A Real-Time Analysis of Protein Transport via the Twin Arginine Translocation Pathway in Response to Different Components of the Protonmotive Forcein vivo

Abstract

AbstractThe 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 Δψ component of PMFin vitro. To investigate the comprehensive PMF requirement inEscherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology (NanoBiT) inE. colispheroplasts. 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, ΔpH, in addition to Δψ, contributes energetically to Tat transportin vivoinE. colispheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway.