Membrane topology of aspartate:alanine antiporter AspT from Comamonas testosteroni

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.