J. Biol. Chem., Vol. 263, Issue 15, 7037-7042, May, 1988
Effect of mutation of cysteinyl residues in yeast Cu-metallothionein
AR Thrower, J Byrd, EB Tarbet, RK Mehra, DH Hamer and DR Winge
Department of Medicine and Biochemistry, University of Utah Medical Center, Salt Lake City 84132.
Metallothioneins have been isolated from Saccharomyces cerevisiae CUP1
mutants generated by Wright et al. (Wright, C. F., Hamer, D. H., and
McKenney, K. (1986) Nucleic Acids Res. 14, 8489-8499). In the mutant
metallothioneins, pairs of cysteinyl residues have been converted to seryl
residues. The mutant proteins differ only in the positions of the double
substitutions; each mutant molecule contains 10 cysteinyl residues. Each
mutant protein lacks the first 8 residues at the amino terminus from the
decoded gene sequence of the CUP1 locus. Mutant molecules consist of 53
residues analogous to the wild-type metallothionein and are designated
9/11, 24/26, 36/38, and 49/50 (in reference to the sequence positions of
the Cys----Ser conversions). The properties of the mutant metallothioneins
are vastly different, and host cells harboring the different
plasmid-encoded mutant molecules show marked differences in sensitivity to
CuSO4. Growth inhibition was observed at CuSO4 concentrations up to mM in
cells containing the 9/11, 24/26, and 36/38 molecules, but not for cells
containing protein 49/50. A CuSO4 concentration of 5 mM was required to
inhibit the growth of yeast containing either 49/50 or the wild-type
metallothionein. In the purified proteins the copper binding stoichiometry
of each molecule, except protein 24/26, was nearly 8 mol eq. Protein 24/26
bound 5.5 copper ions/molecule. The Cu(I) chelator bathocuproine
disulfonate reacted with over 50% of the copper ions in proteins 9/11,
24/26, and 36/38, but less than 10% of the copper ions in proteins 49/50
and wild- type metallothionein were reactive. The thiolates in 9/11, 24/26,
and 36/38 were also more reactive in a disulfide exchange reaction with
dithiodipyridine compared with the sulfhydryls in 49/50 and the wild- type
molecules. The four mutant copper proteins are luminescent and exhibit a
similar quantum yield. The cluster structures contributing to the
particular electronic transitions are markedly more sensitive to oxygen in
proteins 9/11, 24/26, and 36/38 compared with 49/50 and the wild-type
molecules. The air-sensitive proteins exhibit a tertiary fold not
recognized by polyclonal antibodies directed to a conformational epitope on
yeast Cu-metallothionein. Protein 49/50 cross-reacts with the antibody in a
concentration-dependent fashion similar to the wild- type protein. Mutation
of 2 cysteinyl residues in the carboxyl portion of metallothionein does not
significantly alter properties of the molecule, whereas mutation of several
cysteines in the amino-terminal portion of the molecule yields a different
conformation.
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