Morimoto Yusuke V, Namba Keiichi, Minamino Tohru
Quantitative Biology Center, RIKEN, Suita, Osaka, Japan.
Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.
Bio Protoc. 2017 Jan 5;7(1):e2092. doi: 10.21769/BioProtoc.2092.
The bacterial flagellar type III export apparatus consists of a cytoplasmic ATPase complex and a transmembrane export gate complex, which are powered by ATP and proton motive force (PMF) across the cytoplasmic membrane, respectively, and transports flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure where their assembly occurs (Minamino, 2014). The export gate complex can utilize sodium motive force in addition to PMF when the cytoplasmic ATPase complex does not work properly. A transmembrane export gate protein FlhA acts as a dual ion channel to conduct both H and Na ( Minamino , 2016 ). Here, we describe how to measure the intracellular Na concentrations in living cells using a sodium-sensitive fluorescent dye, CoroNa Green ( Minamino , 2016 ). Fluorescence intensity measurements of CoroNa Green by epi-fluorescence microscopy allows us to measure the intracellular Na concentration quantitatively.
细菌鞭毛III型输出装置由一个胞质ATP酶复合体和一个跨膜输出门复合体组成，它们分别由ATP和跨细胞质膜的质子动力（PMF）提供动力，并将鞭毛组分蛋白从细胞质运输到生长中的鞭毛结构的远端，在那里进行组装（水见，2014年）。当胞质ATP酶复合体不能正常工作时，输出门复合体除了利用PMF外，还可以利用钠动力。跨膜输出门蛋白FlhA作为一个双离子通道来传导H和Na（水见，2016年）。在这里，我们描述了如何使用钠敏感荧光染料CoroNa Green测量活细胞内的Na浓度（水见，2016年）。通过落射荧光显微镜对CoroNa Green进行荧光强度测量，使我们能够定量测量细胞内的Na浓度。
