Exchange of Aspartate and Alanine

Abstract

We examined the idea that aspartate metabolism by Lactobacillus subsp. M3 is organized as a proton-motive metabolic cycle by using reconstitution to monitor the activity of the carrier, termed AspT, expected to carry out the electrogenic exchange of precursor (aspartate) and product (alanine). Membranes of Lactobacillus subsp. M3 were extracted with 1.25% octyl glucoside in the presence of 0.4% Escherichia coli phospholipid and 20% glycerol. The extracts were then used to prepare proteoliposomes loaded with either aspartate or alanine. Aspartate-loaded proteoliposomes accumulated external [ 3 H]aspartate by exchange with internal substrate; this homologous self-exchange (K t ‫؍‬ 0.4 mM) was insensitive to potassium or proton ionophores and was unaffected by the presence or absence of Na Nutrient transport by bacteria is usually thought of as consuming metabolic energy, since this step is typically driven by an ion-motive gradient (e.g. ⌬ Hϩ or ⌬ Naϩ ) or by hydrolysis of a phosphoester bond (e.g. ATP or PEP) (1, 2). Recently, however, a new class of nutrient transport reactions has been identified, one in which substrate transport is actually used to generate rather than consume energy. The first and best understood of these reactions is found in Oxalobacter formigenes (3, 4), a Gram-negative, obligate anaerobe that exploits the decarboxylation of oxalate to support transmembrane ion-motive gradients (5). This cell mediates the exchange of divalent oxalate with the product of its intracellular decarboxylation, monovalent formate (6), using a membrane transporter named OxlT (4). The one-for-one exchange of oxalate 2Ϫ and formate 1Ϫ polarizes the membrane (electrically negative, inside), while the decarboxylation reaction serves to generate an internal alkalinity, since a single cytosolic proton is consumed during production of formate. As a result, the metabolic sequence, oxalate entry, oxalate decarboxylation, formate exit, acts as a proton pump (3) or "proton-motive metabolic cycle" (Refs. 3 and 4; reviewed in Ref. 7). In the same way and in other bacteria, the transport (vectorial) and decarboxylation (scalar) reactions associated with conversion of malate to lactate (8 -10) or histidine to histamine (11) have been shown to act as proton-motive metabolic cycles. Such precedents suggest a new way of interpreting the relationship between anion transport and decarboxylation reactions in microorganisms. For example, some strains of the Lactobacilli catalyze the decarboxylation of either L-aspartate 1 or L-glutamate, 2 with a near-stoichiometric release of the products, L-alanine or ␥-aminobutyrate (and CO 2 ), respectively 1, 2 . These decarboxylations support ATP synthesis in a manner consistent with the idea that processing of these anions involves a proton-motive metabolic cycle 1, 2 (and see below). As their central element, proton-motive metabolic cycles have a vectorial component(s) that mediates the electrogenic exchange of precursor and product. Accordingly, the specific goal of work reported here was to d...

Membrane Potential-generating Malate (MleP) and Citrate (CitP) Transporters of Lactic Acid Bacteria Are Homologous Proteins

“…14 C]malate or [1,[5][6][7][8][9][10][11][12][13][14] C]citrate was added at a concentration of 13.1 or 4.4 M, respectively. Uptake was stopped at different time intervals by adding 2 ml of ice-cold 0.1 M LiCl to a sample and rapid filtration over 0.45-m pore size cellulose nitrate filters (Schleicher & Schuell).…”

Exchange of glutamate and gamma-aminobutyrate in a Lactobacillus strain

“…A similar phenomenon had been observed in Lactobacillus sp. strain M3, in which excess extracellular alanine (Ͼ300 mM) inhibited ATP synthesis driven by the aspartate/alanine proton motive metabolic cycle (1). This suggests, then, that GABA efflux may be catalyzed by a glutamate/GABA antiporter resembling the electrogenic aspartate/alanine antiporter.…”

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

“…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).…”

“…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)(2)(3). Such decarboxylation reactions are thought to be advantageous for cells because they generate metabolic energy and regulate intracellular pH (4,5).…”