TY - JOUR

T1 - A potential late stage intermediate of twin-arginine dependent protein translocation in Escherichia coli

AU - Geise, Hendrik

AU - Heidrich, Eyleen Sabine

AU - Nikolin, Christoph Stefan

AU - Mehner-Breitfeld, Denise

AU - Brüser, Thomas

N1 - Funding Information: This work was funded by the German Research Foundation (DFG grant BR2285/4-2). Publisher Copyright: Copyright © 2019 Geise, Heidrich, Nikolin, Mehner-Breitfeld and Brüser. 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. Copyright: Copyright 2019 Elsevier B.V., All rights reserved.

PY - 2019/7

Y1 - 2019/7

N2 - 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.

AB - 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.

KW - Escherichia coli

KW - Membrane protein complexes

KW - Photo cross-linking

KW - Protein translocation

KW - Twin-arginine translocation

UR - http://www.scopus.com/inward/record.url?scp=85069493647&partnerID=8YFLogxK

U2 - 10.3389/fmicb.2019.01482

DO - 10.3389/fmicb.2019.01482

M3 - Article

AN - SCOPUS:85069493647

VL - 10

JO - Frontiers in Microbiology

JF - Frontiers in Microbiology

SN - 1664-302X

IS - JULY

M1 - 1482

ER -