Abstract
Anion exchanger 1 (AE1) is the chloride/bicarbonate exchange protein of the erythrocyte membrane. By using a combination of introduced cysteine mutants and sulfhydryl-specific chemistry, we have mapped the topology of the human AE1 membrane domain. Twenty-seven single cysteines were introduced throughout the Leu708-Val911 region of human AE1, and these mutants were expressed by transient transfection of human embryonic kidney cells. On the basis of cysteine accessibility to membrane-permeant biotin maleimide and to membrane-impermeant lucifer yellow iodoacetamide, we have proposed a model for the topology of AE1 membrane domain. In this model, AE1 is composed of 13 typical transmembrane segments, and the Asp807-His834 region is membrane-embedded but does not have the usual alpha-helical conformation. To identify amino acids that are important for anion transport, we analyzed the anion exchange activity for all introduced cysteine mutants, using a whole cell fluorescence assay. We found that mutants G714C, S725C, and S731C have very low transport activity, implying that this region has a structurally and/or catalytically important role. We measured the residual anion transport activity after mutant treatment with the membrane-impermeant, cysteine-directed compound, sodium (2-sulfonatoethyl)methanethiosulfonate) (MTSES). Only two mutants, S852C and A858C, were inhibited by MTSES, indicating that these residues may be located in a pore-lining region.
Highlights
Brain, retina, and heart [1]
Cysteine Accessibility to Biotin Maleimide—The membranepermeant compound biotin maleimide reacts covalently with sulfhydryls to introduce a biotin group, which can be readily detected on a blot, using streptavidin-biotinylated horseradish peroxidase followed by chemiluminescence
Intracellular (Y892C) and extracellular (Y555C) control mutants were readily labeled by biotin maleimide, indicating that differences in reactivity were not related to their intra- or extracellular localization but rather to their exposure to the aqueous environment
Summary
Brain, retina, and heart [1]. All members of the anion exchanger family consist of two domains, an N-terminal cytoplasmic domain and a 55-kDa C-terminal membrane domain. Our goal is to map the topology of the human AE1 membrane domain, using substituted cysteine mutants and sulfhydrylspecific chemistry. Introduced cysteine mutants were incubated with biotin maleimide, with or without a preincubation of the cells with LYIA. By contrast, introduced cysteine residues in the aqueous phase, either intracellular or extracellular, were labeled by biotin maleimide. A preincubation of the cells with the membrane-impermeant compound LYIA had little effect on the biotinylation of intracellular cysteines but impaired the biotinylation of most of the extracellular cysteine residues [4]. Twenty-seven single cysteine mutants of human AE1 were constructed between residues Leu708 and Val911 Analysis of these mutants formed the basis for a topology model of human AE1 membrane domain (Fig. 1). Our finding highlights the importance of the Lys851-Pro868 region for the anion transport function
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