ABSTRACT
The twin-arginine translocation (Tat) system serves to translocate folded proteins across energy-transducing membranes in bacteria, archaea, plastids, and some mitochondria. In Escherichia coli, TatA, TatB, and TatC constitute functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) followed by an amphipathic helix. The TMHs of TatA and TatB generate a hydrophobic mismatch with the membrane, as the helices comprise only 12 consecutive hydrophobic residues; however, the purpose of this mismatch is unclear. Here, we shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both and analyzed effects on translocon function and assembly. We found the WT length helices functioned best, but some variation was clearly tolerated. Defects in function were exacerbated by simultaneous mutations in TatA and TatB, indicating partial compensation of mutations in each by the other. Furthermore, length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residue-length is important but not essential for this interaction and translocon assembly. To also address potential effects of helix length on TatA interactions, we characterized these interactions by molecular dynamics simulations, after having characterized the TatA assemblies by metal-tagging transmission electron microscopy. In these simulations, we found that interacting short TMHs of larger TatA assemblies were thinning the membrane and-together with laterally-aligned tilted amphipathic helices-generated a deep V-shaped membrane groove. We propose the 12 consecutive hydrophobic residues may thus serve to destabilize the membrane during Tat transport, and their conservation could represent a delicate compromise between functionality and minimization of proton leakage.
