Structure of the TatC core of the twin-arginine protein transport system

Rollauer, Sarah E.; Tarry, M.J.; Graham, James E.; Jääskeläinen, Mari; Jäger, Franziska; Johnson, Steven; Krehenbrink, Martin; Liu, Sai Man; Lukey, Michael J.; Marcoux, Julien; McDowell, Melanie A.; Rodriguez, Fernanda; Roversi, Pietro; Stansfeld, Phillip J.; Robinson, Carol V.; Sansom, Mark S. P.; Palmer, Tracy; Högbom, Martin; Berks, Ben C.; Lea, Susan M.

doi:10.1038/nature11683

Cited by 166 publications

(276 citation statements)

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“…This was accomplished with a novel system in which recombinant cpTatC was imported into isolated intact chloroplasts and was analyzed independently of endogenous cpTatC. The results of these studies fit nicely with and extend studies of the bacterial Tat system and map biochemical function onto the recently determined crystal structure for monomeric TatC from Aquifex aeolicus (Rollauer et al, 2012). In addition to providing insight into the roles of cpTatC in folded protein transport, our studies provide a rapid and effective method, unique to chloroplasts, for analyzing the functions of an integral thylakoid membrane protein.…”

Section: Introductionsupporting

confidence: 48%

“…It forms a homooligomer to which Hcf106 (TatB) binds to assemble the receptor complex (Behrendt et al, 2007;Orriss et al, 2007); it binds to the signal peptide Alami et al, 2003;Gérard and Cline, 2006); it probably serves as the organizing center for Tha4 assembly (Fröbel et al, 2011;Rollauer et al, 2012); and it may provide the motive force for transmembrane protein movement (Brüser and Sanders, 2003;Dabney-Smith et al, 2006;Cline and McCaffery, 2007). Yet the molecular determinants of these different functions are largely unknown.…”

Section: Introductionmentioning

confidence: 99%

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Mapping the Signal Peptide Binding and Oligomer Contact Sites of the Core Subunit of the Pea Twin Arginine Protein Translocase

Cline

2013

The Plant Cell

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Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.

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Section: Introductionsupporting

confidence: 48%

Section: Introductionmentioning

confidence: 99%

Mapping the Signal Peptide Binding and Oligomer Contact Sites of the Core Subunit of the Pea Twin Arginine Protein Translocase

Cline

2013

The Plant Cell

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“…To confirm that TatA-YFP association was the result of a productive interaction between substrate proteins and TatBC, we expressed both CueO and TatBC from plasmids in strain AyE, but this time we used a TatC variant containing the substitutions F94A and E103A that are known to block signal peptide binding (21,38). These cells failed to produce TatA-YFP assemblies, demonstrating that TatBC must be capable of binding substrate proteins for TatA association to occur (Fig.…”

Section: Coexpression With Tate Improves the Transport Activity Of Cellsmentioning

confidence: 99%

“…Tat-signal peptides are recognized at the membrane by a TatBC receptor complex (18)(19)(20)(21)(22)(23). Substrate protein binding to TatBC causes a PMF-dependent recruitment of TatA (and presumably TatE) to assemble the active translocation site (18,24).…”

mentioning

confidence: 99%

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

Alcock

Baker

Greene

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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170

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The twin-arginine translocation (Tat) machinery transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. It has been inferred that the Tat translocation site is assembled on demand by substrate-induced association of the protein TatA. We tested this model by imaging YFP-tagged TatA expressed at native levels in living Escherichia coli cells in the presence of low levels of the TatA paralogue TatE. Under these conditions the TatA-YFP fusion supports full physiological Tat transport activity. In agreement with the TatA association model, raising the number of transport-competent substrate proteins within the cell leads to an increase in the number of large TatA complexes present. Formation of these complexes requires both a functional TatBC substrate receptor and the transmembrane proton motive force (PMF). Removing the PMF causes TatA complexes to dissociate, except in strains with impaired Tat transport activity. Based on these observations we propose that TatA assembly reaches a critical point at which oligomerization can be reversed only by substrate transport. In contrast to TatA-YFP, the oligomeric states of TatB-YFP and TatC-YFP fusions are not affected by substrate or the PMF, although TatB-YFP oligomerization does require TatC.

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“…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).…”

mentioning

confidence: 99%

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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Structure of the TatC core of the twin-arginine protein transport system

Cited by 166 publications

References 64 publications

Mapping the Signal Peptide Binding and Oligomer Contact Sites of the Core Subunit of the Pea Twin Arginine Protein Translocase

Mapping the Signal Peptide Binding and Oligomer Contact Sites of the Core Subunit of the Pea Twin Arginine Protein Translocase

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

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

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