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A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli

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, an...

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Autores principales: Geise, Hendrik, Heidrich, Eyleen Sabine, Nikolin, Christoph Stefan, Mehner-Breitfeld, Denise, Brüser, Thomas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2019
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637791/
https://www.ncbi.nlm.nih.gov/pubmed/31354642
http://dx.doi.org/10.3389/fmicb.2019.01482
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author Geise, Hendrik
Heidrich, Eyleen Sabine
Nikolin, Christoph Stefan
Mehner-Breitfeld, Denise
Brüser, Thomas
author_facet Geise, Hendrik
Heidrich, Eyleen Sabine
Nikolin, Christoph Stefan
Mehner-Breitfeld, Denise
Brüser, Thomas
author_sort Geise, Hendrik
collection PubMed
description 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 TatC(I50Bpa) 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 TatBC(I50Bpa) complex was formed of the size of wild-type substrate-containing TatABC complexes, suggesting that TatB occupies TatA-binding sites at TatC(I50Bpa). When substrate binding was abolished by point mutations, this TatBC(I50Bpa) complex shifted analogously to active TatABC(I50Bpa) 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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spelling pubmed-66377912019-07-26 A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli Geise, Hendrik Heidrich, Eyleen Sabine Nikolin, Christoph Stefan Mehner-Breitfeld, Denise Brüser, Thomas Front Microbiol Microbiology 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 TatC(I50Bpa) 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 TatBC(I50Bpa) complex was formed of the size of wild-type substrate-containing TatABC complexes, suggesting that TatB occupies TatA-binding sites at TatC(I50Bpa). When substrate binding was abolished by point mutations, this TatBC(I50Bpa) complex shifted analogously to active TatABC(I50Bpa) 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. Frontiers Media S.A. 2019-07-11 /pmc/articles/PMC6637791/ /pubmed/31354642 http://dx.doi.org/10.3389/fmicb.2019.01482 Text en Copyright © 2019 Geise, Heidrich, Nikolin, Mehner-Breitfeld and Brüser. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Geise, Hendrik
Heidrich, Eyleen Sabine
Nikolin, Christoph Stefan
Mehner-Breitfeld, Denise
Brüser, Thomas
A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli
title A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli
title_full A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli
title_fullStr A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli
title_full_unstemmed A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli
title_short A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli
title_sort potential late stage intermediate of twin-arginine dependent protein translocation in escherichia coli
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637791/
https://www.ncbi.nlm.nih.gov/pubmed/31354642
http://dx.doi.org/10.3389/fmicb.2019.01482
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