Structure and function of prokaryotic glutamate transporters from Escherichia coli and Pyrococcus horikoshii.
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.
Raunser
Stefan
S
Department of Structural Biology, Max-Planck-Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany.
Appel
Matthias
M
Ganea
Constanta
C
Geldmacher-Kaufer
Ulrike
U
Fendler
Klaus
K
Kühlbrandt
Werner
W
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*
D027322:Amino Acid Transport System X-AG; D019843:Archaeal Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D001224:Aspartic Acid