A real-time analysis of protein transport via the twin arginine translocation pathway in response to different components of the protonmotive force

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 chloro-plasts, 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 (Delta psi) 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), Delta pH, in addition to Delta psi, 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.

Automated summary

The twin arginine translocation (Tat) pathway plays an important role in protein transport in bacteria, archaea, and plants. By using a real-time luminescence assay, this study reveals that both Delta psi and Delta pH contribute energetically to Tat transport in Escherichia coli spheroplasts.