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 characterized in the model bacterium Escherichia coli. Each of these is an iron-sulfur 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-sulfur 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 phylum, 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 prerequisite for covalent attachment to the cell wall.