University of Groningen Purification and reconstitution of the glutamate carrier GltT of the thermophilic bacterium Bacillus stearothermophilus Gaillard,

An affinity tag consisting of six adjacent histidine residues followed by an enterokinase cleavage site was genetically engineered at the N-terminus of the glutamate transport protein GltT of the thermophilic bacteriumBacillus stearothermophilus. The fusion protein was expressed in Escherichia coliand shown to transport glutamate. The highest levels of expression were observed in E. coli strain DH5R grown on rich medium. The protein could be purified in a single step by Ni 2+-NTA affinity chromatography after solubilization of the cytoplasmic membranes with the detergent Triton X100. Purified GltT was reconstituted in an active state in liposomes prepared from E. coli phospholipids. The protein was reconstituted in detergent-treated preformed liposomes, followed by removal of the detergent with polystyrene beads. Active reconstitution was realized with a wide range of Triton X100 concentrations. Neither the presence of glycerol, phospholipids, nor substrates of the transporter was necessary during the purification and reconstitution procedure to keep the enzyme in an active state. In B. stearothermophilus , GltT translocates glutamate in symport with protons or sodium ions. In membrane vesicles derived from E. coli cells expressing GltT, the Na + ion dependency seems to be lost [Tolner, B. , Ubbink-Kok, T. , Poolman, B. , & Konings, W. N. (1995) Mol. Microbiol. 18, 123-133], suggesting a role for the lipid environment in the cation specificity. In agreement with the last observation, glutamate transport catalyzed by purified GltT reconstituted inE. coli phospholipid is driven by an electrochemical gradient of H + but not of Na+. Bacillus stearothermophilus is a thermophilic bacterium that grows optimally at 63°C. Proteins of thermophiles are likely to be more stable than those of their mesophilic counterparts. This may be of particular importance in the study of membrane proteins when they are taken out of their natural phospholipid environment. Glutamate uptake in B. stearothermophilusis mediated via a secondary transporter termed GltT. Translocation of glutamate is coupled to translocation of two cations, a proton and a sodium ion, and, therefore, is driven by the proton and sodium ion motive forces (Heyne et al., 1991). The gene coding for the transporter GltT has been cloned and sequenced (Tolner et al., 1992a), and the deduced amino acid sequence showed significant homology with the members of a large family of carboxylic acid transporters in which carriers from related organisms like the glutamate transporter GltP from Escherichia coli are found (Tolner et al., 1992b) but also carriers from eukaryotic origin like the glutamate transporter from mammalian brain [e.g. Pines et al. (1992)]. The transporters in this family differ both in substrate specificity and cation specificity. ThegltT gene was expressed in a strain of E. colideficient in glutamate transport. Studies with membrane vesicles derived from the recombinant strain revealed a surprising result. Expression of GltT inE. coli resulted in complete loss of the sodium ion dependency; glutamate transport was driven only by the proton motive force. Similar studies with membrane vesicles fromB. stearothermophilushowed an increase in uptake activity in the presence of a sodium ion motive force, a feature that was most apparent at elevated temperatures (Tolner et al., 1995). The data suggest that, in addition to the primary sequence, the cation specificity of a carrier may depend also on conformation. Subtle conformational differences of GltT induced by the lipid environment and/or temperature may select for proton or sodium ion as the symported cation. Implicitly, it would mean that the difference between a proton and a sodium ion binding site on the carrier is extremely small. The importance of these findings requires that they are confirmed with the purified protein reconstituted in proteoliposomes. The isolated protein can be reconstituted in phospholipids from different origins and studied with respect to their cation selectivity at different temperatures. In addition, purification of the protein is essential to determine conformational changes by spectroscopic techniques. Here, we report on the first step in these studies, the purification and functional reconstitution of GltT in E. coliphospholipids. After optimization of the expression of the gltT gene inE. coli, the protein is purified in a single step by His tag affinity chromatography (Hochuli et al., 1987) and reconstituted by the detergent titration technique (Rigaud & Pitard, 1995). Specific precautions that have been reported for the suc† This work was supported in part by a grant from the HCM program of the EEC (to I.G.) and by the Ministry of Economic Affairs, the Ministry of Education, Culture and Science, and the Ministry of Agriculture, Nature Management and Fishery in the framework of an industrial relevant research program of the Netherlands Association of Biotechnology Centres in the Netherlands (ABON) (to D.-J. S.). * Corresponding author: Department of Microbiology, University of Groningen, Kerklaan 30, 9751NN Haren, The Netherlands. Phone: (31)503632155. Fax: (31)503632154. E-mail: j.s. lolkema@biol.rug. nl. ‡ The first two authors have equally contributed to this paper. X Abstract published inAdVance ACS Abstracts, May 1, 1996. 6150 Biochemistry1996,35, 6150-6156 S0006-2960(95)03005-4 CCC: $12.00 © 1996 American Chemical Society + +