Roles of the four cysteine residues in the function of the integral inner membrane Hg2+-binding protein, MerC.

The roles of the four cysteine residues of the integral inner membrane Hg2+-binding protein, MerC, have been examined using site-directed mutagenesis. Residues Cys-22 and Cys-25 have previously been predicted to lie within the membrane. Substitution of each of these residues in turn with alanine resulted in complete abolition of specific Hg2+ uptake by vesicles. In contrast, substitution by alanine of the other two cysteine residues, Cys-127 and Cys-132, predicted to lie with within a C-terminal cytoplasmic tail, did not significantly affect Hg2+ uptake. Since previous results indicated that native MerC tends to form intermolecular disulfide-bonded dimers, the effects of these substitutions on dimer formation were also examined. Only the Cys-127 and Cys-132 variants spontaneously formed significant amounts of disulfide-bonded dimer. Further experiments using copper-1,10-phenanthroline indicated that each variant with an unpaired cysteine residue was more susceptible to dimer formation than native MerC. Copyright 1999 Academic Press.