2006 - Structure and function of prokaryotic glutamate transporters from Escherichia coli and Pyrococcus horikoshii

   SURFE²R-technology (custom-built system) publication in Biochemistry (2006)

Authors:
Raunser S., Appel M., Ganea C., Geldmacher-Kaufer U., Fendler K., Kühlbrandt W.

Journal:
Biochemistry (2006) 45(42):12796-805.

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 (K_0.5^glut) is 194 µM.

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