Membrane Topology of Aspartate:Alanine Antiporter AspT from Comamonas testosteroni

Abstract

We cloned the aspT gene encoding the L-aspartate:L-alanine antiporter AspTCt in Comamonas testosteroni genomic DNA. Analysis of the nucleotide sequence revealed that C. testosteroni has an asp operon containing aspT upstream of the l-aspartate 4-decarboxylase gene, and that the gene order of the asp operon of C. testosteroni is the inverse of that of Tetragenococcus halophilus. We used proteoliposomes to confirm the transport processes of AspTCt. To elucidate the two-dimensional structure of AspTCt, we analysed its membrane topology by means of alkaline phosphatase (PhoA) and beta-lactamase (BlaM) fusion methods. The fusion analyses revealed that AspTCt has seven transmembrane segments (TMs), a large cytoplasmic loop containing approximately 200 amino acid residues between TM4 and TM5, a cytoplasmic N-terminus, and a periplasmic C-terminus. These results suggest that the orientation of the N-terminus of AspTCt differs from that of tetragenococcal AspT, even though these two AspT orthologues catalyse the same transport reactions.

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

“…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.…”