Tat translocon assembly states and structures

• Applicant: Professor Dr. Thomas Brüser
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 444687508

The twin-arginine translocation (Tat) system serves to transport folded, often cofactor-containing proteins across energy-transducing membranes in bacteria, archaea, plastids and also some mitochondria. Tat-dependently transported proteins are synthesized with a transport-mediating N-terminal signal peptide that contains the eponymous “twin-arginine motif”. The Tat pathway is essential for photosynthetic and many respiratory redox pathways, and it is also of special interest due to its involvement in virulence of many pathogens, including highly important species such as Mycobacterium tuberculosis or Pseudomonas aeruginosa. A key question of current Tat research is, how the Tat system mechanistically enables transport of proteins that can be even larger in diameter than the membrane thickness. Tat translocons are formed by multiple copies of minimally two distinct proteins, one member of the TatA/B family and a TatC. In the intensively studied model organism Escherichia coli, fully active translocons can be formed by the three components TatA, TatB, and TatC. We analyzed the assembly of active TatABC translocons and were able to distinguish three substrate-free Tat complexes (TC1, TC2, TC3) and two substrate associated complexes (TC1S and TC2S) by blue-native polyacrylamide gel electrophoresis (BN PAGE). Based on these and previous data, we developed a working model for translocon assembly steps and the concomitant translocation events. In this project, we aim to differentiate these distinct Tat complexes by site specific-mutagenesis in combination with site-specific cross-linking. We have already identified several mutations that enrich Tat complexes of certain types, and these changes of the complex distributions now permit the identification of complex-specific cross-links. The first part of the project focuses on these aspects and will extend the cross-link analyses to a larger number of positions, which will result in the assessment of changes of the translocon status under distinct conditions or growth phases. The second part of the project focuses on the biochemical and structural analyses of purified Tat complexes. For purifications in scales that permit analyses of substrate binding parameters, subunit stoichiometry or cryo-EM structure determination, Tat complexes will be stabilized by different approaches, including the use of cross-linking as well as novel detergent-independent solubilization methods. Together, both parts of this project will help to understand the still enigmatic mechanism for the fascinating transport of fully folded proteins across biological membranes.

• DFG Programme: Research Grants