Carboxy-terminal vesicular stomatitis virus G protein-tagged intestinal Na+-dependent glucose cotransporter (SGLT1): maintenance of surface expression and global transport function with selective perturbation of transport kinetics and polarized expression.
The Na+-dependent glucose transporter (SGLT1) mediates absorption of luminal glucose by the intestine. However, available intestinal cell lines that recapitulate a monolayer phenotype only express SGLT1 at low levels. Thus, to facilitate studies of the biology of SGLT1 function in epithelial monolayers, we engineered an epitope-tagged construct containing the YTDIEMNRLGK sequence (from the vesicular stomatitis virus G protein). The tag was placed at the carboxyl terminus since this is the least conserved portion of SGLT1. Transiently transfected COS-1 cells demonstrated surface expression of the immunoreactive protein and enhanced Na+-dependent glucose uptake that was phloridzin-sensitive (a specific competitive inhibitor of SGLT1). However, subsequent detailed analyses of epitope-tagged SGLT1 using stably transfected clones derived from the Caco-2 human intestinal epithelial cell line revealed substantial effects of the epitope on critical functions of SGLT1. When compared with native SGLT1 transfectants, the apparent Km for sugar transport was increased 23-fold (313 microM to 7.37 mM for native versus epitope-tagged SGLT1). In contrast, the apparent KNa for epitope-tagged SGLT1 was similar to that for native SGLT1. Permeabilization studies indicated that the C-terminal epitope tag was intracellular and thus could not directly disrupt extracellular ligand-binding sites. Immunolocalization and functional assays designed to detect polarized surface expression indicated that epitope tagging resulted in loss of apical targeting and enrichment of basolateral expression. Functional isolation of the small apical pool of epitope-tagged SGLT1 (by selective inhibition of basolateral epitope-tagged SGLT1) revealed that, despite the documented kinetic alterations in sugar transport, epitope-tagged SGLT1 could promote absorptive Na+ currents. These data show that 1) the C terminus of SGLT1 is intracellular; 2) disruption of protein structure by addition of a C-terminal tag leads to selective modifications of SGLT1 function; 3) the kinetics of sugar transport can be altered independently of influences on the Na+-binding site of SGLT1; and 4) the weak basolateral targeting sequence present within the epitope tag is dominant over endogenous SGLT1 apical targeting information and can direct polytopic membrane protein localization. The data also caution that subtle effects of foreign sequences must be considered when epitope tagging polytopic membrane proteins.
Turner
J R
JR
Division of Gastrointestinal Pathology, Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Lencer
W I
WI
Carlson
S
S
Madara
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D002522:Chlorocebus aethiops; D003110:Colonic Neoplasms; D004848:Epithelium / Q000502:physiology; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000378:metabolism*; D008757:Methylglucosides / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D015202:Protein Engineering; D021382:Protein Sorting Signals / Q000096:biosynthesis; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D016324:Sequence Tagged Sites; D012964:Sodium / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1; D014162:Transfection; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000378:metabolism*
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; D014759:Viral Envelope Proteins; C027020:methylglucoside; D012964:Sodium; D005947:Glucose