Genetic approaches to structure-function analysis of the E. coli Tat translocon components

Some bacterial proteins operate on the outside of the cell, for example the toxins produced by bacterial pathogens. Since all proteins are made inside the bacterium the extracellular proteins must be moved out of the cell across the normally impermeable cell membrane. This task is carried out by machines termed protein transporters that are located in the cell membrane. One type of transporter moves unfolded proteins, threading them across the membrane like string through the eye of a needle. In contrast, a second type of transporter, which we term the Tat system, moves folded proteins across the membrane. In the bacterium E. coli, the Tat transporter is a large protein complex made up of 3 types of proteins, TatA, TatB and TatC. We wish to understand how each of these proteins functions in the transporter. To do that we want to isolate mutants in the TatA, TatB and TatC proteins that prevent the transporter from functioning. These mutants will give us information about important parts of the Tat protein molecules. We have made mutations throughout the TatA, B and C proteins that introduce an amino acid called cysteine. Cysteine is a special amino acid because if it comes close to a second cysteine residue under certain conditions it can form a covalent bond. This covalent bond locks the proteins and prevents them from moving. Therefore we can examine the parts of the TatA, B and C proteins that need to move in order to allow the Tat system to be active. Finally we believe that part of TatA may need to move across the membrane to allow proteins to be transported. We want to test this by putting special tags on the TatA protein and looking to see if the tag appears or can be labelled at the other side of the membrane when the Tat system is operating.

The Tat protein transport system functions to export folded proteins across the bacterial cytoplasmic membrane. TatA, TatB and TatC are the essential components of the bacterial Tat protein export pathway and interact in a dynamic manner to form the membrane-located protein complexes necessary for substrate transport. Building on our recent successful BBSRC-funded work we will now obtain structural, conformational and topological information on the Tat proteins and map the twin arginine signal peptide binding site. We will undertake saturating screens of large libraries of random mutations in tatA, tatB and tatC for substitutions that block Tat function, and which confer recognition of non canonical Tat signal peptides. We will probe conformational changes in Tat proteins during transport by synthetic lethality or synthetic inactivity of co-expressed cysteine variants. Finally we will probe topological changes of TatA associated with protein transport.