Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT

Abstract

AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of L-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of L-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, L-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes L-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a functional unit.In some strains of the lactic acid bacterium Tetragenococcus halophilus, a proton motive force (PMF) is generated by the combined action of an intracellular L-aspartate decarboxylation reaction catalyzed by an L-aspartate-4-decarboxylase (AspD [EC 4.1. The PMF generated is sufficiently high to drive ATP synthesis via the bacterial F o F 1 ATPase. This combination of PMF and ATP synthesis has been proposed as a proton motive metabolic cycle, and the prototype model is found in Oxalobacter formigenes (3, 9, 30). Such decarboxylation reactions are thought to be advantageous for cells because the reactions generate metabolic energy and regulate the intracellular pH. In previous works using proteoliposomes, we found that the aspartate:alanine exchange catalyzed by AspT is electrogenic (1, 2). The biochemical features of substrate transport by AspT indicate that the protein can be classified as a conventional secondary transport protein and that it is an electrogenic antiporter similar to the prototype precursor:product exchanger OxlT, an oxal...

Substrate Specificity of the Aspartate:Alanine Antiporter (AspT) of Tetragenococcus halophilus in Reconstituted Liposomes

“…Nanatani et al (14) established AspT purification and reconstitution methods and confirmed L-aspartate self-exchange properties. Here, we clearly demonstrated that purified AspT alone is sufficient for the electrogenic Fig.…”

“…The solubilized AspT was incubated with L-aspartate (5, 10, and 20 mM) or L-alanine (50, 100, and 200 mM) at 37°C for several minutes. After the reconstitution, the remaining transport activities of AspT reconstituted into proteoliposomes loaded with 200 mM L-alanine and 50 mM N-methyl-D-glucamine-phosphate (pH 7.0) were examined by using a simple filtration method (14).…”

“…Expression, Solubilization, and Purification of AspT(WT)-HisExpression of histidine-tagged AspT was performed as described previously (14). In brief, a preculture of E. coli XL3 carrying pTrcAsp-His was diluted 100-fold in fresh Luria-Bertani (LB) medium containing 30 mM D-glucose, 30 g/ml of carbenicillin, and 30 g/ml of spectinomycin.…”

“…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…”

“…A. ), the Kato Memorial Bioscience Foundation (to K. N.), and a NISR (Noda Institute for Scientific Research) Research grant (to K. A. glutaraldehyde cross-linking assay (14). Here, we sought to characterize, in detail, the kinetic properties of AspT by analyzing the transport kinetics of reconstituted AspT under various conditions.…”