Topology of the Na(+)/dicarboxylate cotransporter: the N-terminus and hydrophilic loop 4 are located intracellularly.
The current secondary structure model of the Na(+)/dicarboxylate cotransporter, NaDC-1, contains 11 transmembrane domains. The model is based on hydropathy analysis and the extracellular location of the carboxy terminus, which contains an N-glycosylation site. In this study, the model was further tested using indirect immunofluorescence of COS-7 cells. The Flag epitope tag (DYKDDDDK) was fused to the amino terminus of NaDC-1 (Flag-NaDC-1), and a monoclonal antibody against the Flag epitope was used to determine the location of the N-terminus. Hydrophilic loop 4 of NaDC-1 was identified using polyclonal antibodies raised against a fusion protein containing amino acids 164--233 of NaDC-1. The expression of NaDC-1 and Flag-NaDC-1 in COS-7 cells was confirmed by functional assays of succinate transport and by Western blots of cell surface biotinylated proteins. Immunofluorescent labeling of cells expressing both NaDC-1 and Flag-NaDC-1 required permeabilization of the plasma membranes with digitonin whereas no immunofluorescence was visible in intact cells. The results of this study show that both the N-terminus and hydrophilic loop 4 of NaDC-1 are located intracellularly, which supports the current model of NaDC-1 structure.
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0641, USA.
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D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D029382:Dicarboxylic Acid Transporters; D004072:Digitonin; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D029363:Organic Anion Transporters, Sodium-Dependent; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013499:Surface Properties; D027981:Symporters; D014162:Transfection
D002352:Carrier Proteins; D029382:Dicarboxylic Acid Transporters; D000939:Epitopes; D008565:Membrane Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D011993:Recombinant Fusion Proteins; C092524:SLC13A2 protein, human; D027981:Symporters; D004072:Digitonin