title: A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase.
abstract: The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system ofEscherichia coliis made up of TatA, TatB, and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site-specific cross-linking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA-YFP fusion showed that the TatB F13Y substitution resulted in signal peptide-independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase.
authors.affiliation: Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors.forename: Qi
authors.initials: Q
authors.lastname: Huang
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
authors.forename: Felicity
authors.initials: F
authors.lastname: Alcock
authors.affiliation: Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors.forename: Holger
authors.initials: H
authors.lastname: Kneuper
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.|Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
authors.forename: Justin C
authors.initials: JC
authors.lastname: Deme
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.|Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Rollauer
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
authors.forename: Susan M
authors.identifier: 0000-0001-9287-8053
authors.initials: SM
authors.lastname: Lea
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
authors.forename: Ben C
authors.initials: BC
authors.lastname: Berks
authors.affiliation: Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom; t.palmer@dundee.ac.uk.
authors.forename: Tracy
authors.identifier: 0000-0001-9043-2592
authors.initials: T
authors.lastname: Palmer
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D021382:Protein Sorting Signals; D021381:Protein Transport; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; C499697:TatA protein, E coli; C473116:TatB protein, E coli; C461379:TatC protein, E coli; D001120:Arginine
keywords: Tat pathway; genetic suppressor; protein transport; twin arginine signal peptide