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

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.

A Trimeric Quaternary Structure Is Conserved in Bacterial and Human Glutamate Transporters

“…Assuming seven transmembrane helices/subunit, the authors assigned five subunits/transporter. In contrast to this pentameric assembly, a trimeric stoichiometry was reported recently for two prokaryotic glutamate transporters based on cross-linking and in-line laser light scattering, refractive index, and ultraviolet absorption measurements (29) raising the possibility that eukaryotic and prokaryotic transporters might exhibit distinct subunit stoichiometries.…”

Static light scattering to characterize membrane proteins in detergent solution

“…Both analyses gave the same result. The protein molecular mass was 154 kDa, confirming that the GltT forms homotrimers [3]. The mass of detergent micelle attached to the protein was determined by calculating d (from Eqs.…”

“…Other examples can be found in references [2,3,7,[19][20][21][22]. A monomer of Histidinetagged GltT from the bacterium Bacillus stearothermophilus has a molecular mass of 46.9 kDa calculated from the amino acid sequence.…”

“…lipids or carbohydrates [9]). (dn/dc) lipid is similar to (dn/dc) detergent for many phospholipids and detergents and often a single value is taken to calculate the total mass of the attached lipid/ detergent micelle [2,3,7]. It is important to note that the determination of the protein molecular mass M W,protein is not affected by inaccuracies in the determination of the attached mass of detergent/ lipid/carbohydrate if the three-detector method is used.…”

“…The mass ratio of protein to detergent in the protein/detergent micelle can vary dramatically depending on both the protein and the nature of the detergent [1][2][3] (Table 1). Consequently, methods that infer the molecular mass of proteins (and thereby the subunit stoichiometry) from hydrodynamic properties are not suitable for membrane proteins in detergent micelles.…”