Frank Sargent

The Tat protein export pathway

The Tat (twin‐arginine translocation) system is a bacterial protein export pathway with the remarkable ability to transport folded proteins across the cytoplasmic membrane. Preproteins are directed to the Tat pathway by signal peptides that bear a characteristic sequence motif, which includes consecutive arginine residues. Here, we review recent progress on the characterization of the Tat system and critically discuss the structure and operation of this major new bacterial protein export pathway.

Overlapping functions of components of a bacterial Sec‐independent protein export pathway

We describe the identification of two Escherichia coli genes required for the export of cofactor‐containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor‐containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.

An Essential Component of a Novel Bacterial Protein Export System with Homologues in Plastids and Mitochondria

Proteins are transported across the bacterial plasma membrane and the chloroplast thylakoid membrane by means of protein translocases that recognize N-terminal targeting signals in their cognate substrates. Transport of many of these proteins involves the well defined Sec apparatus that operates in both membranes. We describe here the identification of a novel component of a bacterial Sec-independent translocase. The system probably functions in a similar manner to a Sec-independent translocase in the thylakoid membrane, and substrates for both systems bear a characteristic twin-arginine motif in the targeting peptide. The translocase component is encoded inEscherichia coli by an unassigned reading frame,yigU, disruption of which blocks the export of at least five twin-Arg-containing precursor proteins that are predicted to bind redox cofactors, and hence fold, prior to translocation. The Sec pathway remains unaffected in the deletion strain. The gene has been designated tatC (for twin-argininetranslocation), and we show that homologous genes are present in a range of bacteria, plastids, and mitochondria. These findings suggest a central role for TatC-type proteins in the translocation of tightly folded proteins across a spectrum of biological membranes.

Sec-independent Protein Translocation in Escherichia coli A DISTINCT AND PIVOTAL ROLE FOR THE TatB PROTEIN

In Escherichia coli, transmembrane translocation of proteins can proceed by a number of routes. A subset of periplasmic proteins are exported via the Tat pathway to which proteins are directed by N-terminal “transfer peptides” bearing the consensus (S/T)RRXFLK “twin-arginine” motif. The Tat system involves the integral membrane proteins TatA, TatB, TatC, and TatE. Of these, TatA, TatB, and TatE are homologues of the Hcf106 component of the ΔpH-dependent protein import system of plant thylakoids. Deletion of the tatB gene alone is sufficient to block the export of seven endogenous Tat substrates, including hydrogenase-2. Complementation analysis indicates that while TatA and TatE are functionally interchangeable, the TatB protein is functionally distinct. This conclusion is supported by the observation that Helicobacter pylori tatA will complement an E. coli tatA mutant, but not a tatB mutant. Analysis of Tat component stability in various tat deletion backgrounds shows that TatC is rapidly degraded in the absence of TatB suggesting that TatC complexes, and is stabilized by, TatB.

The Tat protein translocation pathway and its role in microbial physiology

The Tat (twin arginine translocation) protein transport system functions to export folded protein substrates across the bacterial cytoplasmic membrane and to insert certain integral membrane proteins into that membrane. It is entirely distinct from the Sec pathway. Here, we describe our current knowledge of the molecular features of the Tat transport system. In addition, we discuss the roles that the Tat pathway plays in the bacterial cell, paying particular attention to the involvement of the Tat pathway in the biogenesis of cofactor-containing proteins, in cell wall biosynthesis and in bacterial pathogenicity.

Coordinating assembly and export of complex bacterial proteins

The Escherichia coli twin‐arginine protein transport (Tat) system is a molecular machine dedicated to the translocation of fully folded substrate proteins across the energy‐transducing inner membrane. Complex cofactor‐containing Tat substrates, such as the model (NiFe) hydrogenase‐2 and trimethylamine N‐oxide reductase (TorA) systems, acquire their redox cofactors prior to export from the cell and require to be correctly assembled before transport can proceed. It is likely, therefore, that cellular mechanisms exist to prevent premature export of immature substrates. Using a combination of genetic and biochemical approaches including gene knockouts, signal peptide swapping, complementation, and site‐directed mutagenesis, we highlight here this crucial ‘proofreading’ or ‘quality control’ activity in operation during assembly of complex endogenous Tat substrates. Our experiments successfully uncouple the Tat transport and cofactor‐insertion activities of the TorA‐specific chaperone TorD and demonstrate unequivocally that TorD recognises the TorA twin‐arginine signal peptide. It is proposed that some Tat signal peptides operate in tandem with cognate binding chaperones to orchestrate the assembly and transport of complex enzymes.

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.

Oligomeric Properties and Signal Peptide Binding by Escherichia coli Tat Protein Transport Complexes

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.

The twin-arginine transport system: moving folded proteins across membranes

The Tat (twin-arginine transport) pathway is a protein-targeting system dedicated to the transmembrane translocation of fully folded proteins. This system is highly prevalent in the cytoplasmic membranes of bacteria and archaea, and is also found in the thylakoid membranes of plant chloroplasts and possibly also in the inner membrane of plant mitochondria. Proteins are targeted to a membrane-embedded Tat translocase by specialized N-terminal twin-arginine signal peptides bearing an SRRXFLK amino acid motif. The genes encoding components of the Tat translocase were discovered approx. 10 years ago, and, since then, research in this area has expanded on a global scale. In this review, the key discoveries in this field are summarized, and recent studies of bacterial twin-arginine signal-peptide-binding proteins are discussed.

Transcriptional regulation in response to oxygen and nitrate of the operons encoding the [NiFe] hydrogenases 1 and 2 of Escherichia coli.

Synthesis of the [NiFe] hydrogenases 1 and 2 of Escherichia coli is induced in response to anaerobiosis and is repressed when nitrate is present in the growth medium. The hydrogenase 1 and hydrogenase 2 enzymes are encoded by the polycistronic hyaABCDEF and hybOABCDEFG operons, respectively. Primer extension analysis was used to determine the initiation site of transcription of both operons. This permitted the construction of single-copy lacZ operon fusions, which were used to examine the transcriptional regulation of the two operons. Expression of both was induced by anaerobiosis and repressed by nitrate, which is in complete accord with earlier biochemical studies. Anaerobic induction of the hyb operon was only partially dependent on the FNR protein and, surprisingly, was enhanced by an arcA mutation. This latter result indicated that ArcA suppresses anaerobic hyb expression and that a further factor, which remains to be identified, is involved in controlling anaerobic induction of operon expression. Nitrate repression of hyb expression was mediated by the NarL/NarX and NarP/NarQ two-component regulatory systems. Remarkably, a narP mutant lacked anaerobic induction of hyb expression, even in the absence of added nitrate. Anaerobic induction of hya expression was dependent on the ArcA and AppY regulators, which confirms earlier observations by other authors. Nitrate repression of the hya operon was mediated by both NarL and NarP. Taken together, these data indicate that although the hya and hyb operons share common regulators, there are important differences in the control of expression of the individual operons.