In some strains of the lactic acid bacterium Tetragenococcus halophilus, a proton-motive force is generated by the combined action of an intracellular L-aspartate decarboxylation reaction, catalyzed by an L-aspartate-4-decarboxylase (AspD, EC 4.1.1.12), and an electrogenic aspartate 1Ϫ :alanine 0 exchange reaction, catalyzed by an aspartate:alanine antiporter (AspT, TC number 2.A.81.1.1):The proton-motive force generated is sufficiently high to drive ATP synthesis via the bacterial F 0 F 1 -ATPase. This combination of proton-motive force and ATP synthesis has been proposed as a protonmotive metabolic cycle, the prototype model of which is found in Oxalobacter formigenes (1-3). Such decarboxylation reactions are thought to be advantageous for cells because they generate metabolic energy and regulate intracellular pH (4, 5).In previous work with proteoliposomes, we found that the aspartate:alanine exchange catalyzed by AspT is electrogenic (4, 6). AspT is classified as a conventional secondary transport protein and belongs to the newly classified aspartate:alanine exchanger family (TC number 2.A.81) of transporters in the system developed by Saier et al. (25,26). Recently, the results of a BLAST (7) search of the nucleotide sequence of the aspT gene and the amino acid sequence of the AspT protein against current nucleotide and protein data bases, respectively, suggested that the aspartate:alanine exchanger transporters are conserved in many bacterial species (8, 9). Very recently, Fukui et al. (10) found SucE1, an aspartate:alanine exchanger transporter from Corynebacterium glutamicum, plays a role in the export of succinate generated during fermentation. The putative broad distribution of AspT orthologs and paralogs in bacteria suggests that further biochemical study of AspT could provide valuable insights into membrane transport.AspT is a membrane protein containing 543 amino acids (57.2 kDa). Its membrane topology has been studied by using alkaline phosphatase and -lactamase fusion methods (11); AspT has also been studied by using the cysteine-substituted accessibility method (12, 13), which uses the impermeant, fluorescent thiol-specific probe Oregon Green 488 maleimide and the impermeant, nonfluorescent thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. These analyses revealed that AspT has a unique topology and that transmembrane domain 3 participates in the formation of a hydrophilic cleft in the membrane, implicating the transmembrane domain in the ligand-induced conformational changes (14). In previous work, we developed a solubilization and purification scheme by using n-dodecyl--D-maltoside (DDM), 3 and we studied the oligomerization of AspT by means of a
