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A Trimeric Quaternary Structure Is Conserved in Bacterial and Human Glutamate Transporters
- Source :
- Journal of Biological Chemistry. 279:39505-39512
- Publication Year :
- 2004
- Publisher :
- Elsevier BV, 2004.
-
Abstract
- 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.
- Subjects :
- Glycosylation
Protein subunit
Molecular Sequence Data
Population
Xenopus
Gene Expression
Kidney
Biochemistry
Evolution, Molecular
Xenopus laevis
Animals
Humans
Amino Acid Sequence
Protein Structure, Quaternary
education
Molecular Biology
Cells, Cultured
education.field_of_study
biology
Escherichia coli Proteins
Metabotropic glutamate receptor 7
Metabotropic glutamate receptor 6
Transporter
Cell Biology
biology.organism_classification
Cross-Linking Reagents
Excitatory Amino Acid Transporter 2
Oocytes
Metabotropic glutamate receptor 1
Electrophoresis, Polyacrylamide Gel
Protein quaternary structure
Protein Processing, Post-Translational
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 279
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....08b5bfe7ad6e4e474eebcd49571bf8f4