Membrane topology structure of human high-affinity, sodium-dependent dicarboxylate transporter.
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.
Bai
Xue-Yuan
XY
Department of Biochemistry and Molecular Biology, Chinese PLA Institute of Nephrology, Chinese PLA General Hospital and Military Medical Postgraduate College, 28 Fuxing Rd., Beijing 100853, China.
Chen
Xiangmei
X
Sun
An-Qiang
AQ
Feng
Zhe
Z
Hou
Kai
K
Fu
Bo
B
D001483:Base Sequence; D001692:Biological Transport; D019921:Biotinylation; D002460:Cell Line; D002952:Citric Acid Cycle; D017931:DNA Primers; D029382:Dicarboxylic Acid Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D007700:Kinetics; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019802:Succinic Acid / Q000378:metabolism; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D029382:Dicarboxylic Acid Transporters; D029363:Organic Anion Transporters, Sodium-Dependent; D011994:Recombinant Proteins; C092524:SLC13A2 protein, human; D027981:Symporters; D019802:Succinic Acid