Enough is enough: TatA demand during Tat-dependent protein transport

Hauer, René Steffen; Schlesier, René; Heilmann, K; Dittmar, Julia; Jakob, Mario; Klösgen, Ralf Bernd

doi:10.1016/j.bbamcr.2013.01.030

Cited by 24 publications

(50 citation statements)

References 44 publications

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“…In order to make sure that a still functionally active protein is analyzed, in thylakoido complementation experiments according to Hauer et al . were performed (Fig. B).…”

Section: Resultsmentioning

confidence: 99%

“…Subsequent membrane translocation of the passenger polypeptide, which requires both TatA and the membrane potential, is still a matter of debate. Three deviating working models are currently discussed: (a) translocation pores with different or variable diameter to facilitate membrane transport of the various Tat substrates, which are all distinct in size , (b) TatA‐induced membrane weakening resulting in translocation of the substrate directly through the lipid bilayer , and (c) a catalytic or regulatory function of TatA facilitating membrane transport by the TatBC complexes .…”

Section: Introductionmentioning

confidence: 99%

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Structure and dynamics of plant TatA in micelles and lipid bilayers studied by solution NMR

Pettersson

Jakob

et al. 2018

The FEBS Journal

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The twin-arginine translocase (Tat) transports folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. In Gram-negative bacteria and chloroplasts, the translocon consists of three subunits, TatA, TatB, and TatC, of which TatA is responsible for the actual membrane translocation of the substrate. Herein we report on the structure, dynamics, and lipid interactions of a fully functional C-terminally truncated 'core TatA' from Arabidopsis thaliana using solution-state NMR. Our results show that TatA consists of a short N-terminal transmembrane helix (TMH), a short connecting linker (hinge) and a long region with propensity to form an amphiphilic helix (APH). The dynamics of TatA were characterized using N relaxation NMR in combination with model-free analysis. The TMH has order parameters characteristic of a well-structured helix, the hinge is somewhat less rigid, while the APH has lower order parameters indicating structural flexibility. The TMH is short with a surprisingly low protection from solvent, and only the first part of the APH is protected to some extent. In order to uncover possible differences in TatA's structure and dynamics in detergent compared to in a lipid bilayer, fast-tumbling bicelles and large unilamellar vesicles were used. Results indicate that the helicity of TatA increases in both the TMH and APH in the presence of lipids, and that the N-terminal part of the TMH is significantly more rigid. The results indicate that plant TatA has a significant structural plasticity and a capability to adapt to local environments.

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“…In order to make sure that a still functionally active protein is analyzed, in thylakoido complementation experiments according to Hauer et al . were performed (Fig. B).…”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Structure and dynamics of plant TatA in micelles and lipid bilayers studied by solution NMR

Pettersson

Jakob

et al. 2018

The FEBS Journal

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“…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).…”

Section: Introductionmentioning

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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“…In thylakoido protein transport experiments with radiolabelled precursor proteins were performed as described (Marques et al, 2003). The inhibition of Tat-dependent protein transport with affinity-purified antibodies against TatA as well as the reconstitution of Tat transport with externally added TatA, which was obtained by in vitro translation in the Rapid Translation System (RTS, 5PRIME, Hamburg, Germany), were carried out according to Hauer et al (2013). Preparation of liposomes and liposome insertion experiments followed published protocols (Hou et al, 2006;Schlesier and Klösgen, 2010).…”

Section: Protein Transport and Liposome Insertion Assaysmentioning

confidence: 99%

“…This processing product, which comprises the five additional residues, is fully resistant to externally added protease in line with a localization in the thylakoid lumen. To clarify whether the formation of the 13 kDa processing product requires a functional Tat machinery, thylakoid vesicles were pretreated with antiTatA antibodies according to Hauer et al (2013) to block intrinsic TatA activity. Under these conditions, thylakoid transport of iΔC88 is largely inhibited because the protein is almost completely degraded by externally added protease ( Figure 1D).…”

Section: Thylakoid Transport Of a Truncated 16/23 Protein Correspondimentioning

confidence: 99%

C-terminal truncation of a Tat passenger protein affects its membrane translocation by interfering with receptor binding

Schlesier

Klösgen

2015

Biological Chemistry

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During thylakoid transport of the chimeric model twin-arginine translocation (Tat) substrate 16/23, two consecutive translocation intermediates with different membrane topology are observed. The early translocation intermediate Ti-1 is bound to the membrane such that almost half of the protein is protected against proteolysis and it was concluded that not only the signal peptide but also part of the passenger protein participates in membrane binding. However, topology studies using a membrane-impermeable thiol-reactive reagent show that most of the passenger remains accessible from the stromal side in Ti-1 conformation. Establishment of such Ti-1 topology at the membrane apparently requires the fully folded passenger protein, as it was not observed with 16/23 truncation derivatives lacking the C-terminal 20, 40, 60, or 88 residues. Thylakoid transport of these mutants, which depends on a fully functional Tat machinery, is progressively reduced with increasing size of the truncated passenger polypeptide. The same holds true also for the interaction with the thylakoidal TatBC complexes, suggesting that in this case receptor binding, which is apparently impaired by extended unfolded or malfolded passenger polypeptides, is the rate-limiting step of Tat-dependent membrane transport.

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Enough is enough: TatA demand during Tat-dependent protein transport

Cited by 24 publications

References 44 publications

Structure and dynamics of plant TatA in micelles and lipid bilayers studied by solution NMR

Structure and dynamics of plant TatA in micelles and lipid bilayers studied by solution NMR

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

C-terminal truncation of a Tat passenger protein affects its membrane translocation by interfering with receptor binding

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