Expression of the rat GLUT1 glucose transporter in the yeast Saccharomyces cerevisiae.
We expressed the rat GLUT1 facilitative glucose transporter in the yeast Saccharomyces cerevisiae with the use of a galactose-inducible expression system. Confocal immunofluorescence microscopy indicated that a majority of this protein is retained in an intracellular structure that probably corresponds to endoplasmic reticulum. Yeast cells expressing GLUT1 exhibited little increase in glucose-transport activity. We prepared a crude membrane fraction from these cells and made liposomes with this fraction using the freeze-thaw/sonication method. In this reconstituted system, D-glucose-transport activity was observed with a Km for D-glucose of 3.4 +/- 0.2 mM (mean +/- S.E.M.) and was inhibited by cytochalasin B (IC50= 0.44 +/- 0.03 microM), HgCl2 (IC50)= 3.5 +/- 0.5 microM), phloretin (IC50= 49 +/- 12 microM) and phloridzin (IC50= 355 +/- 67 microM). To compare these properties with native GLUT1 we made reconstituted liposomes with a membrane fraction prepared from human erythrocytes, in which the Km of D-glucose transport and ICs of these inhibitors were approximately equal to those obtained with GLUT1 made by yeast. When the relative amounts of GLUT1 in the crude membrane fractions were measured by quantitative immunoblotting, the specific activity of the yeast-made GLUT1 was 110% of erythrocyte GLUT1, indicating that GLUT1 expressed in yeast is fully active in glucose transport.
Kasahara
T
T
Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Tokyo, Japan.
Kasahara
M
M
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D018076:DNA, Complementary / Q000235:genetics; D004912:Erythrocytes / Q000378:metabolism; D005947:Glucose / Q000493:pharmacokinetics; D051272:Glucose Transporter Type 1; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D018076:DNA, Complementary; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human; C495679:Slc2a1 protein, rat; D005947:Glucose