FIGURE 3.

Subcellular expression and functional activity of SVCT2 histidine mutants expressed in HEK-293 cells. A, predicted location of histidine residues in SVCT2 two-dimensional 12-helix model. Histidine residues His²⁰³, His²⁰⁶, and His⁴¹³ are exofacial, and histidine residues His²⁹, His¹⁰⁹, and His²⁶⁹ were considered to be endofacially localized. B, confocal microscopy of SVCT2-GFP and the different mutants in transfected HEK-293 cells. An optical slice from the middle section of cells is shown. C, confocal analysis of SVCT2-GFP subcellular localization in HEK-293 cells. Seven equally interspersed optical slices, selected to encompass the complete height of the cells, from top to bottom, are shown. D, surface biotinylation of plasma membrane proteins in transfected HEK-293 cells. Proteins from untreated (Total extract), biotinylated (Total extract + Biotin), and biotin-avidin-treated cells (Biotin-avidin) were separated by SDS-PAGE, transferred to a PVDF membrane, incubated with anti-GFP and a secondary HRP-coupled antibody, and revealed by chemiluminescence. Samples were strictly processed in parallel (biotinylation, avidin selection, separation by SDS-PAGE and transfer to PVDF membrane, incubation with primary and secondary antibodies, and analysis by chemiluminescence). The film containing the final image was cut in pieces and reordered for clarity of presentation. Numbers to the left correspond to the migration of molecular size markers. The arrowheads to the right indicate the migration of the main SVCT2-GFP band at about 90 kDa. E, surface biotinylation of the triple mutant H203Q/H206Q/H413Q. Experimental details were as described in D. F, time course ascorbic acid (AA) transport assay 48 h after transfection in HEK-293 cells expressing SVCT2-GFP or the different mutants. Cells expressing SVCT2-GFP or GFP alone were used as the positive and negative controls, respectively. Transport data correspond to the means ± S.D. (error bars) of one experiment of two performed in triplicate. Other experimental details were as indicated under “Experimental Procedures.”