Probing transmembrane topology of the high-affinity Sodium/Glucose cotransporter (SGLT1) with histidine-tagged mutants.
To reexamine the existing predictions about the general membrane topology of the high-affinity Na+/glucose cotransporter (SGLT1) and in particular of the large loop at the C-terminal region, a small 6 x Histidine-tag was introduced at different positions of the SGLT1 sequence by site-directed mutagenesis. Eleven His-SGLT1 mutants were constructed and were transiently transfected into COS-7 cells. As demonstrated by immunofluorescent labeling with antipeptide antibodies against SGLT1, all mutants were expressed and inserted into the plasma membrane. Only mutants with the tag in the N-terminal region and the C-terminal region retained Na+/glucose cotransport activity at 0.1 mM D-glucose. The arrangement of the His-tag in the membrane was analyzed by indirect immunofluorescence, using a monoclonal antihistidine antibody. In nonpermeabilized cells the His-tag could be detected at the N-terminal end (insertion at aa 5) and at the C-terminal end (replacement between aa 584-589 and between aa 622-627), suggesting that these portions of the polypeptide are accessible from the extracellular space. Furthermore, an epitope-specific antibody directed against aa 606-630 reacted strongly with the cell surface. To support this topology intact stably transfected SGLT1 competent CHO cells were partially digested with an immobilized trypsin and subsequently subjected to electrophoresis and Western blot analysis. The size of the digestion product suggests that extravesicular trypsin removed the extracellular loop that contains the amino acid residues 549-664. Thus our results indicate that the last large loop (about aa 541-aa 639) towards the C-terminal end faces the cell exterior where it might be involved in substrate recognition.
Lin
J
J
Abteilung Epithelphysiologie, Max-Planck-Institut für molekulare Physiologie, Otto-Hahn-Str.11, 44202 Dortmund, Germany.
Kormanec
J
J
Homerová
D
D
Kinne
R K
RK
D000818:Animals; D000917:Antibody Formation; D000918:Antibody Specificity; D016466:CHO Cells / Q000648:ultrastructure; D019556:COS Cells; D006224:Cricetinae; D000939:Epitopes / Q000737:chemistry / Q000648:ultrastructure; D015870:Gene Expression; D006639:Histidine / Q000235:genetics*; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry / Q000648:ultrastructure; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000648:ultrastructure*; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000276:immunology; D011487:Protein Conformation / Q000187:drug effects; D051269:Sodium-Glucose Transporter 1; D014162:Transfection; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology
D000939:Epitopes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D010455:Peptides; D051269:Sodium-Glucose Transporter 1; D006639:Histidine; D014357:Trypsin