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 cotransporte r, NaDC-1, contains 11 transmembrane domains. The model is based on hydropa thy analysis and the extracellular location of the carboxy terminus, which contains an N-glycosylation site. In this study, the model was further test ed 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 mo noclonal antibody against the Flag epitope was used to determine the locati on of the N-terminus. Hydrophilic loop 4 of NaDC-1 was identified using pol yclonal antibodies raised against a fusion protein containing amino acids 1 64-233 of NaDC-1. The expression of NaDC-1 and Flag-NaDC-1 in COS-7 cells w as confirmed by functional assays of succinate transport and by Western blo ts of cell surface biotinylated proteins. Immunofluorescent labeling of cel ls expressing both NaDC-1 and Flag-NaDC-1 required permeabilization of the plasma membranes with digitonin whereas no immunofluorescence was visible i n 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 t he current model of NaDC-1 structure. (C) 2001 Elsevier Science B.V. All ri ghts reserved.