Human erythrocyte glucose transporter: normal asymmetric orientation and function in liposomes.
The transport function and orientation of the reconstituted human erythrocyte glucose transporter was studied with liposomes made with bovine brain lipid or Escherichia coli lipid. Reconstitution was achieved by a simple octyl glucoside dilution method. The reconstituted transporters with either lipid showed identical counterflow transport activity, the same response to various inhibitors, and characteristic cytochalasin B (CB) labeling. Functional location and purification of the glucose transporter was performed by using gel-permeation high-performance liquid chromatography with octyl glucoside-containing buffer. The reconstituted transport activity was associated only with band 4.5 protein (preactin) and not with band 3 protein. Both CB binding and transport function of the reconstituted transporters were resistant to trypsin but susceptible to chymotrypsin digestion. However, both trypsin and chymotrypsin treatment of unsealed ghosts completely eliminated the CB labeling and transport function of the glucose transporter. In our reconstitution system the glucose transporters maintained a normal asymmetrical (right-side-out) orientation and good transport function. A specific monoclonal antibody against the glucose transporter inhibited CB labeling of the transporters on unsealed ghosts. This was not found with the reconstituted system; however, after freeze-thawing there was a significant inhibition of CB binding by the antibody. These findings suggest that the CB-binding site of the reconstituted transporter is on the inner side of the proteoliposomes.
D001665:Binding Sites; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003572:Cytochalasins / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli; D006801:Humans; D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D033703:Nucleoside Transport Proteins
D001798:Blood Proteins; D003572:Cytochalasins; D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033703:Nucleoside Transport Proteins