Structure and Function of Prokaryotic Glutamate Transporters from Escherichia coli and Pyrococcus horikoshii

The glutamate transporters GltPEc from Escherichia coli and GltPPh 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 GltPPh is a trimer in detergent solution. Electron microscopy of negatively stained GltPPh two-dimensional crystals shows that the transporter is a trimer also in the membrane. Gel filtration of GltPEc 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 GltPEc involves the cotransport of protons and depends only on pH, whereas GltPPh catalyzes l-glutamate transport with a cotransport of H+ or Na+. l-Glutamate induces a fast transient current in GltPPh 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 GltPPh 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 GltPPh glutamate transport (K 0.5 glut) is 194 μM.