title: Structure and function of prokaryotic glutamate transporters from Escherichia coli and Pyrococcus horikoshii.
abstract: The glutamate transporters GltP(Ec) from Escherichia coli and GltP(Ph) from Pyrococcus horikoshii were overexpressed in E. coli and purified to homogeneity with a yield of 1-2 mg/L of culture. Single-particle analysis and electron microscopy indicate that GltP(Ph) is a trimer in detergent solution. Electron microscopy of negatively stained GltP(Ph) two-dimensional crystals shows that the transporter is a trimer also in the membrane. Gel filtration of GltP(Ec) indicates a reversible equilibrium of two oligomeric states in detergent solution that we identified as a trimer and hexamer by blue-native gel electrophoresis and cross-linking. The purified transporters were fully active upon reconstitution into liposomes, as demonstrated by the uptake of radioactively labeled L-aspartate or L-glutamate. L-aspartate/L-glutamate transport of GltP(Ec) involves the cotransport of protons and depends only on pH, whereas GltP(Ph) catalyzes L-glutamate transport with a cotransport of H+ or Na+. L-glutamate induces a fast transient current in GltP(Ph) proteoliposomes coupled to a solid supported membrane (SSM). We show that the electric signal depends on the concentration of Na+ or H+ outside the proteoliposomes and that GltP(Ph) does not require K+ inside the proteoliposomes. In addition, the electrical currents are inhibited by TBOA and HIP-B. The half-saturation concentration for activation of GltP(Ph) glutamate transport (K0.5(glut)) is 194 microM.
authors.affiliation: Department of Structural Biology, Max-Planck-Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Raunser
authors.forename: Matthias
authors.initials: M
authors.lastname: Appel
authors.forename: Constanta
authors.initials: C
authors.lastname: Ganea
authors.forename: Ulrike
authors.initials: U
authors.lastname: Geldmacher-Kaufer
authors.forename: Klaus
authors.initials: K
authors.lastname: Fendler
authors.forename: Werner
authors.initials: W
authors.lastname: Kühlbrandt
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019843:Archaeal Proteins / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D003461:Crystallography; D003902:Detergents; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D043902:Pyrococcus horikoshii / Q000378:metabolism*
chemical_list: D027322:Amino Acid Transport System X-AG; D019843:Archaeal Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D001224:Aspartic Acid