title: Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.
abstract: The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. TatC is the largest and most conserved component of the Tat machinery. It forms a multisubunit complex with TatB and binds the signal peptides of Tat substrates. Here we have taken a random mutagenesis approach to identify substitutions in Escherichia coli TatC that inactivate protein transport. We identify 32 individual amino acid substitutions that abolish or severely compromise TatC activity. The majority of the inactivating substitutions fall within the first two periplasmic loops of TatC. These regions are predicted to have conserved secondary structure and results of extensive amino acid insertion and deletion mutagenesis are consistent with these conserved elements being essential for TatC function. Three inactivating substitutions were identified in the fifth transmembrane helix of TatC. The inactive M205R variant could be suppressed by mutations affecting amino acids in the transmembrane helix of TatB. A physical interaction between TatC helix 5 and the TatB transmembrane helix was confirmed by the formation of a site-specific disulphide bond between TatC M205C and TatB L9C variants. This is the first molecular contact site mapped to single amino acid level between these two proteins.
authors: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
authors: Holger
authors: H
authors: Kneuper
authors: Barbara
authors: B
authors: Maldonado
authors: Franziska
authors: F
authors: Jäger
authors: Martin
authors: M
authors: Krehenbrink
authors: Grant
authors: G
authors: Buchanan
authors: Rebecca
authors: R
authors: Keller
authors: Matthias
authors: M
authors: Müller
authors: Ben C
authors: BC
authors: Berks
authors: Tracy
authors: T
authors: Palmer
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; 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*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000235:genetics / Q000502:physiology
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C473116:TatB protein, E coli; C461379:TatC protein, E coli