Cysteine-scanning mutagenesis of transmembrane segment 7 of the GLUT1 glucose transporter.
The human erythrocyte facilitative glucose transporter (Glut1) is predicted to contain 12 transmembrane spanning alpha-helices based upon hydropathy plot analysis of the primary sequence. Five of these helices (3, 5, 7, 8, and 11) are capable of forming amphipathic structures. A model of GLUT1 tertiary structure has therefore been proposed in which the hydrophilic faces of several amphipathic helices are arranged to form a central aqueous channel through which glucose traverses the hydrophobic lipid bilayer. In order to test this model, we individually mutated each of the amino acid residues in transmembrane segment 7 to cysteine in an engineered GLUT1 molecule devoid of all native cysteines (C-less). Measurement of 2-deoxyglucose uptake in a Xenopus oocyte expression system revealed that nearly all of these mutants retain measurable transport activity. Over one-half of the cysteine mutants had significantly reduced specific activity relative to the C-less protein. The solvent accessibility and relative orientation of the residues within the helix was investigated by determining the sensitivity of the mutant transporters to inhibition by the sulfhydryl directed reagent p-chloromercuribenzene sulfonate (pCMBS). Cysteine replacement at six positions (Gln(282), Gln(283), Ile(287), Ala(289), Val(290), and Phe(291)), all near the exofacial side of the cell membrane, produced transporters that were inhibited by incubation with extracellular pCMBS. Residues predicted to be near the cytoplasmic side of the cell membrane were minimally affected by pCMBS. These data demonstrate that the exofacial portion of transmembrane segment 7 is accessible to the external solvent and provide evidence for the positioning of this alpha-helix within the glucose permeation pathway.
Hruz
P W
PW
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Mueckler
M M
MM
D000818:Animals; D001483:Base Sequence; D003545:Cysteine; D051272:Glucose Transporter Type 1; D006801:Humans; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D014982:Xenopus laevis
D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D009841:Oligonucleotides; C495677:SLC2A1 protein, human; D003545:Cysteine