How to achieve Tat transport with alien TatA

Hauer, René Steffen; Freudl, Roland; Dittmar, Julia; Jakob, Mario; Klösgen, Ralf Bernd

doi:10.1038/s41598-017-08818-w

“…The exact sequence of the TMH is not relevant, as an artificial TMH in TatA-NT-(LA)6 had the same effect (Figure 3-5). This fits to the recent observation that the exact sequence of that region is not a determinant for function, as it can be exchanged between E. coli and thylakoid systems (41). Our data now experimentally show the postulated membrane-weakening by the unusual TMH of TatA (18), which was so far supported by molecular dynamics simulations (6).…”

Section: Substrate-inducedsupporting

confidence: 74%

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

³

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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“…It is found operating at the cytoplasmic membranes of bacteria and archaea as well as at the thylakoid membranes of chloroplasts and their progenitors, cyanobacteria (Müller and Klösgen, 2005). Despite some differences in detail, the overall structure of the Tat machinery as well as the presumed mechanism of membrane translocation is largely conserved among all these systems (e.g., Cline et al, 2015;Hauer et al, 2017;Petterson et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…Subsequently, transport was reconstituted by supplementing the assays with soluble TatA obtained from in vitro translation or heterologous overexpression. The antibody approach turned out to be particularly suitable and was used, for example, for the characterization of mutant or chimeric TatA derivatives (Dabney-Smith et al, 2003;Hauer et al, 2017) as well as for the quantification of TatA demand during transport of a model Tat substrate (Hauer et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Functional reconstitution of TatB into the thylakoidal Tat translocase

Zinecker¹,

Jakob²,

Klösgen³

2020

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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We have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twinarginine protein transport (Tat 1 ) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analyzed with the authentic Tat substrate pOEC16, reached routinely 20 -25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutant TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional mutant analyses. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport.

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“…It is found operating at the cytoplasmic membranes of bacteria and archaea as well as at the thylakoid membranes of chloroplasts and their progenitors, cyanobacteria (Müller and Klösgen, 2005). Despite some differences in detail, the overall structure of the Tat machinery as well as the presumed mechanism of membrane translocation is largely conserved among all these systems (e.g., Cline et al, 2015;Hauer et al, 2017;Petterson et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…Subsequently, transport was reconstituted by supplementing the assays with soluble TatA obtained from in vitro translation or heterologous overexpression. The antibody approach turned out to be particularly suitable and was used, for example, for the characterization of mutant or chimeric TatA derivatives (Dabney-Smith et al, 2003;Hauer et al, 2017) as well as for the quantification of TatA demand during transport of a model Tat substrate (Hauer et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Functional reconstitution of TatB into thylakoidal Tat translocase

Zinecker

¹

,

Jakob

²

,

Klösgen

³

2019

Preprint

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We have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twinarginine protein transport (Tat 1 ) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analyzed with the authentic Tat substrate pOEC16, reached routinely 20 -25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutant TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional mutant analyses. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport. Keywords Twin-arginine translocation, thylakoid membrane, transport reconstitution, heterologous overexpression, protein purification 1 The abbreviations used are: Tat, twin-arginine translocation; OEC16, 16 kDa subunit of the oxygen-evolving system associated with photosystem II; RTS, Rapid Translation System; Hepes, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; BrCN, cyanogen bromide -3 -

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How to achieve Tat transport with alien TatA

Cited by 11 publications

References 54 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

Functional reconstitution of TatB into the thylakoidal Tat translocase

Functional reconstitution of TatB into thylakoidal Tat translocase

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