Abstract
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.
Highlights
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
This lack of transport activity is not a consequence of substrate selectivity of TatA because neither plant Tat substrates, like the precursor of the 23 kDa subunit of the oxygen evolving system or the chimeric model substrate 16/23, nor even bacterial Tat substrates, like the chimeric model substrates TorA-MalE or TorA-mCherry[36, 37], show any membrane transport in these assays (Fig. 2A and B). All these proteins are efficiently transported if the assays are complemented with pea TatA which reconfirms earlier observations that bacterial Tat substrates are principally suited for thylakoidal membrane transport[38, 39]
EcoTatA[N8pea], and even E. coli TatA (Fig. 8C,D) despite the fact that the three proteins are barely or not at all active in the thylakoid system (Figs. 2 and 5). From these results we conclude that in the thylakoid system the catalytic transport activity of a given TatA protein is not a simple consequence of its extent of membrane integration but rests instead to a larger degree on processes taking place after membrane binding. It was the goal of this study to find out if chimeric TatA proteins composed of segments derived from both, E. coli TatA and pea TatA, can principally replace the respective authentic TatA proteins in bacterial and plant Tat transport systems and, if so, to determine which residues of either of the original TatAs are required to render the chimeric proteins catalytically active in the respective heterologous system
Summary
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. The twin-arginine translocation (Tat) pathway, which is found at the thylakoid membrane of chloroplasts and the plasma membranes of bacteria and archaea (for recent reviews see refs 1–4), is engaged by proteins carrying signal peptides with a characteristic twin pair of arginine residues within their N-region which gave rise to the name of the pathway[5, 6]. The energy for their membrane transport is provided solely by the transmembrane potential, notably ΔpH and/or ΔΨ7, 8. Both substoichiometric[25], stoichiometric[25, 31], as well as excess amounts of TatA32 compared with TatB and TatC were described depending on the method used for analysis and/or the plant species studied
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
