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Journal of Biochemistry Advance Access published online on December 11, 2006
Journal of Biochemistry, doi:10.1093/jb/mvm010
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© 2006 The Japanese Biochemical Society

Membrane Topology of Aspartate:Alanine Antiporter AspT from Comamonas testosteroni

Takashi Fujikia, Kei Nanatania, Kei Nishitania, Kyoko Yagia, Fumito Ohnishia, Hiroshi Yoneyamab, Takafumi Uchidaa, Tasuku Nakajimaa and Keietsu Abea,*

aLaboratory of Enzymology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555 JAPAN
bLaboratory of Dairy Microbiology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555 JAPAN

*Corresponding author: Keietsu Abe, Mailing address: Laboratory of Enzymology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiya, Tsutsumi-dori, Sendai, 981-8555 JAPAN, TEL: +81-22-717-8777, FAX: +81-22-717-8778, E-mail: kabe{at}biochem.tohoku.ac.jp


  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 analyzed its membrane topology by means of alkaline phosphatase (PhoA) and ß-lactamase (BlaM) fusion methods. The fusion analyses revealed that AspTCt has seven transmembrane segments (TMs), a large cytoplasmic loop containing ~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 catalyze the same transport reactions.

Key Words: Bacteria, bioenergetics, aspartate, alanine antiporter, membrane topology, fusion methods


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