Orientation of the glucose transporter in the human erythrocyte membrane. Investigation by in situ proteolytic dissection.
Glucose transporter proteins (zone 4.5) which had been photoaffinity labeled with [3H]cytochalasin B in human erythrocyte ghosts were subjected to enzymatic dissection in order to study the transmembrane disposition of the protein in situ. Proteolytic enzymes as well as glycosidases were used to treat unsealed and resealed ghosts in order to explore the various membrane domains of the transporter in a topographically defined manner. Limited digestion of sealed ghosts with trypsin had no effect on the apparent Mr of the transporter (55,000). Similar treatment in unsealed ghosts, however, resulted in the generation of a major fragment of 21.5 kDa, along with several minor fragments. Thermolysin also had no effect on sealed ghosts but caused a complete loss of radiolabel from the zone 4.5 region with no lower-molecular-weight fragments being retained on the gel. Chymotrypsin treatment resulted in the generation of a single peak, Mr = 18,400, in both sealed and unsealed ghosts indicating its action occurs at the outer surface. Digestion with carboxypeptidase and aminopeptidase indicate the C-terminal end of the transporter is located exterior to the membrane with the N terminus located at the cytoplasmic surface. Treatment with endoglycosidase resulted in a shift of mobility of the transporter to a lower Mr of 49,000. The results obtained indicate that the carbohydrate is located near the C-terminal end and that the cytochalasin B-binding site is located near the cytoplasmic N-terminal end.
D000626:Aminopeptidases / Q000378:metabolism; D001665:Binding Sites; D018826:CD13 Antigens; D002268:Carboxypeptidases / Q000378:metabolism; D002352:Carrier Proteins / Q000097:blood*; D020789:Cathepsin C; D002918:Chymotrypsin / Q000378:metabolism; D003571:Cytochalasin B / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000032:analysis*; D006026:Glycoside Hydrolases; D006801:Humans; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D002352:Carrier Proteins; D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase; D002268:Carboxypeptidases; D000626:Aminopeptidases; D018826:CD13 Antigens; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C; D002918:Chymotrypsin; D014357:Trypsin; D013820:Thermolysin