Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI

Signal peptides target protein cargos for secretion from the bacterial cytoplasm. These signal peptides contain a tri-partite structure consisting of a central hydrophobic domain (h-domain), and two flanking polar domains. Using a recently developed in vitro transport assay, we report here that a central h-domain position (C17) of the twin arginine translocation (Tat) substrate pre-SufI is especially sensitive to amino acid hydrophobicity. The C17I mutant is transported more efficiently than wild type, whereas charged substitutions completely block transport. Transport efficiency is well-correlated with Tat translocon binding efficiency. The precursor protein also binds to non-Tat components of the membrane, presumably to the lipids. This lipid-bound precursor can be chased through the Tat translocons under conditions of high proton motive force. Thus, the non-Tat bound form of the precursor is a functional intermediate in the transport cycle. This intermediate appears to directly equilibrate with the translocon-bound form of the precursor.     

Functional reconstitution of TatB into the thylakoidal Tat translocase

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 Twin-arginine protein transport (Tat) 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 analysed 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 mutated 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 analyses of protein variants. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport.     

Protein transport by the bacterial Tat pathway

The twin-arginine translocation (Tat) system accomplishes the remarkable feat of translocating large – even dimeric – proteins across tightly sealed energy-transducing membranes. All of the available evidence indicates that it is unique in terms of both structure and mechanism; however its very nature has hindered efforts to probe the core translocation events. At the heart of the problem is the fact that two large sub-complexes are believed to coalesce to form the active translocon, and ‘capturing’ this translocation event has been too difficult. Nevertheless, studies on the individual components have come a long way in recent years, and structural studies have reached the point where educated guesses can be made concerning the most interesting aspects of Tat. In this article we review these studies and the emerging ideas in this field. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

大肠杆菌Tat转运酶的研究进展

TatA、TatB和TatC是大肠杆菌Tat转运酶的组成成分.研究表明各Tat蛋白具有不同的功能区域, TatA和TatB蛋白功能重要的位点位于N末端的穿膜片断、其后的双极性α-螺旋和铰链区.TatC的序列保守性低,N末端穿膜片断和位于胞质内的第一环区对转运是必需的.Tat转运酶各成分相互结合成复合物形式并相互依赖.TatA在细胞中高表达并自身聚合形成数量不等的同聚物,具有稳定TatBC复合物的作用,TatB有稳定TatC的功能,TatB和TatC两者结合形成二聚体.实验表明,TatA复合物形成转运通道,TatBC复合物通过TatC蛋白识别底物的信号肽并与底物结合, 再在TatB介导下与TatA复合物结合形成具有活性的转运酶.     

Ciprofloxacin interactions with bacterial protein OmpF: modelling of FRET from a multi-tryptophan protein trimer

The outer membrane protein F (OmpF) is known to play an important role in the uptake of fluoroquinolone antibiotics by bacteria. In this study, the degree of binding of the fluoroquinolone antibiotic ciprofloxacin to OmpF in a lipid membrane environment is quantified using a methodology based on F?rster resonance energy transfer (FRET). Analysis of the fluorescence quenching of OmpF is complex as each OmpF monomer presents two tryptophans at different positions, thus sensing two different distributions of acceptors in the bilayer plane. Specific FRET formalisms were derived accounting for the different energy transfer contributions to quenching of each type of tryptophan of OmpF, allowing the recovery of upper and lower boundaries for the ciprofloxacin-OmpF binding constant (K(B)). log (K(B)) was found to lie in the range 3.15-3.62 or 3.58-4.00 depending on the location for the ciprofloxacin binding site assumed in the FRET modelling, closer to the centre or to the periphery of the OmpF trimer, respectively. This methodology is suitable for the analysis of FRET data obtained with similar protein systems and can be readily adapted to different geometries.     

Ciprofloxacin interactions with bacterial protein OmpF: Modelling of FRET from a multi-tryptophan protein trimer

The outer membrane protein F (OmpF) is known to play an important role in the uptake of fluoroquinolone antibiotics by bacteria. In this study, the degree of binding of the fluoroquinolone antibiotic ciprofloxacin to OmpF in a lipid membrane environment is quantified using a methodology based on Förster resonance energy transfer (FRET). Analysis of the fluorescence quenching of OmpF is complex as each OmpF monomer presents two tryptophans at different positions, thus sensing two different distributions of acceptors in the bilayer plane. Specific FRET formalisms were derived accounting for the different energy transfer contributions to quenching of each type of tryptophan of OmpF, allowing the recovery of upper and lower boundaries for the ciprofloxacin-OmpF binding constant (K_B). log (K_B) was found to lie in the range 3.15-3.62 or 3.58-4.00 depending on the location for the ciprofloxacin binding site assumed in the FRET modelling, closer to the centre or to the periphery of the OmpF trimer, respectively. This methodology is suitable for the analysis of FRET data obtained with similar protein systems and can be readily adapted to different geometries.     

Kinetics of Fast Changing Intramolecular Distance Distributions Obtained by Combined Analysis of FRET Efficiency Kinetics and Time-Resolved FRET Equilibrium Measurements

Detailed studies of the mechanisms of macromolecular conformational transitions such as protein folding are enhanced by analysis of changes of distributions for intramolecular distances during the transitions. Time-resolved Förster resonance energy transfer (FRET) measurements yield such data, but the more readily available kinetics of mean FRET efficiency changes cannot be analyzed in terms of changes in distances because of the sixth-power dependence on the mean distance. To enhance the information obtained from mean FRET efficiency kinetics, we combined the analyses of FRET efficiency kinetics and equilibrium trFRET experiments. The joint analysis enabled determination of transient distance distributions along the folding reaction both in cases where a two-state transition is valid and in some cases consisting of a three-state scenario. The procedure and its limits were tested by simulations. Experimental data obtained from stopped-flow measurements of the refolding of Escherichia coli adenylate kinase were analyzed. The distance distributions between three double-labeled mutants, in the collapsed transient state, were determined and compared to those obtained experimentally using the double-kinetics technique. The proposed method effectively provides information on distance distributions of kinetically accessed intermediates of fast conformational transitions induced by common relaxation methods.     

Export of complex cofactor-containing proteins by the bacterial Tat pathway

The twin-arginine (Tat) protein translocase is a highly unusual protein transport machine that is dedicated to the movement of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat pathway by means of N-terminal signal peptides harbouring a distinctive twin-arginine motif. In the model organism Escherichia coli, many of the Tat substrates bind redox cofactors that are inserted into apo-proteins before they engage with the Tat machinery. Here we review recent advances in understanding the events involved in the coordination of cofactor insertion with the export process. Current models for Tat protein transport are also discussed.     

Kinetics of Fast Changing Intramolecular Distance Distributions Obtained by Combined Analysis of FRET Efficiency Kinetics and Time-Resolved FRET Equilibrium Measurements

Detailed studies of the mechanisms of macromolecular conformational transitions such as protein folding are enhanced by analysis of changes of distributions for intramolecular distances during the transitions. Time-resolved Förster resonance energy transfer (FRET) measurements yield such data, but the more readily available kinetics of mean FRET efficiency changes cannot be analyzed in terms of changes in distances because of the sixth-power dependence on the mean distance. To enhance the information obtained from mean FRET efficiency kinetics, we combined the analyses of FRET efficiency kinetics and equilibrium trFRET experiments. The joint analysis enabled determination of transient distance distributions along the folding reaction both in cases where a two-state transition is valid and in some cases consisting of a three-state scenario. The procedure and its limits were tested by simulations. Experimental data obtained from stopped-flow measurements of the refolding of Escherichia coli adenylate kinase were analyzed. The distance distributions between three double-labeled mutants, in the collapsed transient state, were determined and compared to those obtained experimentally using the double-kinetics technique. The proposed method effectively provides information on distance distributions of kinetically accessed intermediates of fast conformational transitions induced by common relaxation methods.     

Probing Tat peptide-TAR RNA interactions by psoralen photo-cross-linking

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. We have used a site-specific cross-linking method based on psoralen photochemistry to determine the effect of core residues from the Tat sequence on the protein orientation in the Tat-TAR complex and on the specificity of Tat-TAR binding. We synthesized two Tat fragments, Tat(42-72) and Tat(37-72), and incorporated a psoralen-modified amino acid at position 41 during solid-phase assembly of the peptides. We used these psoralen-Tat conjugates to form specific complexes with TAR RNA. Upon near-ultraviolet irradiation (360 nm), psoralen-Asp41-Tat(37-72) cross-linked to a single site in the TAR RNA sequence. The RNA-protein complex was purified and the cross-link site on TAR RNA was determined by primer extension analysis, which revealed that Asp41 of Tat is close to U42 of the lower stem region of TAR RNA. Specificity of the RNA-peptide cross-linking reactions was determined by competition experiments. Our results show that the addition of only four residues (Cys37-Thr40) from the Tat core region significantly enhanced the specificity of the Tat peptide-TAR interactions without altering the site or chemical nature of the cross-link. These studies provide new insights into RNA-protein recognition that could be useful in designing peptidomimetics for RNA targeting. Such psoralen-peptide conjugates provide a new class of probes for sequence-specific protein-nucleic acid interactions and could be used to selectively control gene expression or to induce site-directed mutations.     

Neuropeptide precursor processing detected by triple immunolabeling

Peptides that play critical physiological roles are often encoded in precursors that contain several gene products. Differential processing of a polypeptide precursor by cell-specific proteolytic enzymes can yield multiple messengers with diverse distributions and functions. We have isolated SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide from Drosophila melanogaster. The peptides are encoded in the FMRFamide gene and have a common C-terminal FMRFamide but different N-terminal extensions. In order to investigate the regulation of expression of FMRFamide peptides, we generated antisera to distinguish between the structurally related neuropeptides. We established a triple-label immunofluorescence protocol using antisera raised in the same host species and mapped the neural distribution of SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide. Each peptide has a unique, nonoverlapping cellular expression pattern, suggesting that the precursor is differentially processed. Thus, our data indicate that D. melanogaster contains cell-specific proteolytic enzymes to cleave a polypeptide protein precursor, resulting in unique expression patterns of neuropeptides.     

Oligomers of Tha4 organize at the thylakoid Tat translocase during protein transport

The Tat (twin arginine translocation) systems of thylakoids and bacteria transport fully folded protein substrates without breaching the permeability barrier of the membrane. Two components of the thylakoid system, cpTatC and Hcf106, compose a precursor-bound receptor complex. The third component, Tha4, assembles with the precursor-bound receptor complex for the translocation step and is thought to compose at least part of the protein-conducting channel. Here, we used two different cross-linking approaches to explore the organization of Tha4 in the translocase. These cross-linking techniques showed that transition to an active protein transport state resulted in an alignment of the Tha4 amphipathic helix and C-terminal tail domains to form Tha4 oligomers. Oligomerization required functional Tha4, a twin arginine signal peptide, and an active cpTatC-Hcf106 receptor complex. The spectrum of oligomers obtained was independent of the mature folded domain of the precursor. We propose a trapdoor mechanism for translocation whereby aligned oligomers of Tha4 amphipathic helices fold into the membrane to allow formfitting passage of precursor proteins.     

A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase

A group of bacterial exported proteins are synthesized with N-terminal signal peptides containing a SRRxFLK 'twin-arginine' amino acid motif. Proteins bearing twin-arginine signal peptides are targeted post-translationally to the twin-arginine translocation (Tat) system which transports folded substrates across the inner membrane. In Escherichia coli, most integral inner membrane proteins are assembled by a co-translational process directed by SRP/FtsY, the SecYEG translocase, and YidC. In this work we define a novel class of integral membrane proteins assembled by a Tat-dependent mechanism. We show that at least five E. coli Tat substrate proteins contain hydrophobic C-terminal transmembrane helices (or 'C-tails'). Fusions between the identified transmembrane C-tails and the exclusively Tat-dependent reporter proteins TorA and SufI render the resultant chimeras membrane-bound. Export-linked signal peptide processing and membrane integration of the chimeras is shown to be both Tat-dependent and YidC-independent. It is proposed that the mechanism of membrane integration of proteins by the Tat system is fundamentally distinct from that employed for other bacterial inner membrane proteins.     

Imaging molecular interactions in living cells by FRET microscopy

F?rster resonance energy transfer (FRET) is applied extensively in all fields of biological research and technology, generally as a 'nanoruler' with a dynamic range corresponding to the intramolecular and intermolecular distances characterizing the molecular structures that regulate cellular function. The complex underlying network of interactions reflects elementary reactions operating under strict spatio-temporal control: binding, conformational transition, covalent modification and transport. FRET imaging provides information about all these molecular processes with high specificity and sensitivity via probes expressed by or introduced from the external medium into the cell, tissue or organism. Current approaches and developments in the field are discussed with emphasis on formalism, probes and technical implementation.     

Intracellular localization and interaction of mRNA binding proteins as detected by FRET

Background  

A number of RNA binding proteins (BPs) bind to A+U rich elements (AREs), commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the in vivo composition of RNA-BP multiprotein complexes and how their composition is affected by signaling events and intracellular localization. Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts.     

Protein targeting by the bacterial twin-arginine translocation (Tat) pathway

The Tat (twin-arginine translocation) protein export system is found in the cytoplasmic membrane of most prokaryotes and is dedicated to the transport of folded proteins. The Tat system is now known to be essential for many bacterial processes including energy metabolism, cell wall biosynthesis, the nitrogen-fixing symbiosis and bacterial pathogenesis. Recent studies demonstrate that substrate-specific accessory proteins prevent improperly assembled substrates from interacting with the Tat transporter. During the transport cycle itself substrate proteins bind to a receptor complex in the membrane which then recruits a protein-translocating channel to carry out the transport reaction.     

Transport of cytochrome c derivatives by the bacterial Tat protein translocation system

An experimental system developed previously for the heterologous expression of c-type cytochromes in Escherichia coli Q1has been adapted to monitor protein transfer across the bacteria's cytoplasmic membrane. Apocytochrome, lacking the haem cofactor and probably in an unfolded state, was readily transferred across the cytoplasmic membrane when fused to a Sec-specific signal peptide. Furthermore, cytochrome fused to a signal peptide regarded as specific for the twin arginine transport (Tat) system was translocated in an unfolded state by the Sec apparatus. After maturation and folding in the cytoplasm, Tat-mediated transfer of holocytochrome to the periplasm occurred. We conclude that, in addition to the nature of the specific signal peptide, the folding state of a particular protein also governs its acceptance by a given transport system.     

Molecular interactions of the mitochondrial Tim12 translocase subunit

The small Tims are chaperones that facilitate insertion of hydrophobic precursors into the inner mitochondrial membrane. We purified Tim12 and found it forms dimers that bind to Tim9. In this interaction, Tim12 undergoes structural changes that may be important for transport of its substrates in the mitochondrial carrier import pathway.