Item

Abstract

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 (K0.5glut) is 194 μM.