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

Icon N1   SURFE²R-technology (custom-built system) publication in Biochemistry (2006)

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

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


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 GltP(Ph) 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 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 GltPPh glutamate transport (K0.5glut) is 194 µM.

Download here


We use cookies on our website. Some of them are essential for the operation of the site, while others help us to improve this site and the user experience (tracking cookies). You can decide for yourself whether you want to allow cookies or not. Please note that if you reject them, you may not be able to use all the functionalities of the site.