Trimeric subunit stoichiometry of the glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus.
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.
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.
Dinesh
D
Yernool
Olga
O
Boudker
Ewa
E
Folta-Stogniew
Eric
E
Gouaux
D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001407:Bacillus / Q000737:chemistry* / Q000235:genetics; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D001411:Geobacillus stearothermophilus / Q000737:chemistry / Q000235:genetics; D005976:Glutaral / Q000737:chemistry; D007834:Lasers; D008081:Liposomes; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D021381:Protein Transport / Q000235:genetics; D012542:Scattering, Radiation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet
D027322:Amino Acid Transport System X-AG; D003432:Cross-Linking Reagents; D008081:Liposomes; D021122:Protein Subunits; D005976:Glutaral