Bai Fan, Morimoto Yusuke V, Yoshimura Shinsuke D J, Hara Noritaka, Kami-Ike Nobunori, Namba Keiichi, Minamino Tohru
1] Biodynamic Optical Imaging Center, Peking University, Beijing, 100871, China [2] Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan [3].
1] Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan [2] Quantitative Biology Center, RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan [3].
Sci Rep. 2014 Oct 6;4:6528. doi: 10.1038/srep06528.
For construction of the bacterial flagellum, FliI ATPase forms the FliH2-FliI complex in the cytoplasm and localizes to the flagellar basal body (FBB) through the interaction of FliH with a C ring protein, FliN. FliI also assembles into a homo-hexamer to promote initial entry of export substrates into the export gate. The interaction of FliH with an export gate protein, FlhA, is required for stable anchoring of the FliI6 ring to the gate. Here we report the stoichiometry and assembly dynamics of FliI-YFP by fluorescence microscopy with single molecule precision. More than six FliI-YFP molecules were associated with the FBB through interactions of FliH with FliN and FlhA. Single FliI-YFP molecule exchanges between the FBB-localized and free-diffusing ones were observed several times per minute. Neither the number of FliI-YFP associated with the FBB nor FliI-YFP turnover rate were affected by catalytic mutations in FliI, indicating that ATP hydrolysis by FliI does not drive the assembly-disassembly cycle of FliI during flagellar assembly. We propose that the FliH2FliI complex and FliI6 ring function as a dynamic substrate carrier and a static substrate loader, respectively.
为构建细菌鞭毛，FliI ATP酶在细胞质中形成FliH2 - FliI复合物，并通过FliH与C环蛋白FliN的相互作用定位于鞭毛基体（FBB）。FliI还组装成同六聚体以促进输出底物最初进入输出通道。FliH与输出通道蛋白FlhA的相互作用是FliI6环稳定锚定在通道上所必需的。在此，我们通过具有单分子精度的荧光显微镜报告了FliI - YFP的化学计量和组装动力学。超过六个FliI - YFP分子通过FliH与FliN和FlhA的相互作用与FBB相关联。每分钟可观察到单个FliI - YFP分子在定位于FBB的分子和自由扩散的分子之间进行数次交换。与FBB相关联的FliI - YFP数量和FliI - YFP周转速率均不受FliI催化突变的影响，这表明在鞭毛组装过程中，FliI的ATP水解并不驱动FliI的组装 - 拆卸循环。我们提出，FliH2FliI复合物和FliI6环分别作为动态底物载体和静态底物加载器发挥作用。
