Twin arginine translocase pathway and fast-folding lipoprotein biosynthesis in E. coli: interesting implications and applications.

Bacterial lipoproteins, an important class of membrane proteins, are generally thought to be translocated in an unfolded state by the well-studied Sec machinery, whereas the role of TAT, meant for folded proteins, is hardly investigated. Using appropriately engineered fast-folding Enhanced Green Fluorescence Protein (EGFP), as a model, here we show that TAT is essential for not only translocating fast-folding lipoprotein but also its lipid modification. EGFP was lipid-modified and targeted to the outer membrane's outer surface with a prototypical TAT signal sequence containing lipobox but not with the lipoprotein or TAT signal sequence. Justifiably signal sequences of many substrate-binding and co-factor-containing lipoproteins contained both TAT-box and lipobox (Shruthi et al., submitted). Cytoplasmic accumulation of unmodified precursors of engineered EGFP in a tatC mutant implicated this TAT-box-recognizing component in lipid-modification. Similar observations reported earlier with Sec components and murein lipoprotein led us to propose that the translocation-competent and translocase-associated (Sec or TAT) precursor form is prerequisite to initiation of lipid-modification in vivo. The above missing links between translocation and lipid modification machineries in vivo is important to our understanding of bacterial lipoprotein biosynthesis and its utility as a protein engineering tool for potent applications in synthetic biology and nanobiotechnology like display, arrays on bacterial surfaces, vaccines and biosensors.