Dual functionality of twin-arginine signal peptides

Prokaryotes are the simplest truly living organisms known to man. They include the single-celled bacteria and their cousins the archaea, probably the closest surviving examples of the earliest cellular life-forms that ever existed. Many of these organisms can live and grow without oxygen, and instead utilise other chemicals from the environment to generate energy for life and nitrogen-rich chemicals such as nitrates are very commonly used as a replacement across the whole spectrum of prokaryotes. To get energy from nitrate prokaryotes contain special proteins or enzymes, many of which are made-up of lots of different parts, or subunits, which in themselves often contain metal and sulphur atoms. In addition, these enzymes are often found 'outside' on the surface of the cell. How these enzymes get out the cell, and how they are fully assembled with all their metals and subunits attached before that, is the thrust of this research project. Most enzymes that are destined to be located outside the cell are identifiable by the presence of a special 'signal' on them. We have found this signal, which is also made of protein, has two jobs in the cell. First, it helps to assemble the subunits and metals, then second, it helps to locate the finished enzyme outside the cell. We want to study these functions in isolation, without interference from the other one, in order to understand them fully and in detail. We will then look again at the complete system and applying our new knowledge to understand how the two functions work together in harmony. Once we learn in detail how these processes work we may be able to make it work 'better' so that biotechnology companies can use it to make useful everyday products. Or we may be able to learn how design a new antibiotic to stop this system working without harming the environment - some deadly bacteria that cannot perform these tasks are no longer dangerous.

The twin-arginine translocation (Tat) pathway is a remarkable protein transport system dedicated to the transport of fully folded proteins across the energy-tranducing membranes of archaea, eubacteria, and chloroplast thylakoids. All proteins destined for the Tat translocase are synthesised with N-terminal 'twin-arginine' signal peptides bearing a conserved SRRxFLK amino acid motif. The signal peptide engages directly with the Tat translocation machinery and triggers the protein export event. The majority of prokaryotic Tat substrates are complex, multi-subunit, cofactor-containing, redox enzymes with key roles in respiratory or photosynthetic electron transport chains. Pre-export biosynthesis and assembly of such enzymes involves a second activity mediated by the twin-arginine signal peptide completely distinct from that of Tat transport: Tat proofreading. This process involves the direct binding of the twin-arginine signal by a dedicated chaperone until such time as all biosynthetic processes are complete, processes catalysed in part by the self-same chaperone that binds the signal peptide. In an exciting new development, identified here for the first time are remnant twin-arginine signal peptides that have lost the Tat targeting function but still retain affinity for biosynthetic chaperones. These remnant signals are located on model eubacterial non-exported molybdenum enzymes, including the respiratory nitrate reductases central to a broad spectrum of prokaryotic electron transport systems. Interestingly, the remnant signals are readily revivable with respect to Tat transport and therefore represent ideal novel tools to study, in isolation, the molecular basis of the chaperone binding activity of twin-arginine signal peptides, and shed new light on the molecular requirements for the ultimate Tat transport event