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TatB Functions as an Oligomeric Binding Site for Folded Tat Precursor Proteins

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      The TatABC subunits of the twin-arginine translocation machinery allow transport of folded proteins by an unknown mechanism. Here we show that the entire surfaces of folded Tat substrates contact TatB via both of its predicted helices. Our data suggest that TatB forms an oligomeric binding site that transiently accommodates folded Tat precursors.


      Twin-arginine-containing signal sequences mediate the transmembrane transport of folded proteins. The cognate twin-arginine translocation (Tat) machinery of Escherichia coli consists of the membrane proteins TatA, TatB, and TatC. Whereas Tat signal peptides are recognized by TatB and TatC, little is known about molecular contacts of the mature, folded part of Tat precursor proteins. We have placed a photo-cross-linker into Tat substrates at sites predicted to be either surface-exposed or hidden in the core of the folded proteins. On targeting of these variants to the Tat machinery of membrane vesicles, all surface-exposed sites were found in close proximity to TatB. Correspondingly, incorporation of the cross-linker into TatB revealed multiple precursor-binding sites in the predicted transmembrane and amphipathic helices of TatB. Large adducts indicative of TatB oligomers contacting one precursor molecule were also obtained. Cross-linking of Tat substrates to TatB required an intact twin-arginine signal peptide and disappeared upon transmembrane translocation. Our collective data are consistent with TatB forming an oligomeric binding site that transiently accommodates folded Tat precursors.

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      Most cited references 63

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      TatD is a cytoplasmic protein with DNase activity. No requirement for TatD family proteins in sec-independent protein export.

      The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Genes known to be involved in this process include tatA, tatB, and tatC that form an operon with a fourth gene, tatD. The tatD gene product has two homologues in E. coli coded by the unlinked ycfH and yjjV genes. An E. coli strain with in-frame chromosomal deletions in all three of tatD, ycfH, and yjjV exhibits no significant defect in the cellular location of five cofactor-containing enzymes that are synthesized with twin arginine signal peptides. Neither these mutations nor overproduction of the TatD protein cause any discernible effect on the export kinetics of an additional E. coli Tat pathway substrate. It is concluded that proteins of the TatD family have no obligate involvement in protein export by the Tat system. TatD is shown to be a cytoplasmic protein. TatD binds to immobilized Ni(2+) or Zn(2+) affinity columns and exhibits magnesium-dependent DNase activity. Features of the tatA operon that may control TatD expression are discussed.
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        Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli.

        The twin-arginine translocation (Tat) machinery of the Escherichia coli inner membrane is dedicated to the export of proteins harboring a conserved SRRxFLK motif in their signal sequence. TatA, TatB, and TatC are the functionally essential constituents of the Tat machinery, but their precise function is unknown. Using site-specific crosslinking, we have analyzed interactions of the twin-arginine precursor preSufI with the Tat proteins upon targeting to inner membrane vesicles. TatA association is observed only in the presence of a transmembrane H(+) gradient. TatB is found in contact with the entire signal sequence and adjacent parts of mature SufI. Interaction of TatC with preSufI is, however, restricted to a discrete area around the consensus motif. The results reveal a hierarchy in targeting of a Tat substrate such that for the primary interaction, TatC is both necessary and sufficient while a subsequent association with TatB likely mediates transfer from TatC to the actual Tat pore.
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          Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway.

          To examine the relationship between folding and export competence by the twin-arginine translocation (Tat) pathway we analyzed the subcellular localization of fusions between a set of eight putative Tat leader peptides and alkaline phosphatase in isogenic Escherichia coli strains that either allow or disfavor the formation of protein disulfide bonds in the cytoplasm. We show that export by the Tat translocator is observed only in strains that enable oxidative protein folding in the cytoplasm. Further, we show that other disulfide-containing proteins, namely single-chain Fv and heterodimeric F(AB) antibody fragments, are export-competent only in strains having an oxidizing cytoplasm. Functional, heterodimeric F(AB) protein was exported from the cytoplasm by means of a Tat leader peptide fused to the heavy chain alone, indicating that the formation of a disulfide-bonded dimer preceeds export. These results demonstrate that in vivo only proteins that have attained the native conformation are exported by the Tat translocator, indicating that a folding quality-control mechanism is intrinsic to the export process. The ability to export proteins with disulfide bonds and the folding proofing feature of the Tat pathway are of interest for biotechnology applications.

            Author and article information

            Institute of Biochemistry and Molecular Biology, ZBMZ, University of Freiburg, Stefan-Meier-Strasse 17, D-79104 Freiburg, Germany
            Author notes
            Address correspondence to: Matthias Müller ( matthias.mueller@ ).
            Role: Monitoring Editor
            Mol Biol Cell
            Mol. Bio. Cell
            Molecular Biology of the Cell
            The American Society for Cell Biology
            1 December 2010
            : 21
            : 23
            : 4151-4161
            (Monitoring Editor)
            © 2010 by The American Society for Cell Biology

            This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial –Share Alike 3.0 Unported Creative Commons License (

            Membrane Trafficking

            Molecular biology


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