THE ROLE OF CYSTEINE RESIDUES IN THE ERYTHROCYTE PLASMA-MEMBRANE ANION-EXCHANGE PROTEIN, AE1

AE1 (Band 3), a congruent to 110-kDa integral plasma membrane protein, facilitates the electroneutral movement of Cl- and HCO3- across the erythrocyte membrane and serves as the primary attachment site for the erythrocyte spectrin-actin cytoskeleton. In this investigation, we have characterized the role of native cysteines in the function of AE1, We have constructed a mutant version of human AE1 (AE1C(-)) in which all five cysteines of AE1 were replaced with serines. Wild-type and AE1C(-) cDNAs were expressed by transient transfection of human embryonic kidney cells. Two of the mutated cysteines in AE1C(-) are in a region involved in ankyrin binding, and ankyrin binding has previously been shown to be sensitive to the oxidation state of these cysteines. However, the K-D values for ankyrin binding by AE1 and AE1C(-) were indistinguishable, suggesting that AE1 cysteines are not essential components of the ankyrin-binding site. Using size exclusion chromatography, both AE1 and AE1C(-) were found to associate as a mixture of dimers and high molecular mass complexes. The rate of anion exchange by AE1C(-), as measured in a reconstituted microsome sulfate transport assay, was indistinguishable from that by AE1 and was inhibited by 4,4'-diisothiocyanodihydrostilbene- 2,2'-disulfonate. We conclude that the cysteines of AE1 are not required for the anion exchange or cytoskeletal binding roles of the protein.