Biochemical characterization of membrane complexes of the thylakoidal Delta pH-dependent twin arginine translocase (Tat translocase)

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Among the four protein transport pathways operating at the chloroplast thylakoid membrane, the DpH/Tat pathway is unique with respect to its ability to transport folded proteins and its energy source, the transmembrane proton gradient generated upon photosynthesis. From the three components of the DpH/Tat machinery (TatA, TatB, TatC), only TatB and TatC have so far been found in heteromeric complexes. We intend to characterize the DpH/Tat translocase by following approaches: (1) Isolation of the TatB/C and TatA complexes from thylakoid membranes using affinity chromatography based on single chain antibody fragments (scFv). (2) Isolation of individual Tat proteins by different chromatographic methods and mass spectrometry in order to identify possible modifications. (3) Isolation of presumed ligands of the Tat complexes by affinity chromatography. (4) Assessing a potential specificity of the DpH/Tat translocase for the folding state of the substrates using EGFP-containing chimeric precursor proteins. (5) In vitro trapping of the translocase in its active conformation using specifically engineered chimeric substrates. (6) In vivo trapping of the active translocase by transient expression of such chimeric substrates.