Moving folded proteins across membranes. (360G-Wellcome-107929_Z_15_Z)

The Tat (twin arginine translocation) system, which I co-discovered, is a highly unusual protein transport pathway that is able to export folded proteins across the cytoplasmic membrane of bacteria. It is required for the virulence of many common pathogens. A central question in the Tat field is howthe transporter is able to actively transport folded proteins across the membrane bilayer without significant ion leakage. Tat transport involves threetypes of small integral membrane proteins, multiple copies of which dynamically assemble in the presence of a substrate protein to form a transient transport complex. It is this transport complex that is key to understanding the mechanism of Tat transport. Although we have recently determined high resolution structures of the individual Tat components, the assembled translocation site has been considered to be too ephemeral and unstable to be isolated and studied. In major methodological breakthroughs my group has identified ways of following the behaviour of the complex in real time in living cells, of stalling the transport complex in the assembled state, and of solubilizing the translocation complex from its native membrane environment. I now aim to exploit these advances to answer the key questions about the mechanism of Tat transport: (1) What is the structure of the assembled Tat translocation site? (2) What, based on this structural data, is the physical mechanism of substrate transport across the membrane and of energy transduction to drive transport? (3) What are the component steps in the Tat transport cycle, what are the structural changes occurring at each step, and what molecular features control the interconversion between the steps?