Cysteine-directed cross-linking localizes regions of the human erythrocyteanion-exchange protein (AE1) relative to the dimeric interface

Citation
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
Citations number
38
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMICAL JOURNAL
ISSN journal
02646021 → ACNP
Volume
359
Year of publication
2001
Part
3
Pages
661 - 668
Database
ISI
SICI code
0264-6021(20011101)359:<661:CCLROT>2.0.ZU;2-8
Abstract
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.