Coupling of subunit availability to activation of PMF-driven flagellar type III secretion
Coupling of subunit availability to activation of PMF-driven flagellar type III secretion
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亚基可用性与 PMF 驱动的鞭毛 III 型分泌激活的耦合
DOI：
10.1099/acmi.ac2019.po0216
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发表时间：
2019
影响因子：
--
通讯作者：
Bryant O
中科院分区：
文献类型：
--
作者：
Bryant O
细菌鞭毛由数千个蛋白质亚基组装而成，这些蛋白质亚基通过专门的III型分泌系统展开和输出。亚基输出是由质子动力（PMF）促进的细胞质ATP酶复合物，包括FliH，FliI和FliJ，这是进化相关的F1 ATP酶的组件。ATP酶的FliJ茎部分结合输出门蛋白FlhA，使其能够利用Δ fl驱动高效的亚基输出。目前尚不清楚的是，当没有可用的亚基时，如何调节FlhA的FliJ活化以防止组成性质子流入。FliJ介导的出口门激活可以通过结合FliJ的其他蛋白质来调节。我们已经表明，FliJ招募空载出口分子伴侣，将其转移到其同源亚基，以创建分子伴侣-亚基结合的局部循环。为了研究分子伴侣是否也调节FlhA的FliJ活化，我们试图分离在FliJ结合中有缺陷但保留其结合亚基和其他输出组分的能力的分子伴侣变体。伴侣-FliJ结合的破坏减弱运动性和同源亚基输出。为了测试伴侣是否阻断FlhA-FliJ相互作用，我们开发了体外和体内竞争测定。我们的数据显示，伴侣蛋白和FlhA竞争FliJ上的共同结合位点，并且当亚基水平低时将存在于细胞中的未负载的伴侣蛋白破坏FliJ-FlhA相互作用，防止激活输出门。这提供了一种机制，由此仅在子单元可用时才激活导出门。
Bacterial flagella are assembled from thousands of protein subunits that are unfolded and exported via a specialized type III secretion system. Subunit export is fuelled by the proton motive force (PMF) facilitated by a cytoplasmic ATPase complex comprising FliH, FliI and FliJ, which are evolutionarily related to components of the F1 ATPase. The FliJ stalk component of the ATPase binds the export gate protein FlhA, allowing it to utilise ΔΨ to drive highly efficient subunit export. What is unclear is how FliJ activation of FlhA is regulated to prevent constitutive proton influx when there are no subunits available. FliJ-mediated export gate activation could be regulated by other proteins that bind FliJ. We have shown that FliJ recruits unladen export chaperones, transferring them to their cognate subunits to create a local cycle of chaperone-subunit binding. To investigate whether chaperones also regulate FliJ activation of FlhA, we sought to isolate chaperone variants that were defective in FliJ binding but retained their ability to bind subunits and other export components. Disruption of chaperone-FliJ binding attenuated motility and cognate subunit export. To test whether chaperones blocked the FlhA-FliJ interaction, we developedin vitroandin vivocompetition assays. Our data showed that chaperones and FlhA compete for a common binding site on FliJ, and that unladen chaperones, which would be present in the cell when subunit levels are low, disrupt the FliJ-FlhA interaction, preventing activation of the export gate. This provides a mechanism whereby the export gate is only activated when subunits are available.