Towards the membrane topology of the early translocation intermediate Ti-1 during Tat-dependent protein transport

• Applicant: Professor Dr. Ralf Bernd Klösgen
• Subject Area: Plant Biochemistry and Biophysics
• Term: from 2011 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 194404447

Protein transport by the twin-arginine translocation (Tat) pathway, which operates at the thylakoid membrane of chloroplasts and at the plasma membranes of bacteria and archaea, is unique with respect to its property to translocate fully folded proteins across ion-tight membranes. It depends on signal peptides carrying a twin pair of arginine residues and can be divided into four consecutive steps, (i) unassisted binding of the precursor protein to the target membrane, (ii) association with the Tat receptor, (iii) membrane translocation of the passenger protein, and (iv) proteolytic removal of the transport signal. The initial membrane binding step, which leads to the formation of the early translocation intermediate Ti-1, is assumed to result in the insertion of the Tat substrate into the lipid bilayer, although firm data are lacking yet. Therefore, we propose to determine in detail the membrane topology of Ti-1 for the chimeric model substrate 16/23. Employing a set of molecular, biochemical and cell biology approaches we want to examine if the 16/23 chimera develops an integral loop topology within the membrane or if it is partially membrane inserted or even only firmly attached to the lipid bilayer in a conformation that prevents complete proteolytic degradation. Furthermore, we propose to determine the oligomeric state of the Tat substrate in its Ti-1 conformation to examine its relevance for the transport mechanism.

• DFG Programme: Research Grants