The role of cysteine residues in glucose-transporter-GLUT1-mediated transport and transport inhibition.
The role of cysteine residues in transport function of the glucose transporter GLUT1 was investigated by a mutagenesis-expression strategy. Each of the six cysteine residues was individually replaced by site-directed mutagenesis. Expression of the heterologous wild-type or mutant glucose transporters and transport measurements at two hexose concentrations (50 microM and 5 mM) were undertaken in Xenopus oocytes. The catalytic activity of GLUT1 was retained, despite substitution of each single cysteine residue, which indicated that no individual residue is essential for hexose transport. This finding questions the involvement of oligomerization or intramolecular stabilization by a single disulphide bond as a prerequisite for transporter activation under basal conditions. Application of the impermeant mercurial thiol-group-reactive reagent p-chloromercuribenzenesulphonate (pCMBS) to the external or internal surface of plasma membrane demonstrated that cysteine-429, within the sixth external loop, and cysteine-207, at the beginning of the large intracellular loop which connects transmembrane segments 6 and 7, are the residues which are involved in transport inhibition by impermeant thiol-group-reactive reagents from either side of the cell. These data support the predicted membrane topology of the transport protein by transport measurements. If residues other than the cysteines at positions 429 or 207 are exposed to either side of the plasma membrane by conformational changes, they do not contribute to the transport inhibition by pCMBS. Application of pCMBS to one side of the plasma membrane also inhibited transport from the opposite direction, most likely due to the hindrance of sugar-induced interconversion of transporter conformation.
Wellner
M
M
Institut für Pharmakologie, Freien Universität Berlin, Federal Republic of Germany.
Monden
I
I
Keller
K
K
D002731:4-Chloromercuribenzenesulfonate; D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism*; D018076:DNA, Complementary; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D014981:Xenopus
D018076:DNA, Complementary; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D002731:4-Chloromercuribenzenesulfonate; D003545:Cysteine