Synthesis and maturation of the erythrocyte anion transport protein--an internal sequence for membrane insertion

The biosynthesis of the erythrocyte anion transport glycoprotein, Band III (Mr 100,000), is of interest, as its N-terminal half is hydrophilic and faces the cytoplasmic surface; the C-terminal half spans the phospholipid bilayer several times. Band III is synthesized by erythroid precursor cells obtained from the spleens of anaemic mice. Newly synthesized Band III was inserted into rough endoplasmic reticulum membranes with an asymmetric orientation which resembled that of mature Band III in erythrocyte membranes: the N-terminal portion of the molecule facing the cytoplasm. Newly made Band III contained a high-mannose asparagine-linked oligosaccharide, which was susceptible to cleavage by endoglycosidase H. During the next 20-30 min, this oligosaccharide was processed to a form resistant to endoglycosidase H degradation, presumably in the Golgi complex. The processed Band III was subsequently expressed on the cell surface, at about 30-45 min after synthesis. To study the mechanism of insertion of Band III into microsomes, we used erythroid precursor cells from the spleens of anaemic mice as a source of messenger RNA for studies in vitro in the wheat germ and reticulocyte lysate cell-free system containing dog pancreatic microsomes. Immediately after synthesis, Band III was found to be inserted into microsomal membranes in its mature configuration, with the N-terminal portion exposed to the cytoplasm and its hydrophobic C-terminal portion spanning the lipid bilayer. The newly-synthesized Band III was also provided with a high-mannose asparagine-linked oligosaccharide. Band III was found to be inserted into dog pancreatic microsomes in a co-translational manner; in synchronized translation studies microsomes could be added as late as the time when the hydrophilic N-terminal half of the protein had been synthesized and still allow normal trans-membrane insertion and glycosylation. There is no cleavage of any N-terminal peptide during membrane insertion. In many respects, therefore, the biosynthesis of Band III resembles that of co-translationally-inserted proteins whose N-terminal portions are exposed on the exterior of the cell, like vesicular stomatitis virus glycoprotein, HLA-A antigens, and glycophorin. However, our results suggest that Band III contains a sequence near the middle of the protein which directs its insertion into endoplasmic reticulum membranes.