Functional expression of rat GLUT 1 glucose transporter in Dictyostelium discoideum.
To facilitate expression of the rat GLUT 1 glucose transporter cDNA in Dictyostelium discoideum, we mutated the 5' end of the coding sequence such that the codons for the first ten amino acids conformed to preferred Dictyostelium codon usage. As determined by Western-blot analysis, a population of Dictyostelium transformed with the mutated cDNA expressed nonglycosylated GLUT 1 protein. Cell lines expressing GLUT 1 transport radiolabelled 2-deoxy-D-glucose at a rate 6-10 times that of cell lines transformed with vector alone. The initial rate of inward transport of 2-deoxy-D-glucose was stimulated several-fold by the presence of unlabelled glucose in the Dictyostelium cytoplasm, exemplifying the trans-activation of GLUT 1 transport characteristic of GLUT 1 present in erythrocyte membranes. The K(m) and Ki values for 2-deoxy-D-glucose, D-glucose, D-mannose and D-galactose were 3.7 mM, 2.6 mM, 11 mM and 30 mM respectively, similar to the values for GLUT 1 expressed in mammalian cells. L-Glucose and L-galactose, which are not transported by GLUT 1, do not inhibit uptake of 2-deoxy-D-glucose in Dictyostelium expressing GLUT 1. Thus, even though GLUT 1 expressed in Dictyostelium is not N-glycosylated, it transports hexoses normally; this is the first example of functional expression of a mammalian transport protein in this lower eukaryote.
Cohen
N R
NR
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
Knecht
D A
DA
Lodish
H F
HF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004023:Dictyostelium / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D051272:Glucose Transporter Type 1; D006601:Hexoses / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D003062:Codon; D017931:DNA Primers; D018076:DNA, Complementary; D051272:Glucose Transporter Type 1; D006601:Hexoses; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; C495679:Slc2a1 protein, rat