Investigation of the structure and function of the human erythrocyte glucose transporter by proteolytic dissection.
Tryptic and papain digestion have been employed to investigate the structure and function of the human erythrocyte glucose transporter. Trypsin cleaves the native protein into two large, membrane-embedded fragments and a number of small peptides that are released from the membrane. These fragments have been isolated and located within the transporter sequence by fast atom bombardment mass spectrometry and amino acid analysis. The results indicate that the segments of the sequence comprising residues 213-269 and 457-492 are cleaved from the cytoplasmic surface of the membrane by trypsin treatment. These findings are compatible with a model previously proposed for the arrangement of the polypeptide in the membrane (Mueckler, M., et al. (1985) Science 229, 941-945). Despite the loss of these 93 residues, the portion of the protein remaining embedded in the membrane is still able to bind cytochalasin B. This binding is inhibited by D-glucose, indicating that the membrane-embedded fragments retain the substrate-binding site. Fourier transform infrared spectroscopic analysis of the protein before and after proteolytic digestion shows that the intramembranous part of the protein is largely alpha-helical, although some beta-sheet structure appears also to be present. The spectroscopic findings also indicate that the extramembranous, cytoplasmic domain of the transporter, which is removed by trypsin, contains alpha-helical structure.
Department of Biochemistry and Chemistry, Royal Free Hospital School of Medicine, (University of London), U.K.
M T
MT
Cairns
J
J
Alvarez
M
M
Panico
A F
AF
Gibbs
H R
HR
Morris
D
D
Chapman
S A
SA
Baldwin
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003571:Cytochalasin B / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared; D014357:Trypsin
D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D003571:Cytochalasin B; D014357:Trypsin; D010206:Papain; D005947:Glucose