Charged residues in the cytoplasmic loop of MotA are required for stator assembly into the bacterial flagellar motor
Charged residues in the cytoplasmic loop of MotA are required for stator assembly into the bacterial flagellar motor
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DOI：
10.1111/j.1365-2958.2010.07391.x
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发表时间：
2010-12-01
影响因子：
3.6
通讯作者：
Minamino, Tohru
中科院分区：
文献类型：
--
作者：
Morimoto, Yusuke V.;Nakamura, Shuichi;Minamino, Tohru
P>MotA 和 MotB 形成跨膜质子通道，充当细菌鞭毛马达的定子，将质子流与扭矩产生耦合。 MotB 的 C 端周质结构域在将定子锚定到电机方面发挥着作用。然而，目前尚不清楚它们的初始结合位点在哪里。在这里，我们构建了表达 GFP-MotB 和 MotA-mCherry 的沙门氏菌菌株，并通过荧光显微镜研究了它们的亚细胞定位。 MotB 中消除质子流的 D33N 和 D33A 突变以及质子动力的耗尽均不影响 GFP-MotB 组装到电机中，这表明定子组装不需要质子易位活动。 MotA 的过度表达显着抑制野生型的运动，这是由于功能性定子数量的减少。一致地，即使在没有 MotB 的情况下，MotA-mCherry 也被观察到与 GFP-FliG 共定位。这些结果表明 MotA 可以单独安装到电机中。 MotA (MotA(C)) 细胞质环中的 R90E 和 E98K 突变已被证明可以消除与 FliG 的相互作用，显着影响定子组装，表明 MotA(C) 与 FliG 的静电相互作用是转子周围定子的有效组装所必需的。
P>MotA and MotB form a transmembrane proton channel that acts as the stator of the bacterial flagellar motor to couple proton flow with torque generation. The C-terminal periplasmic domain of MotB plays a role in anchoring the stators to the motor. However, it remains unclear where their initial binding sites are. Here, we constructed Salmonella strains expressing GFP-MotB and MotA-mCherry and investigated their subcellular localization by fluorescence microscopy. Neither the D33N and D33A mutations in MotB, which abolish the proton flow, nor depletion of proton motive force affected the assembly of GFP-MotB into the motor, indicating that the proton translocation activity is not required for stator assembly. Overexpression of MotA markedly inhibited wild-type motility, and it was due to the reduction in the number of functional stators. Consistently, MotA-mCherry was observed to colocalize with GFP-FliG even in the absence of MotB. These results suggest that MotA alone can be installed into the motor. The R90E and E98K mutations in the cytoplasmic loop of MotA (MotA(C)), which has been shown to abolish the interaction with FliG, significantly affected stator assembly, suggesting that the electrostatic interaction of MotA(C) with FliG is required for the efficient assembly of the stators around the rotor.