title: Amino acid substitutions at tryptophan 388 and tryptophan 412 of the HepG2 (Glut1) glucose transporter inhibit transport activity and targeting to the plasma membrane in Xenopus oocytes.
abstract: All 6 tryptophan residues in the human HepG2-type glucose transporter (Glut1) were individually altered by site-directed mutagenesis to investigate the role of these residues in transport function. Tryptophan residues in positions 48, 65, 186, 363, 388, and 412 of Glut1 were changed to either a glycine or leucine residue. Mutant mRNAs were synthesized and injected into Xenopus laevis oocytes. Transporter function as assessed by uptake of 2-deoxy-D-[3H]glucose or transport of 3-O-[3H]methylglucose was decreased in the 388 and 412 mutants but was unaltered in all other mutants. The amount of the mutant transporters expressed in total membrane and plasma membrane fractions was measured using Glut1-specific antibodies. Calculation of the intrinsic transport activity of each of the mutants using these data demonstrated that the reduced transport activity of the 412 mutants was caused entirely by a dramatic decrease in the intrinsic activity of the mutant proteins whereas the reduced activity of the 388 mutants was a result of a decreased level of the protein in oocytes, decreased targeting to the plasma membrane, and a modest decrease in the intrinsic activity. Protease/glycosidase mapping of in vitro translation products indicated that the effects of the 388 and 412 point mutations could not be attributed to a disruption in the ability of the mutant proteins to insert properly into the membrane. The ID50 for cytochalasin B inhibition of 2-deoxyglucose uptake was increased from 5 x 10(-7) M for the wild-type Glut1 to 4 x 10(-6) M in the 388 mutants but was unaltered in the 412 mutants. These observations suggest that 1) Trp-412 may comprise part of a hexose binding site or is involved in maintaining a local tertiary structure critical for transport function; 2) Trp-388 is involved in stabilizing the equilibrium binding of cytochalasin B to the transporter. Trp-388 may therefore lie near a substrate binding site and also appears to participate in stabilization of local tertiary structure important for full catalytic activity and efficient targeting to the Xenopus plasma membrane.
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: J C
authors.initials: JC
authors.lastname: Garcia
authors.forename: M
authors.initials: M
authors.lastname: Strube
authors.forename: K
authors.initials: K
authors.lastname: Leingang
authors.forename: K
authors.initials: K
authors.lastname: Keller
authors.forename: M M
authors.initials: MM
authors.lastname: Mueckler
mesh_terms: D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003571:Cytochalasin B / Q000378:metabolism; D004247:DNA / Q000235:genetics; D003847:Deoxyglucose / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010063:Ovum / Q000378:metabolism; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D014364:Tryptophan / Q000378:metabolism*; D014981:Xenopus
chemical_list: D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D003571:Cytochalasin B; D014364:Tryptophan; D004247:DNA; D003847:Deoxyglucose; D006026:Glycoside Hydrolases; D010450:Endopeptidases