TatBC-Independent TatA/Tat Substrate Interactions Contribute to Transport Efficiency

Taubert, Johannes; Hou, Bo; Risselada, Herre Jelger; Mehner, Denise; Lünsdorf, Heinrich; Grubmüller, Helmut; Brüser, Thomas

doi:10.1371/journal.pone.0119761

Cited by 20 publications

(25 citation statements)

References 82 publications

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“…In that study, also MD simulations were carried out that indicated intensive contacts between the APH of TatA and mainly the c-domain of the signal peptide. This fully agrees with the fact that the RR-motif is not recognized by TatA (26). MD simulations of TatA in membranes suggest that only oligomeric TatA assemblies can destabilize the membrane (6).…”

Section: Substrate-inducedsupporting

confidence: 79%

“…We have recently demonstrated that TatA can interact with Tat substrates in a signal peptide-dependent, RRindependent manner (26). In that study, also MD simulations were carried out that indicated intensive contacts between the APH of TatA and mainly the c-domain of the signal peptide.…”

Section: Substrate-inducedmentioning

confidence: 92%

“…An APH interaction with mature domains of Tat substrates had been for the first time experimentally demonstrated by the group of Carole Dabney Smith for the thylakoidal system (46). Also in the E. coli system, mature domains of Tat substrates have been shown to interact with TatA (16,(47)(48)(49), and it has been demonstrated that TatA has the capacity to interact with Tat substrates in a TatBC independent manner (26). For the thylakoid system it has been suggested that the APH interactions relate to nonspecific passive contacts before or during the membrane passage of mature domains, as a crosslink to a position at the end of the APH (Phe-48) was TatB-and PMF-dependent (46).…”

Section: Substrate-inducedmentioning

confidence: 95%

“…Accessibility of this segment in any orientation to the aqueous surface in the course of substrateinduced conformational changes should thus become detectable. Dyes at positions 22,23,26,27,28,32,33,34,35,42, and 45 cover the region from the hinge to the APH and an adjacent conserved negatively charged stretch. Position 51 served as positive control for quenching, as it is already positioned in an exposed region that is not anymore required for functionality (25).…”

Section: A Cell-growth Effect Of the Tata Membrane-anchor That Is Commentioning

confidence: 99%

“…Based on the substrateindependent TatA/TatBC interactions, we currently favor the view that TatA and TatBC always function hand in hand and remain in close proximity. The RR-motif of Tat signal peptides can remain associated with TatC during translocation (15), and Tat substrates are contacting TatA during transport with their mature domains (46,49,26). TatA recognizes the transported domains to initiate the conformational switch, but TatBC recognizes the RR-motif and transport is only achieved when both activities cooperate.…”

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confidence: 99%

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

Hou¹,

Heidrich²,

Mehner-Breitfeld

et al. 2018

Journal of Biological Chemistry

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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 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 membranedestabilizing effects of the short TatA membrane anchor are compensated by the membraneimmersed 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.The Tat system serves to transport folded proteins in bacteria, archaea, plant plastids, and possibly plant mitochondria (1-4). In Escherichia coli, the Tat system consists of TatA, TatB, and TatC components. TatA and TatB are similar, and two-component "minimal" Tat systems exist in which TatA exerts functions of TatA and TatB (5). TatA/B components are N-terminally membraneanchored by a very short hydrophobic transmembrane domain (TMD), which is followed by a short hinge region, an amphipathic helix (APH), and a variable C-terminal domain (6). TatC is a polytopic membrane protein with six TMDs (7,8). TatB tightly interacts with TatC (9, 10). TatA associates with these TatBC complexes and is thought to permeabilize the membrane for protein transport (11)(12)(13)(14). TatBC complexes recognize and tightly bind the signal peptides of the cargo proteins throughout the translocation process (15,16).The mechanism by which this translocation is achieved must be unusual and is not understood (17). A currently favored model suggests that the N-termini of multiple TatA molecules at the translocon site weaken the membrane upon substrate-binding, thereby permitting a TatCmediated pulling of the substrate through the Control of membrane-weakening by TatA 2 destabilized membrane ("membrane-weakening and pulling mechanism", 18). Molecular dynamics simulations support this view ...

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Section: Substrate-inducedsupporting

confidence: 79%

Section: Substrate-inducedmentioning

confidence: 92%

Section: Substrate-inducedmentioning

confidence: 95%

Section: A Cell-growth Effect Of the Tata Membrane-anchor That Is Commentioning

confidence: 99%

mentioning

confidence: 99%

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

Hou¹,

Heidrich²,

Mehner-Breitfeld

et al. 2018

Journal of Biological Chemistry

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Tat transport in Escherichia coli requires zwitterionic phosphatidylethanolamine but no specific negatively charged phospholipid

et al. 2017

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Translocation of folded proteins by the Tat system of Escherichia coli is believed to rely on the presence of phosphatidylethanolamine (PE) and the negatively charged phospholipids cardiolipin (CL) and phosphatidylglycerol (PG). Here we show that while PE is indeed essential for activity, the Tat system is fully functional in a clsA/clsB/clsC deletion strain lacking CL, and in a pgsA deletion strain lacking both PG and CL during aerobic growth on complex media. In contrast to early studies that relied on strains with reduced lipid levels, this study therefore demonstrates that PG and CL are dispensable for Tat transport. The lack of these lipids may be compensated by other anionic phospholipids such as phosphatidic acid, CDP-diacylglycerol, or N-acyl-PE.

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Secretion of recombinant proteins from E. coli

Kleiner-Grote

Risse

Friehs

2018

Engineering in Life Sciences

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The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.

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TatBC-Independent TatA/Tat Substrate Interactions Contribute to Transport Efficiency

Cited by 20 publications

References 82 publications

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

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

Tat transport in Escherichia coli requires zwitterionic phosphatidylethanolamine but no specific negatively charged phospholipid

Secretion of recombinant proteins from E. coli

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