Differential targeting of glucose transporter isoforms heterologously expressed in Xenopus oocytes.
We have examined the subcellular distribution of three members of the human glucose transporter family expressed in oocytes from Xenopus laevis. Following injection of in vitro-transcribed mRNA encoding the transporter isoform to be studied, we have determined the subcellular localization of the expressed protein by immunofluorescence and by subcellular fractionation coupled with immunoblotting using specific anti-peptide antibodies. We have shown that both the liver-type (GLUT 2) and brain-type (GLUT 3) glucose transporters are expressed predominantly in the plasma membranes of oocytes, and in both cases high levels of glucose transport activity are exhibited. In contrast, the insulin-regulatable glucose transporter (GLUT 4) is localized predominantly to an intracellular membrane pool, and the levels of transport activity recorded in oocytes expressing GLUT 4 are correspondingly lower. The localization of the different transporter isoforms to distinct subcellular fractions mirrors the situation observed in their native cell type and thus demonstrates that oocytes may prove to be a useful system with which to study the targeting signals for this important class of membrane proteins. In addition, the determination of the amounts of the transporters expressed per oocyte together with a knowledge of their Km values has allowed us to estimate the turnover numbers of these transporters. Insulin was without effect on glucose transport in oocytes expressing any of these transporter isoforms. Microinjection of guanosine 5'-[gamma-thio]triphosphate into oocytes expressing GLUT 4 was also without effect on the transport rate.
Thomas
H M
HM
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Takeda
J
J
Gould
G W
GW
D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D003847:Deoxyglucose / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D051273:Glucose Transporter Type 2; D051274:Glucose Transporter Type 3; D051275:Glucose Transporter Type 4; D006801:Humans; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D009865:Oocytes / Q000378:metabolism* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D014982:Xenopus laevis
D051273:Glucose Transporter Type 2; D051274:Glucose Transporter Type 3; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C495687:SLC2A3 protein, human; C495692:SLC2A4 protein, human; D003847:Deoxyglucose