Transmembrane folding of the human erythrocyte anion exchanger (AE1, Band 3) determined by scanning and insertional N-glycosylation mutagenesis

The human erythrocyte anion exchanger (AE1, Band 3) contains up to 14 trans membrane segments, with a single site of N-glycosylation at Asn(642) in ext racellular (EC) loop 4. Scanning and insertional N-glycosylation mutagenesi s were used to determine the folding pattern of AE1 in the membrane. Full-l ength AE1, when expressed in transfected human embryonic kidney (HEK)-293 o r COS-7 cells, retained a high-mannose oligosaccharide structure. Scanning N-glycosylation mutagenesis of EC loop 4 showed that N-glycosylation accept or sites (Asn-Xaa-Ser/Thr) spaced 12 residues from the ends of adjacent tra nsmembrane segments could be N-glycosylated. An acceptor site introduced at position 743 in intracellular (IC) loop 5 that could be N-glycosylated in a cell-free translation system was not N-glycosylated in transfected cells. Mutations designed to disrupt the folding of this loop enhanced the level of N-glycosylation at Asn(743) in vitro. The results suggest that this loop might be transiently exposed to the lumen of the endoplasmic reticulum dur ing biosynthesis but normally folds rapidly, precluding N-glycosylation. EC loop 4 insertions into positions 428, 484, 754 and 854 in EC loops 1, 2, 6 and 7 respectively were efficiently N-glycosylated, showing that these reg ions were extracellular. EC loop 4 insertions into positions 731 or 785 wer e poorly N-glycosylated, which was inconsistent with an extracellular dispo sition for these regions of AE1. Insertion of EC loop 4 into positions 599 and 820 in IC loops 3 and 6 respectively were not N-glycosylated in cells, which was consistent with a cytosolic disposition for these loops. Inhibito r-affinity chromatography with 4-acetamido-4'-isothiocyanostilbene-2,2'-dis ulphonate (SITS)-Affi-Gel was used to assess whether the AE1 mutants were i n a native state. Mutants with insertions at positions 428, 484, 599, 731 a nd 785 showed impaired inhibitor binding, whereas insertions at positions 7 54, 820 and 854 retained binding. The results indicate that the folding of the C-terminal region of AE1 is more complex than originally proposed and t hat this region of the transporter might have a dynamic aspect.