Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface.
The human erythrocyte anion-exchanger isoform 1 (AE1) is a dimeric membrane protein that exchanges chloride for bicarbonate across the erythrocyte plasma membrane. Crystallographic studies suggest that the transmembrane anion channel lies at the interface between the two monomers, whereas kinetic analysis provides evidence that each monomer contains an anion channel. We have studied the structure-function relationship of residues at the dimeric interface of AE1 by cysteine-directed cross-linking. Single cysteine mutations were introduced in 16 positions of putative loop regions throughout AE1. The ability of these residues to be chemically cross-linked to their partner within the dimeric protein complex was assessed by mobility of the protein on immunoblots. Introduced cysteine residues in extracellular loops (ECs) 1-4 and intracellular loop 1 formed disulphide cross-linked dimers. Treatment with homobifunctional maleimide cross-linkers of different lengths (6, 10 and 16 A; 1 A identical with 0.1 nm) also cross-linked AE1 with introduced cysteines in EC5 and close to the start of transmembrane segment (TM) 1. On the basis of these data, tentative positional constraints of TMs 1-4 and 6 relative to the dimeric interface are proposed. Neither disulphide- nor maleimide-mediated cross-linking perturbed AE1 transport function, suggesting that loop-loop contacts across the dimeric interface are not primarily responsible for allosteric interactions between monomers within the functional dimeric protein complex.
Taylor
A M
AM
Departments of Physiology and Biochemistry, Membrane Transport Group and CIHR Group in Molecular Biology of Membrane Proteins, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Zhu
Q
Q
Casey
J R
JR
D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D006801:Humans; D008301:Maleimides / Q000737:chemistry; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014162:Transfection
D001457:Anion Exchange Protein 1, Erythrocyte; D003432:Cross-Linking Reagents; D008301:Maleimides; C043592:maleimide; D001710:Biotin; D003545:Cysteine