Tat translocon assembly states and structures
Final Report Abstract
The twin-arginine translocation (Tat) system is used to transport folded, often cofactorcontaining proteins across energized membranes of bacteria, archaea, plant mitochondria and plastids. Tat-dependently transported proteins are synthesized with a transportmediating N-terminal signal peptide, which contains a so-called twin-arginine motif. The Tat pathway is essential for all photosynthetic and many respiratory redox pathways. It is therefore not surprising that the Tat system is also required for the virulence of many pathogenic bacteria, including such important species as Mycobacterium tuberculosis or Pseudomonas aeruginosa. A central question in current Tat research is how the Tat system mechanistically allows the transport of proteins that can even be larger in diameter than membrane thicknesses. Tat translocons are composed of multiple copies of at least two different proteins, one of the TatAB protein family and one TatC. The intensively studied model organism Escherichia coli forms active Tat translocons from the three components TatA, TatB and TatC, of which TatB and TatC form the so-called “Tat receptor”, whereas TatA is recruited to permit the permeabilization of the membrane. Using blue-native polyacrylamide gel electrophoresis (BN-PAGE), three substrate-free Tat complexes (Tat complex TC1, TC2, TC3) and two substrate-associated complexes (TC1S, TC2S) can be distinguished, with TC3 being shown in analyses of certain mutated TatC variants. In this project, we were able for the first time to biochemically differentiate and characterize the two Tat complexes TC1 and TC2. This was made possible because we found mutations in TatC that selectively stabilize TC1 or TC2, or destabilize the respective other complex. This enabled us to purify TC1 and TC2, examine their composition, and further characterize the interactions of their components biochemically. Regarding their composition, we found that the TatBC components in TC2 interact significantly more strongly with TatA than TC1, as TatA is co-purified to a greater extent with TC2. Clusters of multiple TatA protomers largely dissociate from TatBC under the conditions of the BN-PAGE analysis, which supports the idea that TatA clusters laterally associate with the TatBC cores. This agrees with crosslinking analyses, in which we were able to show that the TatC:TatA interaction sites in TatC are the same in TC1 and TC2, suggesting that the TatA association is not accompanied with larger rearrangements of TatBC core complexes. The TatA interaction site remained unaltered upon substrate-binding, and therefore are likely used throughout the transport process. However, substrate-binding lead to sensitization of the TatA-TatC interaction to treatment with the detergent digitonin, which is generally used to solubilize Tat complexes, suggesting that substrate-binding induces conformational changes in TatBC that affect the TatA interaction site. Based on these results, it was possible to classify the two Tat complexes into the assembly and transport processes. This enabled a better mechanistic understanding of Tat transport of folded proteins to be achieved.
Publications
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Towards a functional and structural understanding of membrane-weakening by TatA. Poster on the EMBO Meeting „New challenges in protein translocation across membranes“, 17 – 21 September 2022 in Sant Feliu de Guixols, Spain
Mehner-Breitfeld, D. et al.
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A larger TatBC complex associates with TatA clusters for transport of folded proteins across the bacterial cytoplasmic membrane. Scientific Reports, 14(1).
Werner, Max-Hinrich; Mehner-Breitfeld, Denise & Brüser, Thomas
