Effects of Mutations of the Arginine Motif of Signal Peptide in a Tat Substrate on Translocation Activity in Escherichia Coli

The twin-arginine translocase (Tat) system is a recently discovered pathway which transports proteins in bacteria and chloroplasts. This system only translocates proteins with a cleavable N-terminal signal peptide with the distinctive twin-arginine (RR) motif, which is crucial to Tat function. Here, site-directed mutagenesis of the signal peptide of the Tat substrate TorA-GFP which has a tripletarginine (RRR) instead of the usual twin arginine were attempted and the effect on the translocation of this substrate in Escherichia coli was measured by SDS-PAGE / Western Blot of the cytoplasmic, periplasmic and membrane fractions of the bacteria. The KRK mutant of the signal peptide of TorA-GFP was found to mediate transport less than that of TorA-GFP. The main difference between the Tat and Sec pathways in bacteria is that the Sec pathway transports unfolded proteins in the cytoplasm to the periplasm (where they fold), whilst the Tat pathway transports the proteins fully folded. The Sec pathway in E. coli is reasonably well understood (1) compared to the Tat pathway; its ability to let large transition-metal containing proteins such as trimethylamine N-oxidase (TorA) through a tight membrane is still a mystery. Another major difference is that Tat translocation is driven by the change in pH between the cytoplasm and periplasm (∆pH), whilst the Sec pathway uses the hydrolysis of nucleosides, such as ATP. Also, in the Sec pathway the proteins SecA and SecB act as chaperones which bind to the unfolded protein; there are no known analogues for Tat (although proteins such as TorA have specific enzymes that associate with them to aid cofactor insertions – in this case TorD). The synthesis of proteins by the ribosomal translation of messenger RNA takes place in the cytosol of cells, but to function many are required in other compartments of the cell and hence have to be transported in some way, which involves transmembrane translocation. In Gram-negative bacteria, there are only two compartments – the cytoplasm and periplasm, and the transferral of proteins from cytoplasm to periplasm is usually via the Sec (secretory) pathway (1), which transports these proteins in a largely unfolded state. In E. coli there are three main components in the Tat system, namely TatA, TatB and TatC. There is another component known as TatE, but this has the same role as TatA and only a very small amount of the protein is expressed in the cell. The removal of any of TatA (or TatE), TatB or TatC inactivates the pathway, which implies each of these is essential for Tat function.