title: Properties of the human erythrocyte glucose transport protein are determined by cellular context.
abstract: Human erythrocyte hexose transfer is mediated by the glucose transport protein GLUT1 and is characterized by a complexity that is unexplained by available hypotheses for carrier-mediated sugar transport [Cloherty, E. K., Heard, K. S., and Carruthers, A. (1996) Biochemistry 35, 10411-10421]. The study presented here examines the possibility that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae (RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1), mutant GLUT1 (GLUT1(338)(-)(A3)), or carboxy-terminal hemagglutinin-polyHis-tagged GLUT1 (GLUT1-HA-H6). GLUT1 and GLUT1-HA-H6 are expressed at the yeast cell membrane and restore 2-deoxy-d-glucose, 3-O-methylglucose, and d-glucose transport capacity to RE700A. GLUT1-HA-H6 confers GLUT1-specific sugar transport characteristics to transfected RE700A, including inhibition by cytochalasin B and high-affinity transport of the nonmetabolized sugar 3-O-methylglucose. GLUT1(338)(-)(A3), a catalytically inactive GLUT1 mutant, is expressed but fails to restore RE700A sugar uptake capacity or growth on glucose. In contrast to transport in human red cells, K(m(app)) for 2-deoxy-d-glucose uptake equals K(i(app)) for 2-deoxy-d-glucose inhibition of 3-O-methylglucose uptake. Unlike transport in human red cells or transport in human embryonic kidney cells transfected with GLUT1-HA-H6, unidirectional sugar uptake in RE700A-GLUT1-HA-H6 is not inhibited by reductant and is not stimulated by intracellular sugar. Net uptake of subsaturating 3-O-methylglucose by RE700A-GLUT1-HA-H6 is a simple, first-order process. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, UMass Medical School, 364 Plantation Street, Worcester, Massachusetts 01605, USA.
authors.forename: Kara B
authors.initials: KB
authors.lastname: Levine
authors.forename: Trista K
authors.initials: TK
authors.lastname: Robichaud
authors.forename: Stephanie
authors.initials: S
authors.lastname: Hamill
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Sultzman
authors.forename: Anthony
authors.initials: A
authors.lastname: Carruthers
mesh_terms: D019943:Amino Acid Substitution; D001693:Biological Transport, Active; D002451:Cell Compartmentation; D004912:Erythrocytes / Q000378:metabolism*; D017353:Gene Deletion; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D051272:Glucose Transporter Type 1; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D009004:Monosaccharide Transport Proteins / Q000097:blood* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D014162:Transfection
chemical_list: D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; C495677:SLC2A1 protein, human; D005947:Glucose