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Journal of Bacteriology, October 2007, p. 7089-7097, Vol. 189, No. 19
0021-9193/07/$08.00+0 doi:10.1128/JB.00088-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Topology of AspT, the Aspartate:Alanine Antiporter of Tetragenococcus halophilus, Determined by Site-Directed Fluorescence Labeling
,
Kei Nanatani,1
Takashi Fujiki,1
Kazuhiko Kanou,2
Mayuko Takeda-Shitaka,2
Hideaki Umeyama,2
Liwen Ye,3
Xicheng Wang,3
Tasuku Nakajima,1
Takafumi Uchida,1
Peter C. Maloney,3 and
Keietsu Abe1*
Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555 Japan,1 Department of Biomolecular Design, School of Pharmaceutical Sciences, Kitasato University, Tokyo, 108-8641 Japan,2 Department of Physiology, Johns Hopkins School of Medicine, Baltimore, Maryland 212053
Received 17 January 2007/ Accepted 17 July 2007
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-ß-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.
* Corresponding author. Mailing address: Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555 Japan. Phone: 81-22-717-8777. Fax: 81-22-717-8778. E-mail: kabe{at}biochem.tohoku.ac.jp
Published ahead of print on 27 July 2007.
Supplemental material for this article may be found at http://jb.asm.org/.
Journal of Bacteriology, October 2007, p. 7089-7097, Vol. 189, No. 19
0021-9193/07/$08.00+0 doi:10.1128/JB.00088-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
This article has been cited by other articles:
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Nanatani, K., Maloney, P. C., Abe, K.
(2009). Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT. J. Bacteriol.
191: 2122-2132
[Abstract] [Full Text]
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