Twin-arginine Translocation System Research Articles

Analysis of twin-arginine translocation system gene homology and transcription in Vibrio species

To determine the function of twin-arginine translocation system (Tat) and gene cluster in Vibrio strains and to analyze the homology of tat gene cluster among different Vibrio spp. strains based on N16961 and tatABC mutant strains N169-dtat. Different serotypes of biotype strains of Vibrio spp. were selected to detect the transcription of 4 genes of Tat transport system and upstream ubi aarF gene and downstream cyt551 gene by the total RNA reverse transcription and homologicity of the gene cluster by sequencing analysis. Our results showed that the 4 genes of tat cluster (tatA, tatB, tatC, and tatE) were intragenic and co-transcribed. We found that ubi aarF gene could be co-transcribed with tatA, tatB, but not with tatC. The electron transport chain and energy metabolism-related genes, cytochrome C551 peroxidase gene, and 4 genes located at upstream of tatABC operon were not transcribed with tatABC. Although the co-transcription between ubi aarF and tatAB was blocked in N169-dtat strain, they were still transcribed separately. Homologous analysis of genes of tat cluster in different types of Vibrio cholerae showed that tat gene cluster was a very conservative. The ubi and aarF gene might be co-transcribed with genes of tat cluster in Vibrio cholerae, which and the close relationship showed that they might play a key function in Vibrio cholerae.

The twin-arginine protein translocation pathway.

The twin-arginine translocation (Tat) system, found in prokaryotes, chloroplasts, and some mitochondria, allows folded proteins to be moved across membranes. How this transport is achieved without significant ion leakage is an intriguing mechanistic question. Tat transport is mediated by complexes formed from small integral membrane proteins from just two protein families. Atomic-resolution structures have recently been determined for representatives of both these protein families, providing the first molecular-level glimpse of the Tat machinery. I review our current understanding of the mechanism of Tat transport in light of these new structural data.

Formate dehydrogenase localization and activity are dependent on an intact twin arginine translocation system (Tat) in Campylobacter jejuni 81-176.

The oxidation of formate constitutes a main energy source for Campylobacter jejuni, an important foodborne pathogen. Formate occurs in the hosts' gut as a byproduct of fermentation, which may contribute to C. jejuni's adaptation to that niche. C. jejuni possesses a periplasmic formate dehydrogenase (Fdh) that facilitates the metabolism of formate. In C. jejuni NCTC-11168, a poorly invasive strain, Fdh localizes to the periplasm via the twin arginine translocation (Tat) system, and the fdhA encodes the signature Tat signal motif. However, screening the genome of C. jejuni 81-176, a highly invasive strain, showed that the Tat signal motif was encoded on a separate gene (CJJ81176_1504) directly upstream of the fdhA (CJJ81176_1503). This possibly suggested a difference in formate metabolism between these two strains. Therefore, we investigated whether the Fdh was Tat dependent in C. jejuni 81-176. For this purpose, we measured formate respiration in C. jejuni 81-176 (wild-type), a Tat mutant (ΔtatC-81-176), and a complementation strain (C-ΔtatC-81-176) using BIOLOG-AN plates. We also used a viologen-based assay to specifically measure the Fdh enzymatic activity in whole cells and periplasmic preparations of the wild-type and mutant strains. Collectively, our data indicated that the Fdh activity and localization in C. jejuni 81-176 were dependent on a functional Tat system. The proper maturation and maintenance of Fdh are essential for cognate energy production, which might explain the association of the Fdh with the Tat system across different strains. The latter highlights the potential of the Tat system and Fdh as appealing targets for novel anti-C. jejuni therapeutics.

Protein translocation without specific quality control in a computational model of the Tat system

The twin-arginine translocation (Tat) system transports folded proteins of various sizes across both bacterial and plant thylakoid membranes. The membrane-associated TatA protein is an essential component of the Tat translocon, and a broad distribution of different sized TatA-clusters is observed in bacterial membranes. We assume that the size dynamics of TatA clusters are affected by substrate binding, unbinding, and translocation to associated TatBC clusters, where clusters with bound translocation substrates favour growth and those without associated substrates favour shrinkage. With a stochastic model of substrate binding and cluster dynamics, we numerically determine the TatA cluster size distribution. We include a proportion of targeted but non-translocatable (NT) substrates, with the simplifying hypothesis that the substrate translocatability does not directly affect cluster dynamical rate constants or substrate binding or unbinding rates. This amounts to a translocation model without specific quality control. Nevertheless, NT substrates will remain associated with TatA clusters until unbound and so will affect cluster sizes and translocation rates. We find that the number of larger TatA clusters depends on the NT fraction f. The translocation rate can be optimized by tuning the rate of spontaneous substrate unbinding, . We present an analytically solvable three-state model of substrate translocation without cluster size dynamics that follows our computed translocation rates, and that is consistent with in vitro Tat-translocation data in the presence of NT substrates.

The TatBC Complex of the Tat Protein Translocase in Escherichia coli and Its Transition to the Substrate-Bound TatABC Complex

The twin-arginine translocation (Tat) system serves to transport folded proteins across membranes of prokaryotes and plant plastids. In Escherichia coli, a complex consisting of multiple copies of TatB and TatC initiates the transport by binding the signal peptides of the Tat substrates. Using blue-native polyacrylamide gel electrophoresis, bands of TatBC-containing complexes can be detected at molecular masses of 440 and 580 kDa. We systematically analyzed the formation of Tat complexes with TatB or TatC variants that carried point mutations at selected positions. Several mutations resulted in specific disassembly patterns and alterations in the 440 kDa:580 kDa complex ratios. The 440 kDa complex contains only TatBC, whereas the 580 kDa complex consists of TatABC. Substrate binding results in a TatBC-Tat substrate complex at ~500 kDa and a TatABC-Tat substrate complex at ~600 kDa. Only the ~600 kDa complex was detected with nonrecombinant substrate levels and thus could be the physiologically most relevant species. The results suggest that some TatA is usually associated with TatBC, regardless of substrate binding.

Solution structure of the TatB component of the twin-arginine translocation system

The twin-arginine protein transport (Tat) system translocates fully folded proteins across lipid membranes. In Escherichia coli, the Tat system comprises three essential components: TatA, TatB and TatC. The protein translocation process is proposed to initiate by signal peptide recognition and substrate binding to the TatBC complex. Upon formation of the TatBC–substrate protein complex, the TatA subunits are recruited and form the protein translocation pore. Experimental evidences suggest that TatB forms a tight complex with TatC at 1:1 molar ratio and the TatBC complex contains multiple copies of both proteins. Cross-linking experiments demonstrate that TatB functions in tetrameric units and interacts with both TatC and substrate proteins. However, structural information of the TatB protein is still lacking, and its functional mechanism remains elusive. Herein, we report the solution structure of TatB in DPC micelles determined by Nuclear Magnetic Resonance (NMR) spectroscopy. Overall, the structure shows an extended ‘L-shape’ conformation comprising four helices: a transmembrane helix (TMH) α1, an amphipathic helix (APH) α2, and two solvent exposed helices α3 and α4. The packing of TMH and APH is relatively rigid, whereas helices α3 and α4 display notably higher mobility. The observed floppiness of helices α3 and α4 allows TatB to sample a large conformational space, thus providing high structural plasticity to interact with substrate proteins of different sizes and shapes.

Twin-Arginine Translocation System in Helicobacter pylori: TatC, but Not TatB, Is Essential for Viability

ABSTRACTThe twin-arginine translocation (Tat) system, needed to transport folded proteins across biological membranes, has not been characterized in the gastric pathogen Helicobacter pylori. Analysis of all H. pylori genome sequences available thus far reveals the presence of single copies of tatA, tatB, and tatC needed for the synthesis of a fully functional Tat system. Based on the presence of the twin-arginine hallmark in their signal sequence, only four H. pylori proteins appear to be Tat dependent: hydrogenase (HydA), catalase-associated protein (KapA), biotin sulfoxide reductase (BisC), and the ubiquinol cytochrome oxidoreductase Rieske protein (FbcF). In the present study, targeted mutations were aimed at tatA, tatB, tatC, or queA (downstream gene control). While double homologous recombination mutations in tatB and queA were easily obtained, attempts at disrupting tatA proved unsuccessful, while deletion of tatC led to partial mutants following single homologous recombination, with cells retaining a chromosomal copy of tatC. Double homologous recombination tatC mutants were obtained only when a plasmid-borne, isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible copy of tatC was introduced prior to transformation. These conditional tatC mutants could grow only in the presence of IPTG, suggesting that tatC is essential in H. pylori. tatB and tatC mutants had lower hydrogenase and catalase activities than the wild-type strain did, and the ability of tatC mutants to colonize mouse stomachs was severely affected compared to the wild type. Chromosomal complementation of tatC mutants restored hydrogenase and catalase activities to wild-type levels, and additional expression of tatC in wild-type cells resulted in elevated Tat-dependent enzyme activities. Unexpectedly, the tat strains had cell envelope defects.

The Twin Arginine Translocation System Is Essential for Aerobic Growth and Full Virulence of Burkholderia thailandensis

The twin arginine translocation (Tat) system in bacteria is responsible for transporting folded proteins across the cytoplasmic membrane, and in some bacteria, Tat-exported substrates have been linked to virulence. We report here that the Tat machinery is present in Burkholderia pseudomallei, B. mallei, and B. thailandensis, and we show that the system is essential for aerobic but not anaerobic growth. Switching off of the Tat system in B. thailandensis grown anaerobically resulted in filamentous bacteria, and bacteria showed increased sensitivity to some β-lactam antibiotics. In Galleria mellonella and zebrafish infection models, the Tat conditional mutant was attenuated. The aerobic growth-restricted phenotype indicates that Tat substrates may play a functional role in oxygen-dependent energy conservation. In other bacteria, aerobic growth restriction in Tat mutants has been attributed to the inability to translocate PetA, the Rieske iron-sulfur protein which forms part of the quinol-cytochrome c oxidoreductase complex. Here, we show that PetA is not responsible for aerobic growth restriction in B. thailandensis. However, we have identified an operon encoding 2 proteins of unknown function (BTH_I2176 and BTH_I2175) that play a role in aerobic growth restriction, and we present evidence that BTH_I2176 is Tat translocated.

Investigating the interaction between peptides of the amphipathic helix of Hcf106 and the phospholipid bilayer by solid-state NMR spectroscopy

The chloroplast twin arginine translocation (cpTat) system transports highly folded precursor proteins into the thylakoid lumen using the protonmotive force as its only energy source. Hcf106, as one of the core components of the cpTat system, is part of the precursor receptor complex and functions in the initial precursor-binding step. Hcf106 is predicted to contain a single amino terminal transmembrane domain followed by a Pro-Gly hinge, a predicted amphipathic α-helix (APH), and a loosely structured carboxy terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes. To better understand the membrane active capabilities of Hcf106, we used solid-state NMR spectroscopy to investigate those properties of the APH. In this study, synthesized peptides of the predicted Hcf106 APH (amino acids 28–65) were incorporated at increasing mol.% into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and POPC/MGDG (monogalactosyldiacylglycerol; mole ratio 85:15) multilamellar vesicles (MLVs) to probe the peptide–lipid interaction. Solid-state 31P NMR and 2H NMR spectroscopic experiments revealed that the peptide perturbs the headgroup and the acyl chain regions of phospholipids as indicated by changes in spectral lineshape, chemical shift anisotropy (CSA) line width, and 2H order SCD parameters. In addition, the comparison between POPC MLVs and POPC/MGDG MLVs indicated that the lipid bilayer composition affected peptide perturbation of the lipids, and such perturbation appeared to be more intense in a system more closely mimicking a thylakoid membrane.

Solid-state NMR investigations of peptide–lipid interactions of the transmembrane domain of a plant-derived protein, Hcf106

The chloroplast twin arginine translocation system transports highly folded precursor proteins across the thylakoid using the protonmotive force as its only energy source. Hcf106 and another thylakoid protein, cpTatC compose the precursor receptor complex. Hcf106 is predicted to contain a single amino terminal transmembrane domain (TMD) followed by a Pro–Gly hinge, an amphipathic α-helix, and a loosely structured carboxyl terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes; however, how this occurs is not understood. To investigate how Hcf106 inserts itself into the membrane unassisted, solid-state NMR spectroscopy was used to investigate the membrane activity of the TMD. A synthetic peptide of the Hcf106 TMD was incorporated into multilamellar vesicles made of 100% 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) or 85%:15% ratio with monogalactosyl diacylglycerol (POPC/MGDG) to probe peptide–lipid interaction. Solid-state 31P NMR and 2H NMR spectroscopic techniques were used to reveal peptide perturbations of the phospholipid membranes. Changes in spectral lineshape, chemical shift anisotropy width, 31P T1 relaxation time and SCD order parameters demonstrated that the Hcf106 TMD peptide interacted with the phospholipids. Furthermore, the comparison between POPC and POPC/MGDG multilamellar vesicles indicated that lipid bilayer composition affected the peptide–lipid interaction with the peptide interacting preferentially with vesicles that more closely mimic the thylakoid.

Presenting a foreign antigen on live attenuated Edwardsiella tarda using twin-arginine translocation signal peptide as a multivalent vaccine

The twin-arginine translocation (Tat) system is a major pathway for transmembrane translocation of fully folded proteins. In this study, a multivalent vaccine to present foreign antigens on live attenuated vaccine Edwardsiella tarda WED using screened Tat signal peptide was constructed. Because the Tat system increases the yields of folded antigens in periplasmic space or extracellular milieu, it is expected to contribute to the production of conformational epitope-derived specific antibodies. E. tarda Tat signal peptides fused with the green fluorescent protein (GFP) was constructed under the control of an in vivo inducible dps promoter. The resulting plasmids were electroporated into WED and the subcellular localizations of GFP were analyzed with Western blotting. Eight signal peptides with optimized GFP translocation efficiency were further fused to a protective antigen glyceraldehyde-3-phosphate dehydrogenase (GapA) from a fish pathogen Aeromonas hydrophila. Signal peptides of DmsA, NapA, and SufI displayed high efficiency for GapA translocation. The relative percent survival (RPS) of turbot was measured with a co-infection of E. tarda and A. hydrophila, and the strain with DmsA signal peptide showed the maximal protection. This study demonstrated a new platform to construct multivalent vaccines using optimized Tat signal peptide in E. tarda.

Twin‐arginine translocation system (tat) mutants of Salmonella are attenuated due to envelope defects, not respiratory defects

The twin-arginine translocation system (Tat) transports folded proteins across the cytoplasmic membrane and is critical to virulence in Salmonella and other pathogens. Experimental and bioinformatic data indicate that 30 proteins are exported via Tat in Salmonella Typhimurium. However, there are no data linking specific Tat substrates with virulence. We inactivated every Tat-exported protein and determined the virulence phenotype of mutant strains. Although a tat mutant is highly attenuated, no single Tat-exported substrate accounts for this virulence phenotype. Rather, the attenuation is due primarily to envelope defects caused by failure to translocate three Tat substrates, the N-acetylmuramoyl-l-alanine amidases, AmiA and AmiC, and the cell division protein, SufI. Strikingly, neither the amiA amiC nor the sufI mutations alone conferred any virulence defect. Although AmiC and SufI have previously been localized to the divisome, the synthetic phenotypes observed are the first to suggest functional overlap. Many Tat substrates are involved in anaerobic respiration, but we show that a mutant completely deficient in anaerobic respiration retains full virulence in both the oral and systemic phases of infection. Similarly, an obligately aerobic mutant is fully virulent. These results suggest that in the classic mouse model of infection, S. Typhimurium is replicating only in aerobic environments.

High‐level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin‐arginine translocation system in Escherichia coli

The twin-arginine translocation (Tat) system transports folded proteins across the plasma membrane in bacteria, and heterologous proteins can be exported by this pathway if a Tat-type signal peptide is present at the N-terminus. The system thus has potential for biopharmaceutical production in Escherichia coli, where export to the periplasm is often a favoured approach. Previous studies have shown that E. coli cells can export high levels of protein by the Tat pathway, and the protein product accummulates almost exclusively in the periplasm. In this study, we analysed E. coli cells that express the Bacillus subtilis TatAdCd system in place of the native TatABC system. We show that a heterologous model protein, comprising the TorA signal peptide linked to green fluorescent protein (TorA-GFP), is efficiently exported by the TatAdCd system. However, whereas the GFP is exported initially to the periplasm during batch fermentation, the mature protein is increasingly found in the extracellular culture medium. By the end of a 16-h fermentation, ~ 90% of exported GFP is present in the medium as active mature protein. The total protein profiles of the medium and periplasm are essentially identical, confirming that the outer membrane becomes leaky during the fermentation process. The cells are otherwise intact, and there is no large-scale release of cytoplasmic contents. Export levels are relatively high, with ~ 0.35 g GFP·L⁻¹ culture present in the medium. This system thus offers a means of producing recombinant protein in E. coli and harvesting directly from the medium, with potential advantages in terms of ease of purification and downstream processing.

A Multicopper Oxidase Is Required for Copper Resistance in Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most important bacterial pathogens. Recent work has revealed that the natural bactericidal properties of copper are utilized by the host immune system to combat infections with bacteria, including M. tuberculosis. However, M. tuberculosis employs multiple mechanisms to reduce the internal copper amount by efflux and sequestration, which are required for virulence of M. tuberculosis. Here, we describe an alternative mechanism of copper resistance by M. tuberculosis. Deletion of the rv0846c gene increased the susceptibility of M. tuberculosis to copper at least 10-fold, establishing Rv0846c as a major component of copper resistance in M. tuberculosis. In vitro assays showed that Rv0846c oxidized organic substrates and Fe(II). Importantly, mutation of the predicted copper-coordinating cysteine 486 resulted in inactive Rv0846c protein which did not protect M. tuberculosis against copper stress. Hence, Rv0846c is a multicopper oxidase of M. tuberculosis and was renamed mycobacterial multicopper oxidase (MmcO). MmcO is membrane associated, probably by lipidation after export across the inner membrane by the twin-arginine translocation system. However, mutation of the lipidation site did not affect the oxidase activity or the copper protective function of MmcO. Our study revealed MmcO as an important copper resistance mechanism of M. tuberculosis, which possibly acts by oxidation of toxic Cu(I) in the periplasm.

Characterization of a pre-export enzyme–chaperone complex on the twin-arginine transport pathway

The Tat (twin-arginine translocation) system is a protein targeting pathway utilized by prokaryotes and chloroplasts. Tat substrates are produced with distinctive N-terminal signal peptides and are translocated as fully folded proteins. In Escherichia coli, Tat-dependent proteins often contain redox cofactors that must be loaded before translocation. Trimethylamine N-oxide reductase (TorA) is a model bacterial Tat substrate and is a molybdenum cofactor-dependent enzyme. Co-ordination of cofactor loading and translocation of TorA is directed by the TorD protein, which is a cytoplasmic chaperone known to interact physically with the TorA signal peptide. In the present study, a pre-export TorAD complex has been characterized using biochemical and biophysical techniques, including SAXS (small-angle X-ray scattering). A stable, cofactor-free TorAD complex was isolated, which revealed a 1:1 binding stoichiometry. Surprisingly, a TorAD complex with similar architecture can be isolated in the complete absence of the 39-residue TorA signal peptide. The present study demonstrates that two high-affinity binding sites for TorD are present on TorA, and that a single TorD protein binds both of those simultaneously. Further characterization suggested that the C-terminal ‘Domain IV’ of TorA remained solvent-exposed in the cofactor-free pre-export TorAD complex. It is possible that correct folding of Domain IV upon cofactor loading is the trigger for TorD release and subsequent export of TorA.

Leaving home ain't easy: protein export systems in Gram-positive bacteria

Transport of proteins into or across biological membranes is catalyzed by membrane-bound transport machineries. In Gram-positive bacteria, the vast majority of proteins are exported out of the cytosol by the conserved general secretion (Sec) system or, alternatively, by the twin-arginine translocation (Tat) system, that closely resemble their well-studied counterparts in Gram-negative bacteria. Besides these common major export routes, additional unique protein export systems (such as accessory Sec2 systems and/or type VII/WXG100 secretion systems) exist in some Gram-positive bacteria that are specifically involved in the secretion of limited subsets of proteins.

Fluorescence imaging of GFP-fused periplasmic components of Na+-driven flagellar motor using Tat pathway in Vibrio alginolyticus

The twin-arginine translocation (Tat) system works to export folded proteins across the cytoplasmic membrane via specific signal peptides harbouring a twin-arginine motif. In Escherichia coli, a functional GFP is exported to the periplasm through the Tat pathway by fusion of the signal peptide of TorA, which is one of the periplasmic proteins exported by the Tat pathway. In this study, we fused the signal peptide of Vibrio alginolyticus TorA (TorASP) to GFP and demonstrate the export of functional GFP to the periplasm of V. alginolyticus. We also made fusions of TorASP-GFP with MotX, MotY and FlgT, which are periplasmic components of the Na(+)-driven flagellar motor. Those fusion proteins were localized to the flagellar motor independent of the Na(+) concentration in the environment.

Investigating the Interaction Between Hcf106 Peptides and the Phospholipid Bilayer by Solid-State NMR Spectroscopy

In plant cells most thylakoid lumen proteins are synthesized in the cytosol as higher molecular weight precursors containing targeting sequences, which are imported into chloroplasts and localized to thylakoid lumen for function. Two thylakoid transport systems direct precursors to the thylakoid lumen. Our lab focuses on one of those transport systems: cpTat (chloroplast Twin arginine translocation) system, which transports fully folded proteins utilizing the trans-thylakoidal proton motive force as its only energy source. The cpTat pathway consists of three membrane-bound subunits, Tha4, Hcf106, and cpTatC. cpTatC and Hcf106 form a receptor complex where the precursor binds to initiate the translocation events, whereas Tha4 is initially found as a separate complex that joins the receptor once the precursor has bound. Hcf106 is predicted to contain a N-terminal transmembrane domain (TMD), followed by an amphipathic helix (APH) and an unstructured C-terminus. However, detailed structural information of Hcf106 remains unknown. In this study, peptides encompassing the Hcf106 TMD or APH domain were incorporated into POPC and POPC/MGDG vesicles and analyzed by solid-state NMR spectroscopy. Using 31P NMR we show that Hcf106 TMD/APH interact with the phospholipid headgroups. 2H-NMR revealed the perturbation of the deuterated acyl chain by the peptides. Lastly we used CD spectroscopy to analyze the secondary structure of Hcf106 TMD and APH peptides. The experimental results suggest: (1) Hcf106 TMD and APH peptides interact with both POPC and POPC/MGDG lipids with the lipid composition influencing the peptide-lipid interaction. (2) Hcf106 TMD and APH form alpha helical structures to varying degrees under the same conditions. (3) Hcf106 APH appears to be parallel to the membrane surface with a slight tilt into the hydrophobic region of phospholipids, whereas the TMD peptide appears to be perpendicular to the membrane surface.

Yersinia enterocolitica and Photorhabdus asymbiotica β-lactamases BlaA are exported by the twin-arginine translocation pathway

In general, β-lactamases of medically important Gram-negative bacteria are Sec-dependently translocated into the periplasm. In contrast, β-lactamases of Mycobacteria spp. (BlaC, BlaS) and the Gram-negative environmental bacteria Stenotrophomonas maltophilia (L2) and Xanthomonas campestris (BlaXCC-1) have been reported to be secreted by the twin-arginine translocation (Tat) system. Yersinia enterocolitica carries 2 distinct β-lactamase genes (blaA and blaB) encoding BlaAYe and the AmpC-like β-lactamase BlaB, respectively. By using the software PRED-TAT for prediction and discrimination of Sec from Tat signal peptides, we identified a functional Tat signal sequence for Yersinia BlaAYe. The Tat-dependent translocation of BlaAYe could be clearly demonstrated by using a Y. enterocolitica tatC-mutant and cell fractionation. Moreover, we could demonstrate a unique unusual temperature-dependent activity profile of BlaAYe ranging from 15 to 60°C and a high ‘melting temperature’ (TM=44.3°) in comparison to the related Sec-dependent β-lactamase TEM-1 (20–50°C, TM=34.9°C). Strikingly, the blaA gene of Y. enterocolitica is present in diverse environmental Yersinia spp. and a blaA homolog gene could be identified in the closely related Photorhabdus asymbiotica (BlaAPa; 69% identity to BlaAYe). For BlaAPa of P. asymbiotica, we could also demonstrate Tat-dependent secretion. These results suggest that Yersinia BlaA-related β-lactamases may be the prototype of a large Tat-dependent β-lactamase family, which originated from environmental bacteria.

Direct Interaction between a Precursor Mature Domain and Transport Component Tha4 during Twin Arginine Transport of Chloroplasts

Proteins destined for the thylakoid lumen of chloroplasts must cross three membranes en route. The chloroplast twin arginine translocation (cpTat) system facilitates the transport of about one-half of all proteins that cross the thylakoid membrane in chloroplasts. Known mechanistic features of the cpTat system are drastically different from other known translocation systems, notably in its formation of a transient complex to transport fully folded proteins utilizing only the protonmotive force generated during photosynthesis for energy. However, key details, such as the structure and composition of the translocation pore, are still unknown. One of the three transmembrane cpTat components, Tha4, is thought to function as the pore by forming an oligomer. Yet, little is known about the topology of Tha4 in thylakoid, and little work has been done to detect precursor-Tha4 interactions, which are expected if Tha4 is the pore. Here, we present evidence of the interaction of the precursor with Tha4 under conditions leading to transport, using cysteine substitutions on the precursor and Tha4 and disulfide bond formation in pea (Pisum sativum). The mature domain of a transport-competent precursor interacts with the amphipathic helix and amino terminus of functional Tha4 under conditions leading to transport. Detergent solubilization of thylakoids post cross linking and blue-native polyacrylamide gel electrophoresis analysis shows that Tha4 is found in a complex containing precursor and Hcf106 (i.e. the cpTat translocase). Affinity precipitation of the cross-linked complex via Tha4 clearly demonstrates that the interaction is with full-length precursor. How these data suggest a role for Tha4 in cpTat transport is discussed.