Topology of the region surrounding Glu681 of human AE1 protein, the erythrocyte anion exchanger.
AE1 protein transports Cl- and HCO3- across the erythrocyte membrane by an electroneutral exchange mechanism. Glu681 of human AE1 may form part of the anion translocation apparatus and the permeability barrier. We have therefore studied the structure of the sequence surrounding Glu681, using scanning cysteine mutagenesis. Residues of the Ser643 (adjacent to the glycosylation site) to Ser690 region of cysteineless mutant (AE1C-) were replaced individually with cysteine. The ability of mutants to mediate Cl-/HCO3- exchange in transfected HEK293 cells revealed that extracellular mutants, W648C, I650C, P652C, L655C, and F659C have an important role in transport. By contrast, only transmembrane mutation E681C fully blocked anion exchange activity. The topology of the region was investigated by comparing cysteine labeling with the membrane-permeant cysteine-directed reagent 3-(N-maleimidylpropionyl)biocytin, with or without prior labeling with membrane-impermeant lucifer yellow iodoacetamide (LYIA). Two regions readily label with 3-(N-maleimidylpropionyl)biocytin (Ser643-Met663 and Ile684-Ser690). We propose that poorly labeled Met664-Gln683 corresponds to transmembrane segment 8 of AE1. Regions Ser643-Met663 and Ile684-Ser690 localize, respectively, to extracellular and intracellular sites on the basis of accessibility to LYIA. On the basis of LYIA accessibility, we propose that the Arg656-Met663 region forms a "vestibule" that leads anions to the transport channel. Glu681 is located 3 amino acids from the C terminus of transmembrane segment 8, which places the membrane permeability barrier within 5 A of the intracellular surface of the membrane.
Tang
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XB
Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Fujinaga
J
J
Kopito
R
R
Casey
J R
JR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D014867:Water / Q000737:chemistry
D001457:Anion Exchange Protein 1, Erythrocyte; D014867:Water; D018698:Glutamic Acid; D003545:Cysteine