Specificity of Signal Peptide Recognition in Tat-Dependent Bacterial Protein Translocation (original) (raw)
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European Journal of Biochemistry, 1999
The periplasmic, NADP-containing glucose-fructose oxidoreductase of the gram-negative bacterium Zymomonas mobilis belongs to a class of redox cofactor-dependent enzymes which are exported with the aid of a signal peptide containing a so-called twin-arginine motif. In this paper we show that the replacement of one or both arginine residues results in drastically reduced translocation of glucose-fructose oxidoreductase to the periplasm, showing that this motif is essential. Mutant proteins which, in contrast to wild-type glucose-fructose oxidoreductase, bind NADP in a looser and dissociable manner, were severely affected in the kinetics of plasma membrane translocation. These results strongly suggest that the translocation of glucose-fructose oxidoreductase into the periplasm uses a Sec-independent apparatus which recognizes, as an additional signal, a conformational change in the structure of the protein, most likely triggered by cofactor binding. Furthermore, these results suggest that glucose-fructose oxidoreductase is exported in a folded form. A glucose-fructose oxidoreductase:b-galactosidase fusion protein is not lethal to Z. mobilis cells and leads to the accumulation of the cytosolic preform of wild-type glucose-fructose oxidoreductase expressed in trans but not of a typical Sec-substrate (OmpA), indicating that the glucose-fructose oxidoreductase translocation apparatus can be blocked without interfering with the export of essential proteins via the Sec pathway.
Archives of Microbiology, 2001
The Escherichia coli Tat protein export pathway transports folded proteins synthesized with N-terminal twin-arginine signal peptides. Twin-arginine signal sequences contain a conserved SRRxFLK "twin-arginine" amino acid sequence motif which is required for protein export by the Tat pathway. The E. coli trimethylamine N-oxide reductase (TorA) is a Tat-dependent periplasmic molybdoenzyme that facilitates anaerobic respiration with trimethylamine N-oxide as terminal electron acceptor. Here, we describe mutant strains constructed with modified TorA twin-arginine signal peptides. Substitution of the second arginine residue of the TorA signal peptide twin-arginine motif with either lysine or aspartate, or the simultaneous substitution of both arginines with lysine residues, completely abolished export. In each case, the now cytoplasmically localised TorA retained full enzymatic activity with the artificial electron donor benzyl viologen. However, the mutant strains were incapable of anaerobic growth with trimethylamine N-oxide and the non-fermentable carbon-source glycerol. The growth phenotype of the mutant strains was exploited in a genetic screen with the aim of identifying second-site suppressor mutations that allowed export of the modified TorA precursors.
Export Pathway Selectivity of Escherichia coli Twin Arginine Translocation Signal Peptides
Journal of Biological Chemistry, 2007
The Escherichia coli genome encodes at least 29 putative signal peptides containing a twin arginine motif characteristic of proteins exported via the twin arginine translocation (Tat) pathway. Fusions of the putative Tat signal peptides plus six to eight amino acids of the mature proteins to three reporter proteins (short-lived green fluorescent protein, maltose-binding protein (MBP), and alkaline phosphatase) and also data from the cell localization of epitope-tagged full-length proteins were employed to determine the ability of the 29 signal peptides to direct export through the Tat pathway, through the general secretory pathway (Sec), or through both. 27/29 putative signal peptides could export one or more reporter proteins through Tat. Of these, 11 signal peptides displayed Tat specificity in that they could not direct the export of Sec-only reporter proteins. The rest (16/27) were promiscuous and were capable of directing export of the appropriate reporter either via Tat (green fluorescent protein, MBP) or via Sec (PhoA, MBP). Mutations that conferred a ≥ +1 charge to the N terminus of the mature protein abolished or drastically reduced routing through the Sec pathway without affecting the ability to export via the Tat pathway. These experiments demonstrate that the charge of the mature protein N terminus affects export promiscuity, independent of the effect of the folding state of the mature protein.
FEBS Letters, 1999
Glucose-fructose oxidoreductase (GFOR), a periplasmic protein of Zymomonas mobilis, is synthesized as a precursor polypeptide with a twin-R signal peptide for Sec-independent protein export in bacteria. In higher plant chloroplasts, twin-R signal peptides are specific targeting signals for the Secindependent v vpH pathway of the thylakoid membrane system. In agreement with the assumed common phylogenetic origin of the two protein transport mechanisms, GFOR can be efficiently translocated by the v vpH-dependent pathway when analyzed with isolated thylakoid membranes. Transport is sensitive to the ionophore nigericin and competes with specific substrates for the v vpH-dependent transport route. In contrast, neither sodium azide nor enzymatic destruction of the nucleoside triphosphates in the assays affects thylakoid transport of GFOR indicating that the Sec apparatus is not involved in this process. Mutagenesis of the twin-R motif in the GFOR signal peptide prevents membrane translocation of the protein emphasizing the importance of these residues for the transport process.
A novel Sec-independent periplasmic protein translocation pathway in Escherichia coli
The EMBO Journal, 1998
The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a soluble periplasmic molybdoenzyme. The precursor of this enzyme possesses a cleavable N-terminal signal sequence which contains a twin-arginine motif. By using various moa, mob and mod mutants defective in different steps of molybdocofactor biosynthesis, we demonstrate that acquisition of the molybdocofactor in the cytoplasm is a prerequisite for the translocation of the TMAO reductase. The activation and translocation of the TMAO reductase precursor are post-translational processes, and activation is dissociable from translocation. The export of the TMAO reductase is driven mainly by the proton motive force, whereas sodium azide exhibits a limited effect on the export. The most intriguing observation is that translocation of the TMAO reductase across the cytoplasmic membrane is independent of the SecY, SecE, SecA and SecB proteins. Depletion of Ffh, a core component of the signal recognition particle of E.coli, appears to have a slight effect on the export of the TMAO reductase. These results strongly suggest that the translocation of the molybdoenzyme TMAO reductase into the periplasm uses a mechanism fundamentally different from general protein translocation.
European Journal of Biochemistry, 1997
G1ucose:fructose oxidoreductase (GFOR) of the gram-negative bacterium Zyrnornonas rnobilis is a periplasmic enzyme with tightly bound cofactor NADP. The preprotein carries an unusually long Nterminal signal peptide of 52 amino acid residues. Expression of the afo gene in cells of Escherichia coli K12, under the control of a tuc promoter, led to immunologically detectable proteins in western blots, and to the formation of an enzymatically active precursor form (preGFOR), located in the cytosol. Processing of preGFOR to the mature form was not observed in E. coli. Replacement of the authentic GFOR signal peptide by the shorter signal peptides of PhoA or OmpA from E. coli led to processing of the respective GFOR precursor proteins. However, the processed proteins were unstable and rapidly degraded in the periplasm unless an E. coli mutant was used that carried a triple lesion for periplasmic and outermembrane proteases. When fusion-protein export was inhibited by sodium azide or carboxylcyanide rnchlorophenylhydrazone, the cytoplasmic precursor forms of the respective preGFOR were not degraded. A major protease-resistant GFOR peptide from the OmpA-GFOR fusion was found within spheroplasts of E. coli to which NADP had been added externally. The formation of this peptide did not occur in the presence of NAD. It is concluded that NADP is required for GFOR to fold into its native conformation and that its absence from the E. coli periplasm is responsible for failure to form a stable periplasmic protein. The results strongly suggest that, in Z. rnobilis, additional protein factors are required for the transport of NADP across the plasma membrane andor incorporation of NADP into the GFOR apoenzyme.
Journal of Biological Chemistry, 2007
The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across the cytoplasmic membrane of bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive arginine residues that are thought to be involved in the recognition of the signal peptides by the Tat translocase. Here, we have analyzed the specificity of Tat signal peptide recognition by using a genetic approach. Replacement of the two arginine residues in a Tat-specific precursor protein by lysine-glutamine resulted in an export-defective mutant precursor that was no longer accepted by the wild-type translocase.
Molecular Microbiology, 2012
The twin-arginine translocation (Tat) pathway is a protein targeting system present in many prokaryotes. The physiological role of the Tat pathway is the transmembrane translocation of fully-folded proteins, which are targeted by N-terminal signal peptides bearing conserved SRRxFLK 'twin-arginine' amino acid motifs. In Escherichia coli the majority of Tat targeted proteins bind redox cofactors and it is important that only mature, cofactor-loaded precursors are presented for export. Cellular processes have been unearthed that sequence these events, for example the signal peptide of the periplasmic nitrate reductase (NapA) is bound by a cytoplasmic chaperone (NapD) that is thought to regulate assembly and export of the enzyme. In this work, genetic, biophysical and structural approaches were taken to dissect the interaction between NapD and the NapA signal peptide. A NapD binding epitope was identified towards the N-terminus of the signal peptide, which overlapped significantly with the twin-arginine targeting motif. NMR spectroscopy revealed that the signal peptide adopted a a-helical conformation when bound by NapD, and substitution of single residues within the NapA signal peptide was sufficient to disrupt the interaction. This work provides an increased level of understanding of signal peptide function on the bacterial Tat pathway.