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

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Raunser,  Stefan
Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Appel,  Matthias
Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Ganea,  Constanta
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Geldmacher-Kaufer,  Ulrike
Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Fendler,  Klaus
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Kühlbrandt,  Werner       
Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Raunser, S., Appel, M., Ganea, C., Ganea, C., Geldmacher-Kaufer, U., Fendler, K., et al. (2006). Structure and Function of Prokaryotic Glutamate Transporters from Escherichia coli and Pyrococcus horikoshii. Biochemistry, 45, 12796-12805. doi:10.1021/bi061008+.


Cite as: https://hdl.handle.net/11858/00-001M-0000-0024-D980-6
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.