Topology of the membrane domain of human erythrocyte anion exchange protein, AE1

Anion exchanger 1 (AE1) is the chloride/bicarbonate exchange protein of the erythrocyte membrane. By using a combination of introduced cysteine mutant s and sulfhydryl-specific chemistry, we have mapped the topology of the hum an AE1 membrane domain. Twenty-seven single cysteines were introduced throu ghout the Leu(708)-Val(911) region of human AE1, and these mutants were exp ressed by transient transfection of human embryonic kidney cells. On the ba sis 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 Asp(807)-His(834) region is memb rane-embedded but does not have the usual alpha-helical conformation. To id entify 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 transpo rt activity after mutant treatment with the membrane-impermeant, cysteine-d irected 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.