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 alpha-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).
Xia
Xiaobing
X
Beijing Institute for Infectious Diseases, Beijing 100039, China. xiaobingmpi@yahoo.com
Wang
Gang
G
Peng
Yanchun
Y
Jen
Jimmy
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1; D013329:Structure-Activity Relationship
D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; D003545:Cysteine