Abstract
Twin-arginine translocation (Tat) is a unique protein transport pathway in bacteria, archaea, and plastids. It mediates the transmembrane transport of fully folded proteins, which harbor a consensus twin-arginine motif in their signal sequences. In Gram-negative bacteria and plant chloroplasts, three membrane proteins, named TatA, TatB, and TatC, are required to enable Tat translocation. Available data suggest that TatA assembles into oligomeric pore-like structures that might function as the protein conduit across the lipid bilayer. Using site-specific photo-cross-linking, we have investigated the molecular environment of TatA under resting and translocating conditions. We find that monomeric TatA is an early interacting partner of functionally targeted Tat substrates. This interaction with TatA likely precedes translocation of Tat substrates and is influenced by the proton-motive force. It strictly depends on the presence of TatB and TatC, the latter of which is shown to make contacts with the transmembrane helix of TatA.
Highlights
The membrane proteins, TatA, TatB, and TatC, enable the transmembrane passage of folded precursor proteins
Radiolabeled RR precursor proteins synthesized by cellfree transcription/translation were allowed to bind to, and translocate into, these vesicles, and cross-links that formed upon UV irradiation of the samples were analyzed by SDSPAGE and phosphorimaging
In chloroplasts the three components of the Tat translocase clearly partition into two separable complexes, a hetero-oligomeric TatBC complex and a homo-oligomeric TatA complex [14], TatBC complexes when purified from E. coli were often found to contain TatA [10, 11]
Summary
The membrane proteins, TatA, TatB, and TatC, enable the transmembrane passage of folded precursor proteins. This interaction with TatA likely precedes translocation of Tat substrates and is influenced by the proton-motive force It strictly depends on the presence of TatB and TatC, the latter of which is shown to make contacts with the transmembrane helix of TatA. The prevailing model for how Tat-specific translocation occurs suggests that RR precursors target the membrane via the TatBC complex (8, 9, 13, 14, 18 –21) and that the RR consensus motif is initially recognized by TatC [19, 22,23,24]. To learn more about the functions and properties of TatA, we have generated multiple E. coli TatA variants carrying a sitespecific photo-activated cross-linker within both its helices and its unstructured C terminus This approach allowed characterizing intermolecular contacts between TatA and Tat substrates as well as between TatA and the other subunits of the Tat translocase
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