Cys351 and Cys361 of the Na+/glucose cotransporter are important for both function and cell-surface expression

Here, we identify Cys351 and Cys361 as novel residues critical for the function and plasma membrane targeting of the Na+/glucose transporter-1 (SGLT1). HEK-293 cells expressing the C351A and C361A mutants showed no detectable Na+-coupled uptake for α-methyl glucoside (AMG). Cell-surface biotinylation and Western blot revealed that the two mutants were overexpressed in 293 cells; however, none of them exhibited normal cell-surface expression. When reconstituted in proteoliposomes, mutant SGLT1s demonstrated significantly lower affinity for AMG compared with the wild-type transporter. Incubation with the reducing agent dithiothreitol did not alter the catalytic activity of wild-type protein, but surprisingly, it nearly restored the ability of SGLT1-C351A and -C361A to bind and translocate AMG. Thus, the C351A and C361A mutations might cause a global reorganization of the disulfide bonds of SGLT1. Furthermore, we showed that a double mutation (C351A/C361A) restored the cell-surface expression of the single C-to-A mutants (C351A and C361A).