Journal of Biological Chemistry
Volume 279, Issue 38, 17 September 2004, Pages 39505-39512
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Membrane Transport, Structure, Function, and Biogenesis
A Trimeric Quaternary Structure Is Conserved in Bacterial and Human Glutamate Transporters*

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Neuronal and glial glutamate transporters play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian central nervous system. They are known to be multimers; however, the number of subunits forming a functional transporter is controversial. We studied the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and a bacterial glutamate transporter from Escherichia coli, ecgltP. Using blue native polyacrylamide gel electrophoresis, analysis of concatenated transporters, and chemical cross-linking, we demonstrated that human and prokaryotic glutamate transporters expressed in Xenopus laevis oocytes or in mammalian cells are assembled as trimers composed of three identical subunits. In an inducible mammalian cell line expressing hEAAT2 the glutamate uptake currents correlate to the amount of trimeric transporters. Overexpression and purification of ecgltP in E. coli resulted in a homogenous population of trimeric transporters that were functional after reconstitution in lipid vesicles. Our results indicate that an evolutionarily conserved trimeric quaternary structure represents the sole native and functional state of glutamate transporters.

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*

This work was supported by Deutsche Forschungsgemeinschaft Grants FOR450/1 (TP1 and TP4 to P. H. and Ch. F., respectively) and SCHM 536/6-1 (to G. S.) and by a START grant of the medical faculty of the Rheinisch-Westfälische Technische Hochschule (to P. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Both authors contributed equally to this work.

These authors contributed equally to this work.