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 KD 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.
Casey
J R
JR
Department of Biological Sciences, Stanford University, California 94305-5020, USA.
Ding
Y
Y
Kopito
R R
RR
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000838:Anions; D017487:Ankyrins / Q000378:metabolism; D001692:Biological Transport; D002460:Cell Line; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D017487:Ankyrins; D003545:Cysteine