FliL association with flagellar stator in the sodium-driven Vibrio motor characterized by the fluorescent microscopy.
FliL association with flagellar stator in the sodium-driven Vibrio motor characterized by the fluorescent microscopy.
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DOI：
10.1038/s41598-018-29447-x
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发表时间：
2018-07-24
影响因子：
4.6
通讯作者：
Lo CJ
中科院分区：
文献类型：
--
作者：
Lin TS;Zhu S;Kojima S;Homma M;Lo CJ
细菌鞭毛马达（bacterial flagellar motor，BFM）是一种蛋白质复合物，参与细菌的运动和趋化过程，参与能量转换、扭矩产生和转换。FliL是一种与鞭毛运动功能相关的单跨膜蛋白。我们进行了生物化学和生物物理学的方法来研究FliL与定子单位相关的功能作用。首先，我们发现FliL的周质区是其极性定位的关键。此外，定子单元的插入突变影响了FliL的极性定位，这意味着定子单元的激活对于FliL的募集是重要的。其次，我们应用单分子荧光显微镜来研究FliL在定子单元组装中的作用。通过光漂白分子计数，我们发现在一个功能马达中定子单元和FliL蛋白的化学计量比为1：1。此外，在没有FliL的情况下，定子单元的周转时间略有增加。通过进一步研究蛋白质在膜上的动力学，我们发现定子单元和FliL的扩散是独立的。令人惊讶的是，没有定子单元的FliL扩散速率出乎意料地慢，表明蛋白质复合物形成事件。研究结果表明，FliL在BFM中对定子起支撑作用。
Bacterial flagellar motor (BFM) is a protein complex used for bacterial motility and chemotaxis that involves in energy transformation, torque generation and switching. FliL is a single-transmembrane protein associated with flagellar motor function. We performed biochemical and biophysical approaches to investigate the functional roles of FliL associated with stator-units. Firstly, we found the periplasmic region of FliL is crucial for its polar localization. Also, the plug mutation in stator-unit affected the polar localization of FliL implying the activation of stator-unit is important for FliL recruitment. Secondly, we applied single-molecule fluorescent microscopy to study the role of FliL in stator-unit assembly. Using molecular counting by photobleaching, we found the stoichiometry of stator-unit and FliL protein would be 1:1 in a functional motor. Moreover, the turnover time of stator-units are slightly increased in the absence of FliL. By further investigation of protein dynamics on membrane, we found the diffusions of stator-units and FliL are independent. Surprisingly, the FliL diffusion rate without stator-units is unexpectedly slow indicating a protein-complex forming event. Our results suggest that FliL plays a supporting role to the stator in the BFM.
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