A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase

The thylakoid ΔpH-dependent/Tat pathway is a novel system with the remarkable ability to transport tightly folded precursor proteins using a transmembrane ΔpH as the sole energy source. Three known components of the transport machinery exist in two distinct subcomplexes. A cpTatC–Hcf106 complex serves as precursor receptor and a Tha4 complex is required after precursor recognition. Here we report that Tha4 assembles with cpTatC–Hcf106 during the translocation step. Interactions among components were examined by chemical cross-linking of intact thylakoids followed by immunoprecipitation and immunoblotting. cpTatC and Hcf106 were consistently associated under all conditions tested. In contrast, Tha4 was only associated with cpTatC and Hcf106 in the presence of a functional precursor and the ΔpH. Interestingly, a synthetic signal peptide could replace intact precursor in triggering assembly. The association of all three components was transient and dissipated upon the completion of protein translocation. Such an assembly–disassembly cycle could explain how the ΔpH/Tat system can assemble translocases to accommodate folded proteins of varied size. It also explains in part how the system can exist in the membrane without compromising its ion and proton permeability barrier.

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Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation

10.1101/2021.03.19.436180 ◽

2021 ◽

Author(s):

Umesh K Bageshwar ◽

Antara DattaGupta ◽

Siegfried M Musser

Keyword(s):

Signal Peptide ◽

Protein Translocation ◽

Binding Interaction ◽

Twin Arginine Translocation ◽

Tat Pathway ◽

Hand Off ◽

Dependent Protein ◽

Tat System ◽

Folded Proteins ◽

High Concentrations

The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitrotransport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD > 50 M), and this monomer binds reversibly to spTorA (KD 1 M). While TorD binds to membranes (KD 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.

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Functional Analysis of the Twin-Arginine Translocation Pathway in Corynebacterium glutamicum ATCC 13869

Applied and Environmental Microbiology ◽

10.1128/aem.01528-06 ◽

2006 ◽

Vol 72 (11) ◽

pp. 7183-7192 ◽

Cited By ~ 34

Author(s):

Yoshimi Kikuchi ◽

Masayo Date ◽

Hiroshi Itaya ◽

Kazuhiko Matsui ◽

Long-Fei Wu

Keyword(s):

Corynebacterium Glutamicum ◽

Signal Peptide ◽

Fluorescent Protein ◽

Peptide Sequence ◽

Signal Peptide Sequence ◽

Gram Negative ◽

Tat Pathway ◽

Tat System ◽

Folded Proteins ◽

Green Fluorescent

ABSTRACT Compared to those of other gram-positive bacteria, the genetic structure of the Corynebacterium glutamicum Tat system is unique in that it contains the tatE gene in addition to tatA, tatB, and tatC. The tatE homologue has been detected only in the genomes of gram-negative enterobacteria. To assess the function of the C. glutamicum Tat pathway, we cloned the tatA, tatB, tatC, and tatE genes from C. glutamicum ATCC 13869 and constructed mutants carrying deletions of each tat gene or of both the tatA and tatE genes. Using green fluorescent protein (GFP) fused with the twin-arginine signal peptide of the Escherichia coli TorA protein, we demonstrated that the minimal functional Tat system required TatA and TatC. TatA and TatE provide overlapping function. Unlike the TatB proteins from gram-negative bacteria, C. glutamicum TatB was dispensable for Tat function, although it was required for maximal efficiency of secretion. The signal peptide sequence of the isomaltodextranase (IMD) of Arthrobacter globiformis contains a twin-arginine motif. We showed that both IMD and GFP fused with the signal peptide of IMD were secreted via the C. glutamicum Tat pathway. These observations indicate that IMD is a bona fide Tat substrate and imply great potential of the C. glutamicum Tat system for industrial production of heterologous folded proteins.

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Specificity of Signal Peptide Recognition in Tat-Dependent Bacterial Protein Translocation

Journal of Bacteriology ◽

10.1128/jb.183.2.604-610.2001 ◽

2001 ◽

Vol 183 (2) ◽

pp. 604-610 ◽

Cited By ~ 50

Author(s):

Natascha Blaudeck ◽

Georg A. Sprenger ◽

Roland Freudl ◽

Thomas Wiegert

Keyword(s):

Fusion Protein ◽

Signal Peptide ◽

Protein Translocation ◽

Signal Peptides ◽

E Coli ◽

Amino Terminal ◽

Twin Arginine Translocation ◽

Tat Pathway ◽

Dependent Protein ◽

Folded Proteins

ABSTRACT The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) ofZymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC andtatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.

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Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation

PLoS ONE ◽

10.1371/journal.pone.0256715 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0256715

Author(s):

Umesh K. Bageshwar ◽

Antara DattaGupta ◽

Siegfried M. Musser

Keyword(s):

Signal Peptide ◽

Protein Translocation ◽

Binding Interaction ◽

Twin Arginine Translocation ◽

Hand Off ◽

Dependent Protein ◽

Tat System ◽

Folded Proteins ◽

High Concentrations

The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitro transport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD > 50 μM), and this monomer binds reversibly to spTorA (KD ≈ 1 μM). While TorD binds to membranes (KD ≈ 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.

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Genetic Analysis of Pathway Specificity during Posttranslational Protein Translocation across the Escherichia coli Plasma Membrane

Journal of Bacteriology ◽

10.1128/jb.185.9.2811-2819.2003 ◽

2003 ◽

Vol 185 (9) ◽

pp. 2811-2819 ◽

Cited By ~ 62

Author(s):

Natascha Blaudeck ◽

Peter Kreutzenbeck ◽

Roland Freudl ◽

Georg A. Sprenger

Keyword(s):

Escherichia Coli ◽

Signal Peptide ◽

Protein Translocation ◽

Alternative Possibilities ◽

Amino Acid Residues ◽

Sec Pathway ◽

Twin Arginine Translocation ◽

Tat Pathway ◽

Arginine Residues ◽

Dependent Protein

ABSTRACT In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the “Sec avoidance signal,” the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.

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Channel crossing: how are proteins shipped across the bacterial plasma membrane?

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2015.0025 ◽

2015 ◽

Vol 370 (1679) ◽

pp. 20150025 ◽

Cited By ~ 44

Author(s):

Ian Collinson ◽

Robin A. Corey ◽

William J. Allen

Keyword(s):

Free Energy ◽

Plasma Membrane ◽

Atp Hydrolysis ◽

Protein Translocation ◽

Transmembrane Protein ◽

Conducting Channel ◽

Outstanding Problem ◽

Unfolded State ◽

Tat System ◽

Folded Proteins

The structure of the first protein-conducting channel was determined more than a decade ago. Today, we are still puzzled by the outstanding problem of protein translocation—the dynamic mechanism underlying the consignment of proteins across and into membranes. This review is an attempt to summarize and understand the energy transducing capabilities of protein-translocating machines, with emphasis on bacterial systems: how polypeptides make headway against the lipid bilayer and how the process is coupled to the free energy associated with ATP hydrolysis and the transmembrane protein motive force. In order to explore how cargo is driven across the membrane, the known structures of the protein-translocation machines are set out against the background of the historic literature, and in the light of experiments conducted in their wake. The paper will focus on the bacterial general secretory (Sec) pathway (SecY-complex), and its eukaryotic counterpart (Sec61-complex), which ferry proteins across the membrane in an unfolded state, as well as the unrelated Tat system that assembles bespoke channels for the export of folded proteins.

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Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC–Hcf106 complex before Tha4-dependent transport

The Journal of Cell Biology ◽

10.1083/jcb.200105149 ◽

2001 ◽

Vol 154 (4) ◽

pp. 719-730 ◽

Cited By ~ 220

Author(s):

Kenneth Cline ◽

Hiroki Mori

Keyword(s):

Specific Binding ◽

Signal Peptides ◽

Hydrophobic Core ◽

Precursor Proteins ◽

Peptide Precursors ◽

Folded Proteins ◽

Dependent Transport ◽

Dependent Pathway ◽

Blue Native ◽

Chemical Cross Linking

The thylakoid ΔpH-dependent pathway transports folded proteins with twin arginine–containing signal peptides. Identified components of the machinery include cpTatC, Hcf106, and Tha4. The reaction occurs in two steps: precursor binding to the machinery, and transport across the membrane. Here, we show that a cpTatC–Hcf106 complex serves as receptor for specific binding of twin arginine–containing precursors. Antibodies to either Hcf106 or cpTatC, but not Tha4, inhibited precursor binding. Blue native gel electrophoresis and coimmunoprecipitation of digitonin-solubilized thylakoids showed that Hcf106 and cpTatC are members of an ∼700-kD complex that lacks Tha4. Thylakoid-bound precursor proteins were also associated with an ∼700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106. Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane. Precursor binding to the cpTatC–Hcf106 complex required both the twin arginine and the hydrophobic core of the signal peptide. Precursors remained bound to the complex when Tha4 was sequestered by antibody, even in the presence of ΔpH. These results indicate that precursor binding to the cpTatC–Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.

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A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615056114 ◽

2017 ◽

Vol 114 (10) ◽

pp. E1958-E1967 ◽

Cited By ~ 19

Author(s):

Qi Huang ◽

Felicity Alcock ◽

Holger Kneuper ◽

Justin C. Deme ◽

Sarah E. Rollauer ◽

...

Keyword(s):

Signal Peptide ◽

Protein Translocation ◽

Signal Sequence ◽

Transmembrane Helix ◽

Peptide Binding ◽

Signal Peptides ◽

Arginine Residues ◽

Tat System ◽

Twin Arginine Translocase

The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system ofEscherichia coliis made up of TatA, TatB, and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site-specific cross-linking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA–YFP fusion showed that the TatB F13Y substitution resulted in signal peptide-independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase.

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Signal Peptide Hydrophobicity Modulates Interaction with the Twin-Arginine Translocase

mBio ◽

10.1128/mbio.00909-17 ◽

2017 ◽

Vol 8 (4) ◽

Cited By ~ 12

Author(s):

Qi Huang ◽

Tracy Palmer

Keyword(s):

Signal Peptide ◽

Secretory Pathway ◽

Recognition Site ◽

Signal Peptides ◽

Tat Pathway ◽

Folded Proteins ◽

Twin Arginine Translocase ◽

Critical Requirement ◽

Highly Hydrophobic ◽

Peptide Hydrophobicity

ABSTRACT The general secretory pathway (Sec) and twin-arginine translocase (Tat) operate in parallel to export proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Substrates are targeted to their respective machineries by N-terminal signal peptides that share a tripartite organization; however, Tat signal peptides harbor a conserved and almost invariant arginine pair that is critical for efficient targeting to the Tat machinery. Tat signal peptides interact with a membrane-bound receptor complex comprised of TatB and TatC components, with TatC containing the twin-arginine recognition site. Here, we isolated suppressors in the signal peptide of the Tat substrate, SufI, that restored Tat transport in the presence of inactivating substitutions in the TatC twin-arginine binding site. These suppressors increased signal peptide hydrophobicity, and copurification experiments indicated that they restored binding to the variant TatBC complex. The hydrophobic suppressors could also act in cis to suppress substitutions at the signal peptide twin-arginine motif that normally prevent targeting to the Tat pathway. Highly hydrophobic variants of the SufI signal peptide containing four leucine substitutions retained the ability to interact with the Tat system. The hydrophobic signal peptides of two Sec substrates, DsbA and OmpA, containing twin lysine residues, were shown to mediate export by the Tat pathway and to copurify with TatBC. These findings indicate that there is unprecedented overlap between Sec and Tat signal peptides and that neither the signal peptide twin-arginine motif nor the TatC twin-arginine recognition site is an essential mechanistic feature for operation of the Tat pathway. IMPORTANCE Protein export is an essential process in all prokaryotes. The Sec and Tat export pathways operate in parallel, with the Sec machinery transporting unstructured precursors and the Tat pathway transporting folded proteins. Proteins are targeted to the Tat pathway by N-terminal signal peptides that contain an almost invariant twin-arginine motif. Here, we make the surprising discovery that the twin arginines are not essential for recognition of substrates by the Tat machinery and that this requirement can be bypassed by increasing the signal peptide hydrophobicity. We further show that signal peptides of bona fide Sec substrates can also mediate transport by the Tat pathway. Our findings suggest that key features of the Tat targeting mechanism have evolved to prevent mistargeting of substrates to the Sec pathway rather than being a critical requirement for function of the Tat pathway.

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