Abstract: | |
The twin-arginine translocation (Tat) system transports folded proteins across membranes of prokaryotes, plant plastids, and some mitochondria. According to blue-native polyacrylamide gel electrophoresis after solubilization with digitonin, distinct interactions between the components TatA, TatB, and TatC result in two major TatBC-containing complexes in Escherichia coli that can bind protein substrates. We now report the first detection of a TatABC complex that likely represents the state at which transport occurs. This complex was initially found when the photo cross-linking amino acid p-benzoyl-L-phenylalanine (Bpa) was introduced at position I50 on the periplasmic side of the first trans-membrane domain of TatC. Cross-linking of TatCI50Bpa resulted in TatC-TatC-cross-links, indicating a close proximity to neighboring TatC in the complex. However, the new complex was not caused by cross-links but rather by non-covalent side chain interactions, as it was also detectable without UV-cross-linking or with an I50Y exchange. The new complex did not contain any detectable substrate. It was slightly upshifted relative to previously reported substrate-containing TatABC complexes. In the absence of TatA, an inactive TatBCI50Bpa complex was formed of the size of wild-type substrate-containing TatABC complexes, suggesting that TatB occupies TatA-binding sites at TatCI50Bpa. When substrate binding was abolished by point mutations, this TatBCI50Bpa complex shifted analogously to active TatABCI50Bpa complexes, indicating that a defect substrate-binding site further enhances TatB association to TatA-binding sites. Only TatA could shift the complex with an intact substrate-binding site, which explains the TatA requirement for substrate transport by TatABC systems.
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License of this version: | CC BY 4.0 Unported - https://creativecommons.org/licenses/by/4.0/ |
Publication type: | Article |
Publishing status: | publishedVersion |
Publication date: | 2019 |
Keywords english: | Escherichia coli, Membrane protein complexes, Photo cross-linking, Protein translocation, Twin-arginine translocation, digitonin, n benzoylphenylalanine derivative, protein translocase, TATA binding protein, TATA binding protein related factor, affinity chromatography, Article, bacterial translocation, binding site, Escherichia coli, gene expression, gene mutation, immunoblotting, mutagenesis, nonhuman, phase contrast microscopy, plasmid, point mutation, polyacrylamide gel electrophoresis, protein binding, protein cross linking, protein expression, size exclusion chromatography, Western blotting |
DDC: | 570 | Biowissenschaften, Biologie |
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