Title : Topology of the region surrounding
Glu681 of human
AE1 protein , the erythrocyte anion exchanger
Abstract :
- 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