Functional reconstitution of thylakoidal Tat translocase (Twin arginine translocase) into lipid membranes

• Applicant: Professor Dr. Ralf Bernd Klösgen
• Subject Area: Plant Biochemistry and Biophysics
• Term: from 2012 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 218735884

The Tat (twin arginine translocation) pathway, which is capable of transporting fully folded proteins across ion-tight membranes, is operating at the thylakoid membrane of chloroplasts as well as at the cytoplasmic membranes of bacteria and archaea. In chloroplasts, Tat machinery is composed of the three membrane proteins TatA, TatB, and TatC. TatB and TatC constitute a set of heteromeric complexes of high molecular weight, some of which provide the membrane receptors for substrate proteins carrying Tat-specific signal peptides. TatA interacts with these receptor complexes and is required for the actual membrane translocation of the Tat substrates. Three models are currently discussed, (a) translocation pores with different or variable diameter, (b) TatA-induced membrane weakening enabling translocation directly through the lipid bilayer, and (c) a catalytic or regulatory function of TatA inducing transport by the TatBC complexes. Despite extensive efforts from numerous labs it was not possible so far to clarify the transport mechanism with the established analytical systems. Therefore, we propose to reconstitute functional Tat translocation machinery in liposomes starting from heterologously expressed, purified Tat subunits. Liposomes are free from potentially interfering components of the thylakoid membrane and should thus allow to determine (i) the properties of the Tat subunits to self-assemble into heteromeric membrane complexes, (ii) the exact (and possibly variable) stoichiometry of Tat subunits within these Tat complexes, (iii) the potential role of lipid composition for the assembly and functionality of the complexes, and (iv) the involvement of either of the subunits in each step of the transport process. In the long term, these experiments will provide the basis also for detailed structural analyses of this exceptional protein transport machinery.

• DFG Programme: Research Grants