Abstract：
Protein transport across the cytoplasmic membrane is coupled to energy derived from ATP hydrolysis or the proton motive force. A sophisticated, multi-component type III secretion system (T3SS) exports substrate proteins of both the bacterial flagellum and virulence-associated injectisome system of many Gram-negative pathogens. The T3SS is primarily a proton motive force-driven protein exporter. Here, we describe a method to investigate the export of substrate proteins of the flagellar T3SS into the culture supernatant under conditions that manipulate the proton motive force. Further, we describe methods to precisely quantify flagellar protein export into the culture supernatant using a split NanoLuc luciferase, and how fluorescence labeling of the extracellular flagellar filament can bring insights into the protein export rate of individual flagellar T3SS.
摘要：
穿过细胞质膜的蛋白质转运与源自ATP水解或质子动力的能量偶联。一个复杂的，多组分III型分泌系统（T3SS）输出细菌鞭毛和许多革兰氏阴性病原体的毒力相关注射系统的底物蛋白。T3SS主要是质子动力驱动的蛋白质出口国。这里,我们描述了一种在操纵质子动力的条件下研究鞭毛T3SS的底物蛋白向培养上清液中的输出的方法。Further,我们描述了使用分裂的NanoLuc荧光素酶精确量化鞭毛蛋白输出到培养上清液中的方法，以及细胞外鞭毛丝的荧光标记如何为单个鞭毛T3SS的蛋白质输出率带来见解。
