Trimeric Subunit Stoichiometry of the Glutamate Transporters from Bacillus caldotenax and Bacillus stearothermophilus

Dinesh Yernool, Olga Boudker, Ewa Folta-Stogniew, Eric Gouaux

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

Catalysis of glutamate transport across cell membranes and coupling of the concentrative transport to sodium, proton, and potassium gradients are processes fundamental to organisms in all kingdoms of life. In bacteria, glutamate transporters participate in nutrient uptake, while in eukaryotic organisms, the transporters clear glutamate from the synaptic cleft. Even though glutamate transporters are crucial to the viability of many life forms, little is known about their structure and quaternary organization. In particular, the subunit stoichiometry of these polytopic integral membrane proteins has not been unequivocally defined. Determination of the native molecular mass of membrane proteins is complicated by their lability in detergent micelles and by their association with detergent and/or lipid molecules. Here we report the purification of glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus in a monodisperse, detergent-solubilized state. Characterization of both transporters either by chemical cross-linking and mass spectrometry or by size-exclusion chromatography and in-line laser light scattering, refractive index, and ultraviolet absorption measurements shows that the transporters have a trimeric quaternary structure. Limited proteolysis further defines regions of primary structure that are exposed to aqueous solution. Together, our results define the subunit stoichiometry of high-affinity glutamate transporters from B. caldotenax and B. stearothermophilus and localize exposed and accessible elements of primary structure. Because of the close amino acid sequence relationship between bacterial and eukaryotic transporters, our results are germane to prokaryotic and eukaryotic glutamate and neutral amino acid transporters.

Original languageEnglish (US)
Pages (from-to)12981-12988
Number of pages8
JournalBiochemistry
Volume42
Issue number44
DOIs
StatePublished - Nov 11 2003
Externally publishedYes

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Amino Acid Transport System X-AG
Geobacillus stearothermophilus
Bacilli
Stoichiometry
Bacillus
Detergents
Glutamic Acid
Membrane Proteins
Neutral Amino Acid Transport Systems
Proteolysis
Size exclusion chromatography
Molecular mass
Micelles
Cell membranes
Refractometry
Light scattering
Catalysis
Nutrients
Purification
Mass spectrometry

ASJC Scopus subject areas

  • Biochemistry

Cite this

Trimeric Subunit Stoichiometry of the Glutamate Transporters from Bacillus caldotenax and Bacillus stearothermophilus. / Yernool, Dinesh; Boudker, Olga; Folta-Stogniew, Ewa; Gouaux, Eric.

In: Biochemistry, Vol. 42, No. 44, 11.11.2003, p. 12981-12988.

Research output: Contribution to journalArticle

Yernool, Dinesh ; Boudker, Olga ; Folta-Stogniew, Ewa ; Gouaux, Eric. / Trimeric Subunit Stoichiometry of the Glutamate Transporters from Bacillus caldotenax and Bacillus stearothermophilus. In: Biochemistry. 2003 ; Vol. 42, No. 44. pp. 12981-12988.
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