The Chloroplast Twin Arginine Transport (Tat) Component, Tha4, Undergoes Conformational Changes Leading to Tat Protein Transport

Aldridge, Cassie; Storm, Amanda R.; Cline, Kenneth; Dabney‐Smith, Carole

doi:10.1074/jbc.m112.385666

Cited by 38 publications

(57 citation statements)

References 39 publications

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“…Both scenarios are conceivable and a direct substrate interaction with the APH has been already demonstrated (46). Also the substrate induced accessibility differences that have been detected in the thylakoid system were considered to potentially indicate a steric hindrance by substrate (33). In this context, one significant difference of our data to the data reported by Aldridge et al (33) may be highly important: the movement of the hinge region preceding the APH was not detected in that study.…”

Section: Substrate-inducedcontrasting

confidence: 56%

“…Also the substrate induced accessibility differences that have been detected in the thylakoid system were considered to potentially indicate a steric hindrance by substrate (33). In this context, one significant difference of our data to the data reported by Aldridge et al (33) may be highly important: the movement of the hinge region preceding the APH was not detected in that study. We think that this difference is caused by the much larger size of the modifying agent (methyl-PEG12)3-PEG4-maleimide (2360 Da) that had to be used to detect modifications by shifts in SDS-PAGE analyses.…”

Section: Substrate-inducedcontrasting

confidence: 55%

“…The group of Ken Cline demonstrated in seminal studies substrate-induced TatA-TatBC and TatATatA cross-links that can be explained by TatA recruitments, rearrangements or conformational transitions (44,12,45). Later, a substrate-induced conformational transition of the APH that is similar to the one that we now show for the E. coli system had been clearly demonstrated for the thylakoid system by Aldridge et al (33). The data of that study are largely consistent with our data and in this study it was already hypothesized that a moved APH could induce a membrane weakening.…”

Section: Substrate-inducedmentioning

confidence: 85%

“…NMR studies (32) and biochemical accessibility studies (33) already indicated that the APH of TatA can adopt a tilted orientation at the membrane surface. The APH could directly contribute to membrane thickness at the membrane-anchor sites.…”

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

confidence: 99%

“…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%

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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-inducedcontrasting

confidence: 56%

Section: Substrate-inducedcontrasting

confidence: 55%

Section: Substrate-inducedmentioning

confidence: 85%

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

confidence: 99%

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

confidence: 99%

See 3 more Smart Citations

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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Thylakoid‐integrated recombinant Hcf106 participates in the chloroplast twin arginine transport system

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New

et al. 2018

Plant Direct

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The chloroplast twin arginine transport (cpTat) system distinguishes itself as a protein transport pathway by translocating fully folded proteins, using the proton‐motive force (PMF) as the sole source of energy. The cpTat pathway is evolutionarily conserved with the Tat pathway found in the plasma membrane of many prokaryotes. The cpTat (Escherichia coli) system uses three proteins, Tha4 (TatA), Hcf106 (TatB), and cpTatC (TatC), to form a transient translocase allowing the passage of precursor proteins. Briefly, cpTatC and Hcf106, with Tha4, form the initial receptor complex responsible for precursor protein recognition and binding in an energy‐independent manner, while a separate pool of Tha4 assembles with the precursor‐bound receptor complex in the presence the PMF. Analysis by blue‐native polyacrylamide gel electrophoresis (BN‐PAGE) shows that the receptor complex, in the absence of precursor, migrates near 700 kDa and contains cpTatC and Hcf106 with little Tha4 remaining after detergent solubilization. To investigate the role that Hcf106 may play in receptor complex oligomerization and/or stability, systematic cysteine substitutions were made in positions from the N‐terminal transmembrane domain to the end of the predicted amphipathic helix of the protein. BN‐PAGE analysis allowed us to identify the locations of amino acids in Hcf106 that were critical for interacting with cpTatC. Oxidative cross‐linking allowed us to map interactions of the transmembrane domain and amphipathic helix region of Hcf106. In addition, we showed that in vitro expressed, integrated Hcf106 can interact with the precursor signal peptide domain and imported cpTatC, strongly suggesting that a subpopulation of the integrated Hcf106 is participating in competent cpTat complexes.

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Protein Routing Processes in the Thylakoid

Dabney‐Smith

Storm

2014

Plastid Biology

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The Chloroplast Twin Arginine Transport (Tat) Component, Tha4, Undergoes Conformational Changes Leading to Tat Protein Transport

Cited by 38 publications

References 39 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

Thylakoid‐integrated recombinant Hcf106 participates in the chloroplast twin arginine transport system

Protein Routing Processes in the Thylakoid

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