Effect of mutation of cysteinyl residues in yeast Cu-metallothionein.
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.
Thrower
A R
AR
Department of Medicine and Biochemistry, University of Utah Medical Center, Salt Lake City 84132.
Byrd
J
J
Tarbet
E B
EB
Mehra
R K
RK
Hamer
D H
DH
Winge
D R
DR
D002352:Carrier Proteins; D003300:Copper / Q000378:metabolism / Q000494:pharmacology*; D003545:Cysteine; D005796:Genes; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008668:Metallothionein / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development
C500769:CUP1-1 protein, S cerevisiae; D002352:Carrier Proteins; C007121:copper thionein; D003300:Copper; D008668:Metallothionein; D003545:Cysteine