title: Escherichia coli TatA and TatB proteins have N-out, C-in topology in intact cells.
abstract: The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. A complex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport.
authors.affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Koch
authors.forename: Maximilian J
authors.initials: MJ
authors.lastname: Fritsch
authors.forename: Grant
authors.initials: G
authors.lastname: Buchanan
authors.forename: Tracy
authors.initials: T
authors.lastname: Palmer
mesh_terms: D058947:Bacterial Secretion Systems / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000502:physiology
chemical_list: D058947:Bacterial Secretion Systems; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C499697:TatA protein, E coli; C473116:TatB protein, E coli