Minamino Tohru, Kinoshita Miki, Namba Keiichi
Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Quantitative Biology Center, RIKEN, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Methods Mol Biol. 2017;1593:3-16. doi: 10.1007/978-1-4939-6927-2_1.
The flagellar type III export apparatus utilizes ATP and proton motive force (PMF) across the cytoplasmic membrane as the energy sources and transports flagellar component proteins from the cytoplasm to the distal growing end of the growing structure to construct the bacterial flagellum beyond the cellular membranes. The flagellar type III export apparatus coordinates flagellar protein export with assembly by ordered export of substrates to parallel with their order of the assembly. The export apparatus is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase complex. Since the ATPase complex is dispensable for flagellar protein export, PMF is the primary fuel for protein unfolding and translocation. Interestingly, the export gate complex can also use sodium motive force across the cytoplasmic membrane in addition to PMF when the ATPase complex does not work properly. Here, we describe experimental protocols, which have allowed us to identify the export substrate class and the primary fuel of the flagellar type III protein export apparatus in Salmonella enterica serovar Typhimurium.
鞭毛III型输出装置利用ATP和跨细胞质膜的质子动力（PMF）作为能量来源，将鞭毛组成蛋白从细胞质运输到生长结构的远端生长端，以构建超出细胞膜的细菌鞭毛。鞭毛III型输出装置通过有序输出底物使其与组装顺序平行，从而协调鞭毛蛋白输出与组装。该输出装置由PMF驱动的跨膜输出门复合体和细胞质ATP酶复合体组成。由于ATP酶复合体对于鞭毛蛋白输出并非必需，PMF是蛋白质解折叠和转运的主要能量来源。有趣的是，当ATP酶复合体不能正常工作时，除了PMF外，输出门复合体还可以利用跨细胞质膜的钠动力。在此，我们描述了一些实验方案，这些方案使我们能够鉴定肠炎沙门氏菌鼠伤寒血清型中鞭毛III型蛋白输出装置的输出底物类别和主要能量来源。
