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 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.
Fujinaga
J
J
Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Tang
X B
XB
Casey
J R
JR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D014162:Transfection
D001457:Anion Exchange Protein 1, Erythrocyte; D017920:Antiporters; D001639:Bicarbonates; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides