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Roles of the Four Cysteine Residues in the Function of the Integral Inner Membrane Hg2+-Binding Protein, MerC,☆☆

https://doi.org/10.1006/bbrc.1999.0192Get rights and content

Abstract

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.

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    Abbreviations used: SDS, sodium dodecyl sulfate; TRIS-Cl, Tris-(hydroxymethyl)aminomethane hydrochloride

    ☆☆

    Muller, F.

    1

    Present address: c/o J. Powlowski, Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Blvd., W., Montreal, Quebec, H3G 1M8 Canada. Fax: 514-848-2868; E-mail:[email protected].

    2

    To whom correspondence should be addressed.

    3

    Present address: Department of Surgery, University of Umeå, S-901 87 Umeå, Sweden.

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