Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-8602, Japan.
Sci Rep. 2018 Aug 14;8(1):12115. doi: 10.1038/s41598-018-30531-5.
FlhF controls the number and position of the polar flagellar formation of Vibrio species. FlhF, is a paralog of FtsY, a GTPase acting in the Sec membrane transport system of bacteria, and localizes at the cell pole. Mutations in the conserved GTPase motif of FlhF lost polar localization capability and flagellar formation. Vibrio FlhF has not, until now, been purified as soluble protein. Here, we report that addition of MgCl and GTP or GDP at the step of cell lysis greatly improved the solubility of FlhF, allowing us to purify it in homogeneity. Purified FlhF showed GTPase activity only in the presence of FlhG. Of twelve FlhF GTPase motif mutants showing reduced function, eleven were recovered as precipitate after the cell disruption. The E440K substitution could be purified and showed no GTPase activity even in the presence of FlhG. Interestingly an FlhF substitution in the putative catalytic residue for GTP hydrolysis, R334A, allowed normal flagellar formation although GTPase activity of FlhF was completely abolished. Furthermore, size exclusion chromatography of purified FlhF revealed that it forms dimers in the presence of GTP but exists as monomer in the presence of GDP. We speculate that the GTP binding allows FlhF to dimerize and localize at the pole where it initiates flagellar formation, and the GDP-bound form diffuses as monomer.
FlhF 控制着弧菌属物种的极生鞭毛形成的数量和位置。FlhF 是 FtsY 的一个旁系同源物，FtsY 是一种在细菌 Sec 膜转运系统中起作用的 GTP 酶，定位于细胞极。FlhF 中的保守 GTP 酶基序突变丧失了极定位能力和鞭毛形成。到目前为止，Vibrio FlhF 尚未被纯化作为可溶性蛋白。在这里，我们报告说，在细胞裂解步骤中添加 MgCl 和 GTP 或 GDP 极大地提高了 FlhF 的可溶性，使我们能够以均相纯化它。纯化的 FlhF 仅在存在 FlhG 的情况下显示 GTPase 活性。在十二个显示功能降低的 FlhF GTP 酶基序突变体中，有十一个在细胞破裂后沉淀回收。E440K 取代可以被纯化，即使在存在 FlhG 的情况下也没有 GTPase 活性。有趣的是，在 FlhF 的假定 GTP 水解催化残基中，R334A 的取代允许正常的鞭毛形成，尽管 FlhF 的 GTPase 活性完全被废除。此外，纯化的 FlhF 的分子筛层析表明，它在 GTP 的存在下形成二聚体，但在 GDP 的存在下以单体形式存在。我们推测，GTP 结合允许 FlhF 二聚化并定位在起始鞭毛形成的极，而 GDP 结合形式作为单体扩散。
