Proteolytic cleavages of cytochalasin B binding components of band 4.5 proteins of the human red blood cell membrane.
The putative hexose transport component of Band 4.5 protein of the human erythrocyte membrane was covalently photolabelled with [3H]cytochalasin B. Its transmembrane topology was investigated by electrophoretically monitoring the effect of proteinases applied to intact erythrocytes, unsealed ghosts, and a reconstituted system. Band 4.5 was resistant to proteolytic digestion at the extracellular face of the membrane in intact cells at both high and low ionic strengths. Proteolysis at the cytoplasmic face of the membrane in ghosts or reconstituted vesicles resulted in cleavage of the transporter into two membrane-bound fragments, a peptide of about 30 kDa that contained its carbohydrate moiety, and a 20 000 kDa nonglycosylated peptide that bore the cytochalasin B label. Because it is produced by a cleavage at the cytoplasmic face and because the carbohydrate moiety is known to be exposed to the outside, the larger fragment must cross the bilayer. It has been reported that the Band 4.5 sugar transporter may be derived from Band 3 peptides by endogenous proteolysis, but the cleavage pattern found in the present study differs markedly from that previously reported for Band 3. Minimization of endogenous proteolysis by use of fresh cells, proteinase inhibitors, immediate use of ghosts and omission of the alkaline wash resulted in no change in the incorporation of [3H]cytochalasin B into Band 4.5, and no labelling of Band 3 polypeptides. These results suggest that the cytochalasin B binding component of Band 4.5 is not the product of proteolytic degradation of a Band 3 component.
D001798:Blood Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D003571:Cytochalasin B / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D033703:Nucleoside Transport Proteins; D011402:Pronase / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D001798:Blood Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033703:Nucleoside Transport Proteins; D003571:Cytochalasin B; D002918:Chymotrypsin; D014357:Trypsin; D011402:Pronase