A trimeric quaternary structure is conserved in bacterial and human glutamate transporters.
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.
Gendreau
Sandra
S
Department of Molecular Pharmacology, Rheinisch-Westfälische Technische Hochschule Aachen, 52057 Aachen, Germany.
Voswinkel
Stephan
S
Torres-Salazar
Delany
D
Lang
Niklas
N
Heidtmann
Hannelore
H
Detro-Dassen
Silvia
S
Schmalzing
Günther
G
Hidalgo
Patricia
P
Fahlke
Christoph
C
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D027342:Excitatory Amino Acid Transporter 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D014982:Xenopus laevis
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D027342:Excitatory Amino Acid Transporter 2