Integration of tail-anchored proteins by twin-arginine translocase

Abstract

The Tat pathway transports folded proteins across the cytoplasmic membranes of bacteria. Tat substrates have N-terminal signal peptides containing a conserved twinarginine motif and are exported in a folded form. While most Tat substrates are soluble periplasmic proteins, a small fraction are anchored to the membrane by a single C-terminal transmembrane helix. In the model organism Escherichia coli, five Tat substrates are tail-anchored membrane proteins. However, the repertoire of tailanchored Tat substrates across prokaryotes is currently unclear. To identify new C-tail anchored Tat dependent proteins, a database search of bacterial proteins was carried out by Dr Govind Chandra, identifying many candidate proteins with both a predicted twin-arginine signal peptide and a hydrophobic stretch close to the C-terminus. A custom-written python code, utilising regular expression techniques was designed to classify the extracted proteins. From this around 80 distinct protein families were classified. To confirm the prediction that some of these represent new families of Tat-dependent C-tail proteins, the twinarginine signal sequences and C-tail encoding regions of a small subset were fused to E. coli reporter proteins to assess for Tat-dependence and membrane integration. From this several new Tat-dependent tail-anchored substrates were verified. The mechanism by which C-tails are integrated into the membrane is currently unknown. I designed genetic constructs with the aim to investigate this process. These constructs harboured full length SufI fused to the C-tail of FdnH, which was fused in frame to either maltose binding protein or β-lactamase. Experiments to determine the membrane localisation and Tat-dependence of these indicated that both fusion proteins were integrated into the membrane Tat-independently. Modification of the β-lactamase fusion through addition of linkers around the transmembrane domain improved stability and membrane integration. However, further modifications would be necessary before the fusion protein could be used as a reporter for Tat-dependent C-tail integration.