Topology of transmembrane segments 1–4 in the human chloride/bicarbonate anion exchanger 1 (AE1) by scanning N-glycosylation mutagenesis

Abstract

Human AE1 (anion exchanger 1), or Band 3, is an abundant membrane glycoprotein found in the plasma membrane of erythrocytes. The physiological role of the protein is to carry out chloride/bicarbonate exchange across the plasma membrane, a process that increases the carbon-dioxide-carrying capacity of blood. To study the topology of TMs (transmembrane segments) 1-4, a series of scanning N-glycosylation mutants were created spanning the region from EC (extracellular loop) 1 to EC2 in full-length AE1. These constructs were expressed in HEK-293 (human embryonic kidney) cells, and their N-glycosylation efficiencies were determined. Unexpectedly, positions within putative TMs 2 and 3 could be efficiently glycosylated. In contrast, the same positions were very poorly glycosylated when present in mutant AE1 with the SAO (Southeast Asian ovalocytosis) deletion (DeltaA400-A408) in TM1. These results suggest that the TM2-3 region of AE1 may become transiently exposed to the endoplasmic reticulum lumen during biosynthesis, and that there is a competition between proper folding of the region into the membrane and N-glycosylation at introduced sites. The SAO deletion disrupts the proper integration of TMs 1-2, probably leaving the region exposed to the cytosol. As a result, engineered N-glycosylation acceptor sites in TM2-3 could not be utilized by the oligosaccharyltransferase in this mutant form of AE1. The properties of TM2-3 suggest that these segments form a re-entrant loop in human AE1.

NBCe1-A Transmembrane Segment 1 Lines the Ion Translocation Pathway

“…The 3-fold periodicity of biotinylation in this region suggests it may have an extended ␤-strand structure. In human AE1, TM2 and TM3 are initially expressed in the endoplasmic reticulum lumen and then fold back into the protein complex at a later stage of maturation (26). We speculate that this highly conserved region may act as a hinge for the proper folding of TMs during protein maturation.…”

“…ning mutagenesis study of human AE1 proposed the C-terminal end of TM1 is at residue Phe-423 (26) and that the GGLLG stretch is part of the first extracellular loop. To define the location of Thr-442 and the stretch of 421 ITFGGLLG 428 in NBCe1-A protein, we assessed the accessibility of introduced cysteine mutants to biotin maleimide (BM), a membrane-permeant sulfhydryl reactive reagent that was used previously for AE1 topology analysis (5).…”

A SLC4-like anion exchanger from renal tubules of the mosquito (Aedes aegypti): evidence for a novel role of stellate cells in diuretic fluid secretion

“…3A shows a hydrophobicity plot of the TM domain of human AE1, for which a detailed topology map exists (13,93,94), aligned with that of AeAE. The similar hydropathy profiles shared between the TM domains of AeAE and human AE1 indicate that these proteins likely share a similar membrane topology.…”

“…The similar hydropathy profiles shared between the TM domains of AeAE and human AE1 indicate that these proteins likely share a similar membrane topology. On the basis of the topology model for human AE1 (13,93,94), we assigned 13 putative TM segments (shaded bars in Fig. 3A) and 2 reentrant loops (R in Fig.…”

Topology of NBCe1 Protein Transmembrane Segment 1 and Structural Effect of Proximal Renal Tubular Acidosis (pRTA) S427L Mutation

“…The topology of NBCe1-A-TM1 was previously proposed based on the human anion exchanger 1 (AE1) (see Fig. 1B), and indeed, the C-terminal extracellular end of NBCe1-A-TM1 strongly matches that of AE1-TM1 (13,14). The AE1-TM1 is predicted to consist of 20 amino acids ranging from Gln-404 to Phe-423 (see Fig.…”