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

J Biol Chem. 1999 Mar 5;274(10):6626-33. doi: 10.1074/jbc.274.10.6626.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Anion Exchange Protein 1, Erythrocyte / metabolism
  • Antiporters / chemistry*
  • Antiporters / genetics*
  • Antiporters / metabolism
  • Bicarbonates / metabolism
  • Chloride-Bicarbonate Antiporters
  • Chlorides / metabolism
  • Erythrocyte Membrane / metabolism
  • Erythrocytes / metabolism*
  • Humans
  • Molecular Sequence Data
  • Mutation
  • Peptide Mapping
  • Transfection

Substances

  • Anion Exchange Protein 1, Erythrocyte
  • Antiporters
  • Bicarbonates
  • Chloride-Bicarbonate Antiporters
  • Chlorides