Department of Plant Biology, University of California, Davis, California, USA.
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, USA.
J Biol Chem. 2023 Nov;299(11):105286. doi: 10.1016/j.jbc.2023.105286. Epub 2023 Sep 22.
The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the transmembrane electrical potential (Δψ) component of PMF in vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions (pH = 6.3), ΔpH, in addition to Δψ, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway.
双精氨酸转运（Tat）途径在细菌、古菌中运输折叠蛋白穿过细胞质膜，在植物中穿过类囊体膜，在一些线粒体中穿过内膜。在植物叶绿体中，Tat 途径利用质子动力势（PMF）来驱动蛋白转运。然而，在细菌中，已经表明 Tat 运输仅依赖于 PMF 的跨膜电势（Δψ）成分在体外。为了研究大肠杆菌中全面的 PMF 需求，我们开发了第一个实时测定法，利用 NanoLuc 二元技术在大肠杆菌原生质体中监测 Tat 运输。这种发光测定法允许对 Tat 运输进行高分辨率的连续监测，从而可以观察到对不同处理的运输的细微变化。通过应用 NanoLuc 测定法，我们报告在酸性条件下（pH = 6.3），除了 Δψ 之外，ΔpH 也有助于大肠杆菌原生质体中 Tat 运输的能量利用。这些结果为 Tat 途径的能量利用机制提供了新的见解。
