TY - JOUR

T1 - The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

AU - Hou, Bo

AU - Heidrich, Eyleen S.

AU - Mehner-Breitfeld, Denise

AU - Brüser, Thomas

N1 - Funding Information: This work was supported by DFG Grant BR2285/4-2. The authors declare that they have no conflicts of interest with the contents of this article. We thank Sybille Traupe and Inge Reupke for technical support. Funding Information: This work was supported by DFG Grant BR2285/4-2. The authors declare that they have no conflicts of interest with the contents of this article. This article contains Fig. S1. 1 Both authors contributed equally to this work. 2 To whom correspondence should be addressed. Tel.: 49-511-762-5945; Fax: 49-511-762-5287; E-mail: brueser@ifmb.uni-hannover.de. 3 The abbreviations used are: Tat, twin-arginine translocation; APH, amphipathic helix; HiPIP, high-potential iron sulfur protein; IMV, inner membrane vesicle; NT, N-terminal domain; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; TMR, tetramethylrhodamine; TMH, transmembrane helix; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; YFP, yellow fluorescent protein.

PY - 2018/5/18

Y1 - 2018/5/18

N2 - The twin-arginine translocation (Tat) system that comprises the TatA, TatB, and TatC components transports folded proteins across energized membranes of prokaryotes and plant plastids. It is not known, however, how the transport of this protein cargo is achieved. Favored models suggest that the TatA component supports transport by weakening the membrane upon full translocon assembly. Using Escherichia coli as a model organism, we now demonstrate in vivo that the N terminus of TatA can indeed destabilize the membrane, resulting in a lowered membrane energization in growing cells. We found that in full-length TatA, this effect is counterbalanced by its amphipathic helix. Consistent with these observations, the TatA N terminus induced proton leakage in vitro, indicating membrane destabilization. Fluorescence quenching data revealed that substrate binding causes the TatA hinge region and the N-terminal part of the TatA amphipathic helix to move toward the membrane surface. In the presence of TatBC, substrate binding also reduced the exposure of a specific region in the amphipathic helix, indicating a participation of TatBC. Of note, the substrate-induced reorientation of the TatA amphipathic helix correlated with detectable membrane weakening. We therefore propose a two-state model in which membrane-destabilizing effects of the short TatA membrane anchor are compensated by the membrane-immersed N-terminal part of the amphipathic helix in a resting state. We conclude that substrate binding to TatABC complexes switches the position of the amphipathic helix, which locally weakens the membrane on demand to allow substrate translocation across the membrane.

AB - The twin-arginine translocation (Tat) system that comprises the TatA, TatB, and TatC components transports folded proteins across energized membranes of prokaryotes and plant plastids. It is not known, however, how the transport of this protein cargo is achieved. Favored models suggest that the TatA component supports transport by weakening the membrane upon full translocon assembly. Using Escherichia coli as a model organism, we now demonstrate in vivo that the N terminus of TatA can indeed destabilize the membrane, resulting in a lowered membrane energization in growing cells. We found that in full-length TatA, this effect is counterbalanced by its amphipathic helix. Consistent with these observations, the TatA N terminus induced proton leakage in vitro, indicating membrane destabilization. Fluorescence quenching data revealed that substrate binding causes the TatA hinge region and the N-terminal part of the TatA amphipathic helix to move toward the membrane surface. In the presence of TatBC, substrate binding also reduced the exposure of a specific region in the amphipathic helix, indicating a participation of TatBC. Of note, the substrate-induced reorientation of the TatA amphipathic helix correlated with detectable membrane weakening. We therefore propose a two-state model in which membrane-destabilizing effects of the short TatA membrane anchor are compensated by the membrane-immersed N-terminal part of the amphipathic helix in a resting state. We conclude that substrate binding to TatABC complexes switches the position of the amphipathic helix, which locally weakens the membrane on demand to allow substrate translocation across the membrane.

UR - http://www.scopus.com/inward/record.url?scp=85047365599&partnerID=8YFLogxK

U2 - 10.1074/jbc.RA118.002205

DO - 10.1074/jbc.RA118.002205

M3 - Article

C2 - 29535185

AN - SCOPUS:85047365599

VL - 293

SP - 7592

EP - 7605

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 20

ER -