Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling

Kei Nanatani, Takashi Fujiki, Kazuhiko Kanou, Mayuko Takeda-Shitaka, Hideaki Umeyama, Liwen Ye, Xicheng Wang, Tasuku Nakajima, Takafumi Uchida, Peter C. Maloney, Keietsu Abe

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13 Citations (Scopus)


The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO2. The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-β-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium) ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.

Original languageEnglish
Pages (from-to)7089-7097
Number of pages9
JournalJournal of bacteriology
Issue number19
Publication statusPublished - 2007 Oct
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology


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