Structural model for the protein-translocating element of the twin-arginine transport system

Rodriguez, Fernanda; Rouse, Sarah L.; Tait, Claudia E.; Harmer, Jeffrey; Riso, Antonio De; Timmel, Christiane R.; Sansom, Mark S. P.; Berks, Ben C.; Schnell, Jason R.

doi:10.1073/pnas.1219486110

Cited by 102 publications

(171 citation statements)

References 52 publications

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“…TatA is anchored by its Nterminus in the cytoplasmic membrane (20). The Nterminal 6 residues are in the periplasmic surface of the membrane (6). The trans-membrane helix (TMH) starts at Trp-7/Gln-8 on the periplasmic side, and ends at Phe-20 on the cytoplasmic side.…”

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

confidence: 99%

“…The trans-membrane helix (TMH) starts at Trp-7/Gln-8 on the periplasmic side, and ends at Phe-20 on the cytoplasmic side. The hydrophobic residues Leu-9 to Phe-20 actually cross the lipid bilayer (21,6). As the extremely short membrane-spanning region of TatA has been suggested to have the membrane-weakening effect, we tested whether production of the TatA membrane anchor (= TatA-NT, residues 1 to 21) affects membrane stability.…”

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

confidence: 99%

“…Molecular dynamics simulations support this view and suggest that multiple TatA N-termini can cause a local thinning of the membrane (6). However, direct experimental evidence for a thinning stress imposed by the Nterminus of TatA is lacking.…”

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confidence: 92%

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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: 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%

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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 structure of E. coli TatA consists of a transmembrane helix followed by an amphipathic helix and then a natively unstructured tail (30). Phenylalanine 39 at the end of the amphipathic helix is invariant and essential for TatA function (39,40).…”

Section: Coexpression With Tate Improves the Transport Activity Of Cellsmentioning

confidence: 99%

“…Substrateinduced association of TatA protomers could allow stepwise construction of a channel-like element around the substrate until the required size for transport is achieved (27). Alternatively, concentrating TatA might perturb the local membrane structure to allow substrate movement (25,(28)(29)(30). It also is possible that formation of a TatA oligomer stores conformational energy that is then used to drive the substrate across the membrane.…”

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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Structural considerations of folded protein import through the chloroplast TOC/TIC translocons

Ganesan

Theg

2019

FEBS Letters

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Protein import into chloroplasts is carried out by the protein translocons at the outer and inner envelope membranes (TOC and TIC). Detailed structures for these translocons are lacking, with only a low‐resolution TOC complex structure available. Recently, we showed that the TOC/TIC translocons can import folded proteins, a rather unique feat for a coupled double membrane system. We also determined the maximum functional TOC/TIC pore size to be 30–35 Å. Here, we discuss how such large pores could form and compare the structural dynamics of the pore‐forming Toc75 subunit to its bacterial/mitochondrial Omp85 family homologs. We put forward structural models that can be empirically tested and also briefly review the pore dynamics of other protein translocons with known structures.

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Structural model for the protein-translocating element of the twin-arginine transport system

Cited by 102 publications

References 52 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

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

Structural considerations of folded protein import through the chloroplast TOC/TIC translocons

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