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J. Biol. Chem., Vol. 281, Issue 51, 39273-39284, December 22, 2006

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Amino Acid Transporter ATA2 Is Stored at the trans-Golgi Network and Released by Insulin Stimulus in Adipocytes^*

Takahiro Hatanaka, Yasue Hatanaka, Jun-ichi Tsuchida^¶, Vadivel Ganapathy^||, and Mitsutoshi Setou^**1

From the Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida, Tokyo 194-8511, Japan, PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan, the ^¶Mitsubishi Pharma Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-0033, Japan, the ^||Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912-2100, and the ^**National Institute of Physiological Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan

Recently, we cloned the ATA/SNAT transporters responsible for amino acid transport system A. System A is one of the major transport systems for small neutral and glucogenic amino acids represented by alanine and is involved in the metabolism of glucose and fat. Here, we describe the cellular mechanisms that participate in the acute translocation of ATA2 by insulin stimulus in 3T3-L1 adipocytes. We monitored this insulin-stimulated translocation of ATA2 using an expression system of enhanced green fluorescent protein-tagged ATA2. Studies in living cells revealed that ATA2 is stored in a discrete perinuclear site and that the transporter is released in vesicles from this site toward the plasma membrane. In immunofluorescent analysis, the storage site of ATA2 overlapped with the location of syntaxin 6, a marker of the trans-Golgi network (TGN), but not with that of EEA1, a marker of the early endosomes. The ATA2-containing vesicles on or near the plasma membrane were distinct from GLUT4-containing vesicles. Brefeldin A, an inhibitor of vesicular exit from the TGN, caused morphological changes in the ATA2 storage site along with the similar changes in the TGN. In non-transfected adipocytes, brefeldin A inhibited insulin-stimulated uptake of -(methylamino)isobutyric acid more profoundly than insulin-stimulated uptake of 2-deoxy-D-glucose. These data demonstrate that the ATA2 storage site is specifically associated with the TGN and not with the general endosomal recycling system. Thus, the insulin-stimulated translocation pathways for ATA2 and GLUT4 in adipocytes are distinct, involving different storage sites.

Received for publication, May 11, 2006 , and in revised form, October 12, 2006.

^* This work was supported by PRESTO from the Japan Science and Technology Agency and Grant-in-aid WAKATE-A (to M. S.) and Grant-in-aid WAKATE-B (to T. H.) from the Ministry of Education, Culture, Sports, Science, and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida, Tokyo 194-8511, Japan. Tel.: 81-42-724-6259; Fax: 81-42-724-6316; E-mail: setou{at}nips.ac.jp.

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