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.