The twin-arginine translocation (Tat(1)) pathway is unique with respect to its property to translocate proteins in a fully folded conformation across ion-tight membranes. In chloroplasts and Gram-negative bacteria, Tat translocase consists of the integral subunits TatB and TatC, which are assumed to constitute the membrane receptor, and TatA, a bitopic membrane protein being responsible in a yet unknown manner for the membrane translocation step. Antibody inhibition of intrinsic thylakoidal TatA activity and recovery of transport by heterologously expressed, purified TatA allowed to exactly quantify the amount of TatA required to catalyse membrane transport of the model Tat substrate 16/23. We can show that TatA concentrations in the 100nM range are sufficient to efficiently catalyse membrane transport of the protein, which corresponds well to the amount of TatA identified in thylakoids. Furthermore, TatA shows cooperativity in its catalytic activity suggesting that Tat translocase operates as an allosteric enzyme complex.

TatA is an essential and structurally conserved component of all known Twin-arginine transport (Tat) machineries which are able to catalyse membrane transport of fully folded proteins. Here we have investigated if bacterial TatA, or chimeric pea/E. coli TatA derivatives, are capable of replacing thylakoidal TatA in function. While authentic E. coli TatA does not show any transport activity in thylakoid transport experiments, TatA chimeras comprising the transmembrane helix (TMH) of pea TatA are fully active. For minimal catalytic activity it is even sufficient to replace three residues within TMH of E. coli TatA by the corresponding pea residues. Almost any further substitution within TMH gradually raises transport activity in the thylakoid system, while functional characterization of the same set of TatA derivatives in E. coli yields essentially inverse catalytic activities. Closer inspection of the substituted residues suggests that the two transport systems have deviating demands with regard to the hydrophobicity of the transmembrane helix.