The N-terminal Region of the Transmembrane Domain of Human Erythrocyte Band 3

Abstract

We studied the role of the N-terminal region of the transmembrane domain of the human erythrocyte anion exchanger (band 3; residues 361-408) in the insertion, folding, and assembly of the first transmembrane span (TM1) to give rise to a transport-active molecule. We focused on the sequence around the 9-amino acid region deleted in Southeast Asian ovalocytosis (Ala-400 to Ala-408), which gives rise to nonfunctional band 3, and also on the portion of the protein N-terminal to the transmembrane domain (amino acids 361-396). We examined the effects of mutations in these regions on endoplasmic reticulum insertion (using cell-free translation), chloride transport, and cell-surface movement in Xenopus oocytes. We found that the hydrophobic length of TM1 was critical for membrane insertion and that formation of a transport-active structure also depended on the presence of specific amino acid sequences in TM1. Deletions of 2 or 3 amino acids including Pro-403 retained transport activity provided that a polar residue was located 2 or 3 amino acids on the C-terminal side of Asp-399. Finally, deletion of the cytoplasmic surface sequence G 381 LVRD abolished chloride transport, but not surface expression, indicating that this sequence makes an essential structural contribution to the anion transport site of band 3.The red cell anion exchanger (band 3, AE1) is composed of two distinct domains: a C-terminal transmembrane domain predicted to span the membrane up to 14 times and a Nterminal cytosolic domain (1-4). The transmembrane domain mediates chloride-bicarbonate exchange, whereas the cytosolic domain associates peripheral proteins with the membrane.Southeast Asian ovalocytosis (SAO) 1 is caused by the heterozygous presence of an abnormal band 3 (band 3 SAO), which has a deletion of 9 amino acid residues (amino acids 400 -408) at the boundary between the cytosolic domain and the first transmembrane-spanning segment (5-7). SAO is prevalent in areas where malaria is endemic (8) and is thought to confer some protection against cerebral malaria (9). Homozygosity for band 3 SAO is probably lethal (10).Band 3 SAO is expressed at the surface of SAO red cells, but the mutant protein has no anion transport function and does not bind the anion transport inhibitor di-isothiocyanatodihydrostilbene either reversibly or irreversibly (11-13). Changes in di-isothiocyanatodihydrostilbene binding (14), N-glycosylation (15), and blood group antigen expression (16,17) indicate that the SAO deletion results in the misfolding of the transmembrane domain. The ␣-helical content of band 3 SAO is similar to normal band 3 (15, 18), but the transmembrane helices of band 3 SAO are disorganized while remaining folded (18).Transmembrane segment 1 (TM1) of SAO band 3 does not insert into microsomes in an alkali-stable fashion, unlike TM1 of normal band 3 (19). However, TMs 1-3 and larger fragments of SAO band 3 are incorporated stably into microsomal membranes. Lys-430, which is located in the first extracellular loop of band 3 between TM1 and TM2, is l...

Role of NH2 and COOH termini in targeting, stability, and activity of sodium bicarbonate cotransporter 1

“…NBC1 has 10 transmembrane-spanning domains with a long NH 2 and a short COOH terminus located intracellularly (31). Previous studies of other members of the bicarbonate superfamily, anion exchangers (AEs), have provided insights into the role of the NH 2 and the COOH termini on structure and function of the exchangers (14,28,33,37,39). In these studies, the NH 2 and COOH termini play an important role in regulation, localization, dimerization, and function of AEs.…”

“…In these studies, the NH 2 and COOH termini play an important role in regulation, localization, dimerization, and function of AEs. The NH 2 terminus of NBC1 contains conserved amino acids found in anion exchanger 2 (AE2) necessary for pH sensing (28) and contains a stretch of conserved amino acids found in AE1 necessary for activity (14). The NH 2 terminus of AE1 is also necessary for interaction with other cytoplasmic loops of AE1 in erythrocytes (14).…”

“…The NH 2 terminus of AE1 is also necessary for interaction with other cytoplasmic loops of AE1 in erythrocytes (14). Furthermore, crystallographic structure analysis of the NH 2 terminus of AE1 showed that its NH 2 terminus is important for cytoskeletal protein binding and dimerization (37) and is necessary for membrane insertion and transport activity (14).The COOH terminus of NBC1 is very hydrophilic and is believed to be involved in protein-protein interaction and targeting to the plasma membrane (20). It contains 15 consecutive positively charged residues, 12 of which are lysine.…”

“…The NH 2 terminus of AE1 is also necessary for interaction with other cytoplasmic loops of AE1 in erythrocytes (14). Furthermore, crystallographic structure analysis of the NH 2 terminus of AE1 showed that its NH 2 terminus is important for cytoskeletal protein binding and dimerization (37) and is necessary for membrane insertion and transport activity (14).…”

“…The NH 2 terminus of NBC1 contains conserved amino acids found in anion exchanger 2 (AE2) necessary for pH sensing (28) and contains a stretch of conserved amino acids found in AE1 necessary for activity (14). The NH 2 terminus of AE1 is also necessary for interaction with other cytoplasmic loops of AE1 in erythrocytes (14). Furthermore, crystallographic structure analysis of the NH 2 terminus of AE1 showed that its NH 2 terminus is important for cytoskeletal protein binding and dimerization (37) and is necessary for membrane insertion and transport activity (14).…”