Twin arginine translocation (Tat)-dependent export in the apparent absence of TatABC or TatA complexes using modified Escherichia coli TatA subunits that substitute for TatB.

The twin arginine translocation pathway exports folded proteins across the cytoplasmic membrane of many bacteria. In Escherichia coli and other Gram-negative bacteria, TatA, TatB, and TatC are all essential for efficient translocation, and current models suggest that separate TatABC and TatA complexes coalesce at the point of translocation. However, other microbes appear only to possess tatA and tatC genes. In Escherichia coli, virtually no translocation is observed when only TatA and TatC are present, but several mutations at the extreme N terminus of TatA were shown to support translocation. Here we show that these apparently bifunctional mutant TatA variants can function as typical TatA components because translocation is observed when they are co-expressed with TatBC, and they assemble into large, heterogeneous complexes that resemble wild type TatA complexes. However, cells expressing TatC plus the mutant TatA variants do not contain complexes that resemble the expected 370-kDa TatABC complex, clearly indicating that the mutant TatA forms cannot assemble efficiently, or stably, into this complex. The simultaneous expression of wild type TatA furthermore blocks translocation activity, suggesting that the mutant TatA forms preferentially bind to other TatA molecules rather than TatC. Surprisingly, we observe translocation in the absence of detectable free TatA, when translational fusions of the mutant TatAs with TatC are expressed. Transport can thus proceed in the simultaneous absence of TatABC and TatA complexes at detectable levels, and we conclude that the active translocon may be formed from dynamic twin arginine translocation complexes, one or more of which may await characterization.

The entire N-terminal half of TatC is involved in twin-arginine precursor binding.

Translocation of twin-arginine precursor proteins across the cytoplasmic membrane of Escherichia coli requires the three membrane proteins TatA, TatB, and TatC. TatC and TatB were shown to be involved in precursor binding. We have analyzed in vitro a number of single alanine substitutions in tatC that were previously shown to compromise in vivo the function of the Tat translocase. All tatC mutants that were defective in precursor translocation into cytoplasmic membrane vesicles concomitantly interfered with precursor binding not only to TatC but also to TatB. Hence structural changes of TatC that affect precursor targeting simultaneously abolish engagement of the twin-arginine signal sequence with TatB and block the formation of a functional Tat translocase. Since these phenotypes were observed for tatC mutations spread over the first half of TatC, this entire part of the molecule must globally be involved in precursor binding.

Evidence for interactions between domains of TatA and TatB from mutagenesis of the TatABC subunits of the twin-arginine translocase.

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. Three subunits, TatA, B and C, are known to be involved but their modes of action are poorly understood, as are the inter-subunit interactions occurring within Tat complexes. We have generated mutations in the single transmembrane (TM) spans of TatA and TatB, with the aim of generating structural distortions. We show that substitution in TatB of three residues by glycine, or a single residue by proline, has no detectable effect on translocation, whereas the presence of three glycines in the TatA TM span completely blocks Tat translocation activity. The results show that the integrity of the TatA TM span is vital for Tat activity, whereas that of TatB can accommodate large-scale distortions. Near-complete restoration of activity in TatA mutants is achieved by the simultaneous presence of a V12P mutation in the TatB TM span, strongly implying a direct functional interaction between the TatA/B TM spans. We also analyzed the predicted amphipathic regions in TatA and TatB and again find evidence of direct interaction; benign mutations in either subunit completely blocked translocation of two Tat substrates when present in combination. Finally, we have re-examined the effects of previously analyzed TatABC mutations under conditions of high translocation activity. Among numerous TatA or TatB mutations tested, TatA F39A alone blocked translocation, and only substitutions of P48 and F94 in TatC blocked translocation activity.

Mutations in subunits of the Escherichia coli twin-arginine translocase block function via differing effects on translocation activity or tat complex structure.

We have used a combination of blue-native (BN) gel electrophoresis and protein purification to analyze the effects of TatA or TatC mutations on the structures of the primary TatABC and multimeric TatA complexes in Escherichia coli. Expression of wild-type TatABC leads to the production of a single major TatABC complex of 370 kDa and a heterogeneous set of TatA complexes of <100 kDa to approximately 500 kDa. Two TatC mutations that block translocation have different effects on complex structures. P48A causes massive defects in TatABC assembly, including a marked separation of the TatBC subunits and the production of TatB and TatC aggregates. In contrast, TatABC complexes from the inactive TatC F94A mutant are structurally intact, suggesting that this mutation affects translocation activity rather than assembly. Neither TatC mutation affects the separate TatA complexes, showing that assembly of the TatA complexes is independent of TatABC assembly or activity. In contrast, three TatA mutations affect both the TatA and TatABC complexes. F39A assembles into smaller, incorrectly organized TatA complexes and the TatABC complexes contain an incorrect TatB:TatC ratio and unusually large amounts of TatA. A triple mutant in the amphipathic region forms slightly larger TatA complexes that are likewise disorganized, and a mutant containing three glycine substitutions in the transmembrane (TM) span assembles as grossly affected TatA complexes that are much larger than wild-type complexes. These mutants lead to a partial failure of TatB to assemble correctly. The data show that the amphipathic and TM regions play critical roles in TatA complex assembly. All of the TatA mutations lead to partial or substantial defects in TatABC complex formation, demonstrating that the properties of TatA can have a marked influence on the TatABC complex.

The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC complex, spectrum of modular TatA complexes and minor TatAB complex.

The Tat system transports folded proteins across bacterial plasma and plant thylakoid membranes. To date, three key Tat subunits have been identified and mechanistic studies indicate the presence of two types of complex: a TatBC-containing substrate-binding unit and a separate TatA complex. Here, we used blue-native gel electrophoresis and affinity purification to study the nature of these complexes in Escherichia coli. Analysis of solubilized membrane shows that the bulk of TatB and essentially all of the TatC is found in a single 370kDa TatABC complex. TatABC was purified to homogeneity using an affinity tag on TatC and this complex runs apparently as an identical band. We conclude that this is the primary core complex, predicted to contain six or seven copies of TatBC together with a similar number of TatA subunits. However, the data indicate the presence of an additional form of Tat complex containing TatA and TatB, but not TatC; we speculate that this may be an assembly or disassembly intermediate of the translocator. The vast majority of TatA is found in separate complexes that migrate in blue-native gels as a striking ladder of bands with sizes ranging from under 100 kDa to over 500 kDa. Further analysis shows that the bands differ by an average of 34 kDa, indicating that TatA complexes are built largely, but possibly not exclusively, from modules of three or four TatA molecules. The range and nature of these complexes are similar in a TatC mutant that is totally inactive, indicating that the ladder of bands does not stem from ongoing translocation activity, and we show that purified TatA can self-assemble in vitro to form similar complexes. This spectrum of TatA complexes may provide the flexibility required to generate a translocon capable of transporting substrates of varying sizes across the plasma membrane in a folded state.

Quantitative export of a reporter protein, GFP, by the twin-arginine translocation pathway in Escherichia coli.

The Tat system mediates the transport of folded proteins across the bacterial cytoplasmic membrane. To study the properties of the Escherichia coli Tat-system, we used green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). In the presence of arabinose, low levels of this protein rapidly saturate the translocase and cause the accumulation of inactive, membrane-bound TorA-GFP; fluorescence microscopy also showed active TorA-GFP to be distributed throughout the cytoplasm. However, the efficiency of export can be massively increased by alteration of the growth conditions, and further increased by overexpression of the tatABC genes. Under these conditions, the levels of GFP in the periplasm are raised over 20-fold and the export efficiency nears 100%. These results show that the Tat-system is relatively inactive under some growth conditions and the data suggest that the system may be applicable for the larger-scale export of heterologous proteins.

Identification of key regions within the Escherichia coli TatAB subunits.

The twin-arginine translocation (Tat) system catalyzes the transport of folded proteins across the bacterial plasma membrane or the chloroplast thylakoid membrane. In Escherichia coli and most other species, three important tat genes have been identified but the structure and mechanism of this system are poorly understood; the role and location of TatA are particularly unclear. In this report we have used site-specific mutagenesis to probe the significance of conserved features of the related TatA/B subunits. We find that an apparent 'hinge' region between the transmembrane (TM) span and an adjacent amphipathic region is important in both proteins, in that substitution of turn-inducing residues inhibits the export of a natural Tat substrate. Surprisingly, large-scale mutagenesis of the conserved amphipathic regions of TatA and TatB leads only to minor effects on Tat-dependent export suggesting that this particular feature is not central to the translocation mechanism. This domain is, however, critical for the translocation process and we identify Gly/Pro residues in these regions of TatA/B that are essential for efficient export.

Essential cytoplasmic domains in the Escherichia coli TatC protein.

The twin-arginine translocation (Tat) system mediates the transport of proteins across the bacterial plasma membrane and chloroplast thylakoid membrane. Operating in parallel with Sec-type systems in these membranes, the Tat system is completely different in both structural and mechanistic terms, and is uniquely able to catalyze the translocation of fully folded proteins across coupled membranes. TatC is an essential, multispanning component that has been proposed to form part of the binding site for substrate precursor proteins. In this study we have tested the importance of conserved residues on the periplasmic and cytoplasmic face of the Escherichia coli protein. We find that many of the mutations on the cytoplasmic face have little or no effect. However, substitution at several positions in the extreme N-terminal cytoplasmic region or the predicted first cytoplasmic loop lead to a significant or complete loss of Tat-dependent export. The mutated strains are unable to grow anaerobically on trimethylamine N-oxide minimal media and are unable to export trimethylamine-N-oxide reductase (TorA). The same mutants are completely unable to export a chimeric protein, comprising the TorA signal peptide linked to green fluorescent protein, indicating that translocation is blocked rather than cofactor insertion into the TorA mature protein. The data point to two essential cytoplasmic domains on the TatC protein that are essential for export.