Mutation of invariant cysteines of mammalian metallothionein alters metal binding capacity, cadmium resistance, and 113Cd NMR spectrum.

Using a yeast expression vector system, we have expressed both wild type and six mutated Chinese hamster metallothionein coding sequences in a metal-sensitive yeast strain in which the endogenous metallothionein gene has been deleted. The mutant proteins have single or double cysteine to tyrosine replacements (C13Y, C50Y, and C13,50Y), single cysteine to serine replacements (C13S and C50S), or a single cysteine to alanine replacement (C50A). These proteins function in their yeast host in cadmium detoxification to differing extents. Metallothioneins which contain a cysteine mutation at position 50 (C50Y, C50S, C50A, and C13,50Y) conferred markedly less cadmium resistance than wild type metallothionein, or metallothionein with a single cysteine mutation at position 13 (C13Y and C13S). Wild type and three of the mutant Chinese hamster metallothioneins (C13Y, C50Y, and C13,50Y) were purified from yeast grown in subtoxic levels of either CdCl2 or 113CdCl2. All three of the mutant proteins bound less cadmium than the wild type protein when metal-binding stoichiometries were determined. The one-dimensional 113Cd NMR spectrum of the recombinant wild type Chinese hamster metallothionein was compared to the spectra of native rat and rabbit liver metallothioneins. The close correspondence between the 113Cd chemical shifts in these metallothioneins is consistent with the presence of two separate metal clusters, A and B, corresponding, respectively, to the alpha- and beta-domains, in the recombinant metallothionein. The one-dimensional 113Cd NMR spectra recorded on each of the three mutant metallothioneins, on the other hand, provide some indication as to the structural basis for the reduced, by one, metal stoichiometry of each of the mutant metallothioneins. For the C13Y mutant, it appears that the beta-domain now binds a total of two metal ions whereas with the C50Y mutant, the alpha-domain appears metal-deficient. For the double mutant, C13,50Y, the 113Cd resonances are indicative of major structural reorganizations in both domains.     

The CUP2 gene product regulates the expression of the CUP1 gene, coding for yeast metallothionein.

The yeast CUP1 gene codes for a copper-binding protein similar to metallothionein. Copper sensitive cup1s strains contain a single copy of the CUP1 locus. Resistant strains (CUP1r) carry 12 or more multiple tandem copies. We isolated 12 ethyl methane sulfonate-induced copper sensitive mutants in a wild-type CUP1r parental strain, X2180-1A. Most mutants reduce the copper resistance phenotype only slightly. However, the mutant cup2 lowers resistance by nearly two orders of magnitude. We cloned CUP2 by molecular complementation. The smallest subcloned fragment conferring function was approximately 2.1 kb. We show that CUP2, which is on chromosome VII, codes for or controls the synthesis or activity of a protein which binds the upstream control region of the CUP1 gene on chromosome VIII. Mutant cup2 cells produced extremely low levels of CUP1-specific mRNA, with or without added copper ions and lacked a factor which binds to the CUP1 promoter. Integrated at the cup2 site, the CUP2 plasmid restored the basal level and inducibility of CUP1 expression and led to reappearance of the CUP1-promoter binding factor. Taken collectively, our data establish CUP2 as a regulatory gene for expression of the CUP1 metallothionein gene product.     

Structural and functional studies of the amino terminus of yeast metallothionein

Purified yeast copper-metallothionein lacks 8 amino-terminal residues that are predicted from the DNA sequence of its gene. The removed sequence is unusual for metallothionein in its high content of hydrophobic and aromatic residues and its similarity to mitochondrial leader sequences. To study the significance of this amino-terminal cleavage, several mutations were introduced into the metallothionein coding gene, CUP1. One mutant, which deletes amino acid residues 2-8, had a minor effect on the ability of the molecule to confer copper resistance to yeast but did not affect CUP1 gene regulation. A second mutation, which changes two amino acids adjacent to the cleavage site, blocked removal of the extension peptide but had no effect on copper detoxification or gene regulation. Immunofluorescence studies showed that both the wild-type and these two mutant proteins are predominantly cytoplasmic with no evidence for mitochondrial localization. The cleavage site mutation allowed isolation and structural characterization of a full length metallothionein polypeptide. The copper content and luminescent properties of this molecule were identical to those of the truncated wild-type protein indicating a homologous cluster structure. Moreover, the amino-terminal peptide was selectively removed by various endopeptidases and an exopeptidase suggesting that it does not participate in the tertiary fold. These results argue that the amino-terminal peptide is not required for either the structural integrity or biological function of yeast metallothionein.     

Regulation of the yeast metallothionein gene

To study regulation of the yeast CUP1 gene, we have employed plasmids containing the CUP1 regulatory sequences fused to the Escherichia coli galK gene. A comparison of galK expression from low- and high-copy-number CUP1/galK fusion plasmids demonstrated that both basal and induced levels of galactokinase (GalK) increase proportionately with plasmid copy number. Host strains with an amplified, single or deleted CUP1 locus were compared to look for effects of chromosomal CUP1 gene dosage on expression from the episomal CUP1 promoter. Basal GalK levels are similar in CUP1R and cupls hosts, but can be induced to higher levels in the cup1s than the CUP1R host. In contrast, in a strain deleted for the chromosomal copy of CUP1, synthesis of GalK is constitutive but can be induced to yet higher levels by copper. A hybrid vector, placing the CUP1 coding sequence under the control of a constitutive promoter, was constructed. Introduction of this hybrid CUP1 gene into the deletion host containing the CUP1/galK plasmid restores regulation. Thus, metallothionein, in trans, can effect repression of the CUP1 promoter. The possible roles of metallothionein and free copper in CUP1 regulation are discussed.     

The requirement for yeast superoxide dismutase is bypassed through mutations in BSD2, a novel metal homeostasis gene.

Oxygen toxicity in Saccharomyces cerevisiae strains lacking superoxide dismutase can be suppressed through mutations in either the BSD1 or BSD2 gene. In this report, we demonstrate that the BSD2 gene normally functions in the homeostasis of heavy metal ions. A mutation in BSD2 not only reverses the aerobic defects of yeast strains lacking superoxide dismutase but also is associated with an increased sensitivity to copper and cadmium toxicity and an elevation in copper ion accumulation. The BSD2 gene was cloned by functional complementation and is predicted to encode a novel 37.5-kDa protein with three potential transmembrane domains. The mutant bsd2-1 allele was isolated and found to contain a single C-to-T transition changing a centrally located proline to a serine. This substitution results in total inactivation of BSD2, since the bsd2-1 mutation is identical to a bsd2 delta gene deletion in phenotype. BSD2 is expressed in yeast cells as a 1.5-kb mRNA. Although the gene functions in copper detoxification, BSD2 is not induced by copper ions, as is the case with S. cerevisiae metallothioneins. A probable role for copper ions in the bsd2 reversal of oxidative damage is discussed.     

Efficient expression of the yeast metallothionein gene in Escherichia coli.

The yeast metallothionein gene CUP1 was cloned into a bacterial expression system to achieve efficient, controlled expression of the stable, unprocessed protein product. The Escherichia coli-synthesized yeast metallothionein bound copper, cadmium, and zinc, indicating that the protein was functional. Furthermore, E. coli cells expressing CUP1 acquired a new, inducible ability to selectively sequester heavy metal ions from the growth medium.