Demand of TatA during Tat-dependent protein transport across the thylakoid membrane

• Applicant: Professor Dr. Ralf Bernd Klösgen
• Subject Area: Plant Biochemistry and Biophysics
• Term: from 2015 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 268746007

The twin-arginine translocation (Tat) pathway is unique with respect to its property to translocate proteins in a fully folded conformation across ion-tight membranes. It is driven by the membrane potential which can be delta pH and/or delta psi. 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 actual membrane translocation step. TatA is additionally found also in the chloroplast stroma and it was recently shown that such soluble TatA, which was purified after heterologous expression in E. coli, can functionally replace the intrinsic thylakoidal TatA activity. This approach allowed us to exactly quantify the amount of TatA that is required for thylakoid transport of the model Tat substrate 16/23 under standard conditions (Hauer et al., 2013). Here we propose to extend the quantitative analysis of TatA demand during thylakoid transport to a set of precursor proteins that differ from each other in terms of molecular mass, dimensions, and/or folding status of the passenger polypeptide as well as with regard to the signal peptide present. The transport experiments will be performed under different physiological conditions, varying for example temperature and/or light regime, in order to take into account any potential influence of the experimental circumstances on TatA requirement. And finally, the role of the unstructured C-terminal domain of TatA in the transport process is to be studied. For this purpose, different TatA derivatives will be purified after heterologous overexpression in E. coli and analysed in such quantitative reconstitution experiments, namely a chimeric TatA protein having EGFP fused to its C-terminus as well as TatA truncation derivatives lacking parts of the C-terminal domain.

• DFG Programme: Research Grants