞭毛组装耦合起来。
