Abstract
Anion exchanger 1 (AE1) is the major erythrocyte membrane protein that mediates chloride/bicarbonate exchange across the erythrocyte membrane facilitating CO2 transport by the blood, and anchors the plasma membrane to the spectrin-based cytoskeleton. This multi-protein cytoskeletal complex plays an important role in erythrocyte elasticity and membrane stability. An in-frame AE1 deletion of nine amino acids in the cytoplasmic domain in a proximity to the membrane domain results in a marked increase in membrane rigidity and ovalocytic red cells in the disease Southeast Asian Ovalocytosis (SAO). We hypothesized that AE1 has a flexible region connecting the cytoplasmic and membrane domains, which is partially deleted in SAO, thus causing the loss of erythrocyte elasticity. To explore this hypothesis, we developed a new non-denaturing method of AE1 purification from bovine erythrocyte membranes. A three-dimensional (3D) structure of bovine AE1 at 2.4 nm resolution was obtained by negative staining electron microscopy, orthogonal tilt reconstruction and single particle analysis. The cytoplasmic and membrane domains are connected by two parallel linkers. Image classification demonstrated substantial flexibility in the linker region. We propose a mechanism whereby flexibility of the linker region plays a critical role in regulating red cell elasticity.
Highlights
Anion exchanger 1 (AE1) is a major membrane protein in erythrocytes (25 to 30% of the total membrane mass) that mediates anion exchange and participates in control of the erythrocyte shape [1,2,3,4,5,6]
By single particle electron microscopy (EM) reconstruction we show that the AE1 dimer has an elongated structure consisting of a double-humped cytoplasmic domain and an oval-shaped membrane domain tethered by two linkers
Purification of Bovine AE1 AE1 in the erythrocyte membrane exists either in a free state or as a multi-proteitn complex with other erythrocyte proteins. Previous investigators extracted both pools of AE1, and used alkaline and chaotropic agents to strip off accessory proteins that have been shown to significantly change the native structure of AE1 [1,24]
Summary
AE1 is a major membrane protein in erythrocytes (25 to 30% of the total membrane mass) that mediates anion exchange and participates in control of the erythrocyte shape [1,2,3,4,5,6]. The C-terminal membrane domain is involved in mediating anion exchange. The N-terminal cytoplasmic domain provides binding sites for many proteins, including ankyrin, band 4.2 and band 4.1 proteins, glycolytic enzymes, hemoglobin, deoxyhemoglobin, hemichromes, p72syk protein tyrosine kinase, adducin and integrin-linked kinase [2,3,6,14,15]. It is widely accepted that interactions between AE1 and the membrane cytoskeleton play a role in mediating erythrocyte shape control [2,3,14,15,16]. Quantitative deficiency of AE1 resulting in decreased anchoring of the lipid bilayer to the membrane cytoskeleton leads to loss of membrane cohesion and resultant membrane surface area loss and generation of spherocytic red cells in hereditary spherocytosis [17]. A qualitative defect resulting in an in-frame deletion of nine amino acids in the cytoplasmic domain of AE1 [2,18], results in a marked increase in membrane rigidity and ovalocytic red cells in Southeast Asian Ovalocytosis (SAO)
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