Identification of novel tail-anchored membrane proteins integrated by the bacterial twin-arginine translocase
The twin arginine protein transport (Tat) system exports folded proteins across the cytoplasmic membranes of prokaryotes and the energy transducing membranes of plant thylakoids and mitochondria. Proteins are targeted to the Tat machinery by N-terminal signal peptides with a conserved twin arginine motif, and some substrates are exported as heterodimers where the signal peptide is present on one of the partner proteins. A subset of Tat substrates is found in the membrane. Tat-dependent membrane proteins usually have large globular domains and a single transmembrane helix present at the N- or C-terminus. Five Tat substrates that have C-terminal transmembrane helices have previously been characterised in the model bacterium Escherichia coli. Each of these is an iron-sulphur cluster-containing protein involved in electron transfer from hydrogen or formate. Here we have undertaken a bioinformatic search to identify further tail-anchored Tat substrates encoded in bacterial genomes. Our analysis has revealed additional tail-anchored iron-sulphur proteins associated in modules with either a b-type cytochrome or a quinol oxidase. We also identified further candidate tail-anchored Tat substrates, particularly among members of the actinobacterial genus, that are not predicted to contain cofactors. Using reporter assays we show experimentally that six of these have both N-terminal Tat signal peptides and C-terminal transmembrane helices. The newly-identified proteins include a carboxypeptidase and a predicted protease, and four sortase substrates for which membrane integration is a pre-requisite for covalent attachment to the cell wall.