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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Frontiers Media S.A.
2019-07-01
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doaj-c21b47c4c94a4659ab338f097b611beb2020-11-25T00:12:29ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2019-07-011010.3389/fmicb.2019.01482458271A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coliHendrik GeiseEyleen Sabine HeidrichChristoph Stefan NikolinDenise Mehner-BreitfeldThomas BrüserThe 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.https://www.frontiersin.org/article/10.3389/fmicb.2019.01482/fulltwin-arginine translocationmembrane protein complexesprotein translocationEscherichia coliphoto cross-linking |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Hendrik Geise Eyleen Sabine Heidrich Christoph Stefan Nikolin Denise Mehner-Breitfeld Thomas Brüser |
spellingShingle |
Hendrik Geise Eyleen Sabine Heidrich Christoph Stefan Nikolin Denise Mehner-Breitfeld Thomas Brüser A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli Frontiers in Microbiology twin-arginine translocation membrane protein complexes protein translocation Escherichia coli photo cross-linking |
author_facet |
Hendrik Geise Eyleen Sabine Heidrich Christoph Stefan Nikolin Denise Mehner-Breitfeld Thomas Brüser |
author_sort |
Hendrik Geise |
title |
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_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_sort |
potential late stage intermediate of twin-arginine dependent protein translocation in escherichia coli |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Microbiology |
issn |
1664-302X |
publishDate |
2019-07-01 |
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 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. |
topic |
twin-arginine translocation membrane protein complexes protein translocation Escherichia coli photo cross-linking |
url |
https://www.frontiersin.org/article/10.3389/fmicb.2019.01482/full |
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