Membrane topology structure of human high-affinity, sodium-dependent dicarboxylate transporter

被引:13
作者
Bai, Xue-Yuan
Chen, Xiangmei
Sun, An-Qiang
Feng, Zhe
Hou, Kai
Fu, Bo
机构
[1] Chinese Peoples Liberat Army Gen Hosp, Chinese PLA Inst Nephrol, Dept Biochem & Mol Biol, Beijing 100853, Peoples R China
[2] Mil Med Postgrad Coll, Beijing 100853, Peoples R China
[3] Mt Sinai Sch Med, Dept Pediat, New York, NY USA
关键词
Krebs cycle; immunofluorescence; SCAM; Flag; localization;
D O I
10.1096/fj.06-7652com
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
High-affinity, sodium-dependent dicarboxylate transporter (NaDC3) is responsible for transport of Krebs cycle intermediates and may involve in regulation of aging and life span. Hydropathy analysis predicts that NaDC3 contains 11 or 12 hydrophobic transmembrane (TM)domains. However, the actual membrane topological structure of NaDC3 remains unknown. In this study, confocal immunofluorescence microscopy and membrane biotinylation of epitope-tagged N and C termini of NaDC3 provide evidence of an extracellular C terminus and an intracellular N terminus, indicating an odd number of transmembrane regions. The position of hydrophilic loops within NaDC3 was identified with antibodies against the loops domains combined with cysteine accessibility methods. A confocal image of membrane localization and transport activity assay of the cysteine insertion mutants show behavior similar to that of wild-type NaDC3 in transfected HEK293 cells, suggesting that these mutants retain a native protein configuration. We find that NaDC3 contains 11 transmembrane helices. The loops 1, 3, 5, 7, and 9 face the extracellular side, and loops 2, 4, 6, and 10 face the cytoplasmic side. A re-entrant loop-like structure between TM8 and TM9 may protrude into the membrane. Our results support the topography of 11 transmembrane domains with an extracellular C terminus and an intracellular N terminus of NaDC3, and for the first time provide experimental evidence for a novel topological model for NaDC3
引用
收藏
页码:2409 / 2417
页数:9
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