title: Cysteine scanning mutagenesis and topological mapping of the Escherichia coli twin-arginine translocase TatC Component.
abstract: The TatC protein is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. It is a polytopic membrane protein that forms a complex with TatB, together acting as the receptor for Tat substrates. In this study we have constructed 57 individual cysteine substitutions throughout the protein. Each of the substitutions resulted in a TatC protein that was competent to support Tat-dependent protein translocation. Accessibility studies with membrane-permeant and -impermeant thiol-reactive reagents demonstrated that TatC has six transmembrane helices, rather than the four suggested by a previous study (K. Gouffi, C.-L. Santini, and L.-F. Wu, FEBS Lett. 525:65-70, 2002). Disulfide cross-linking experiments with TatC proteins containing single cysteine residues showed that each transmembrane domain of TatC was able to interact with the same domain from a neighboring TatC protein. Surprisingly, only three of these cysteine variants retained the ability to cross-link at low temperatures. These results are consistent with the likelihood that most of the disulfide cross-links are between TatC proteins in separate TatBC complexes, suggesting that TatC is located on the periphery of the complex.
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom.
authors.forename: Claire
authors.initials: C
authors.lastname: Punginelli
authors.forename: Bárbara
authors.initials: B
authors.lastname: Maldonado
authors.forename: Sabine
authors.initials: S
authors.lastname: Grahl
authors.forename: Rachael
authors.initials: R
authors.lastname: Jack
authors.forename: Meriem
authors.initials: M
authors.lastname: Alami
authors.forename: Juliane
authors.initials: J
authors.lastname: Schröder
authors.forename: Ben C
authors.initials: BC
authors.lastname: Berks
authors.forename: Tracy
authors.initials: T
authors.lastname: Palmer
mesh_terms: D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C473116:TatB protein, E coli; C461379:TatC protein, E coli