Am. Taylor et al., Cysteine-directed cross-linking localizes regions of the human erythrocyteanion-exchange protein (AE1) relative to the dimeric interface, BIOCHEM J, 359, 2001, pp. 661-668
The human erythrocyte anion-exchanger isoform. 1 (AE1) is a dimeric membran
e protein that exchanges chloride for bicarbonate across the erythrocyte pl
asma membrane. Crystallographic studies suggest that the transmembrane anio
n channel lies at the interface between the two monomers, whereas kinetic a
nalysis provides evidence that each monomer contains an anion channel. We h
ave studied the structure-function relationship of residues at the dimeric
interface of AE1 by cysteine-directed cross-linking.. Single cysteine mutat
ions were introduced in 16 positions of putative loop regions throughout AE
1. The ability of these residues to be chemically cross-linked to their par
tner within the dimeric protein complex was assessed by mobility of the pro
tein on immunoblots. Introduced cysteine residues in extracellular loops (E
Cs) 1-4 and intracellular loop 1 formed disulphide cross-linked dimers. Tre
atment with homobifunctional maleimide cross-linkers of different lengths (
6, 10 and 16 Angstrom; 1 Angstrom drop 0.1 nm) also cross-linked AE1 with i
ntroduced 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 disulphi
de- nor maleimide-mediated cross-linking perturbed AE1 transport function,
suggesting that loop-loop contacts across the dimeric interface are not pri
marily responsible for allosteric interactions between monomers within the
functional dimeric protein complex.