Functional expression of tagged human Na+-glucose cotransporter in Xenopus laevis oocytes.
1. High-affinity, secondary active transport of glucose in the intestine and kidney is mediated by an integral membrane protein named SGLT1 (sodium glucose cotransporter). Though basic properties of the transporter are now defined, many questions regarding the structure- function relationship of the protein, its biosynthesis and targeting remain unanswered. In order to better address these questions, we produced a functional hSGLT1 protein (from human) containing a reporter tag. 2. Six constructs, made from three tags (myc, haemaglutinin and poly-His) inserted at both the C- and N-terminal positions, were thus tested using the Xenopus oocyte expression system via electrophysiology and immunohistochemistry. Of these, only the hSGLT1 construct with the myc tag inserted at the N-terminal position proved to be of interest, all other constructs showing no or little transport activity. A systematic comparison of transport properties was therefore performed between the myc-tagged and the untagged hSGLT1 proteins. 3. On the basis of both steady-state (affinities for substrate (glucose) and inhibitor (phlorizin) as well as expression levels) and presteady-state parameters (transient currents) we conclude that the two proteins are functionally indistinguishable, at least under these criteria. Immunological detection confirmed the appropriate targeting of the tagged protein to the plasma membrane of the oocyte with the epitope located at the extracellular side. 4. The myc-tagged hSGLT1 was also successfully expressed in polarized MDCK cells. alpha-Methylglucose uptake studies on transfected cells showed an exclusively apical uptake pathway, thus indicating that the expressed protein was correctly targeted to the apical domain of the cell. 5. These comparative studies demonstrate that the myc epitope inserted at the N-terminus of hSGLT1 produces a fully functional protein while other epitopes of similar size inserted at either end of the protein inactivated the final protein.
Bissonnette
P
P
Groupe de Recherche en Transport Membranaire (GRTM), Departement de Physiologie, Montreal, Quebec, Canada. bissonn@poste.umontreal.ca
Noël
J
J
Coady
M J
MJ
Lapointe
J Y
JY
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D006639:Histidine; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008757:Methylglucosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism; D009865:Oocytes; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010695:Phlorhizin / Q000494:pharmacology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1; D014162:Transfection; D014982:Xenopus laevis
D000939:Epitopes; D006388:Hemagglutinins; D008562:Membrane Glycoproteins; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; C033223:polyhistidine; D006639:Histidine; C027020:methylglucoside; D010695:Phlorhizin