Yeast metallothionein function in metal ion detoxification

A genetic approach was taken to test the function of yeast metallothionein in metal ion detoxification. A yeast strain was constructed in which the metallothionein locus was deleted (cup1 delta). The cup1 delta strain was complemented with normal or mutant metallothionein genes under normal or constitutive regulatory control on high copy episomal plasmids. Metal resistance of the cup1 delta strain with and without the metallothionein-expressing vectors was analyzed. The normally regulated metallothionein gene conferred resistance only to copper (1000-fold); constitutively expressed metallothionein conferred resistance to both copper (500-fold) and cadmium (1000-fold), but not to mercury, zinc, silver, cobalt, nickel, gold, platinum, lanthanum, uranium, or tin. Two mutant versions of the metallothionein gene were constructed and tested for their ability to confer metal resistance in the cup1 delta background. The first had a deletion of a highly conserved amino acid sequence (Lys-Lys-Ser-Cys-Cys-Ser). The second was a hybrid gene consisting of the sequences coding for the first 20 amino acids of the yeast protein fused to the monkey metallothionein gene. Expression of these genes under the CUP1 promoter provided significant protection from copper, but none of the other metals tested. These results demonstrate that there is significant flexibility in the structural requirements for metallothionein to function in copper detoxification and that yeast metallothionein is also capable of detoxifying cadmium under conditions of constitutive expression.     

Copper ions and the regulation ofSaccharomyces cerevisiae metallothionein genes under aerobic and anaerobic conditions

We have previously reported that theSaccharomyces cerevisiae CRS5 metallothionein gene is negatively regulated by oxygen. The mechanism of this repression was the focus of the current study. We observed that the aerobic repression ofCRS5 is rapid and occurs within minutes of exposing anaerobic cultures to air. Furthermore, theCUP1 metallothionein gene ofS. cerevisiae was found to be subject to a similar down-regulation of gene expression. We provide evidence that the aerobic repression of yeast metallothioneins involves copper ions and Ace1, the coppertrans-activator ofCUP1 andCRS5 gene expression. A functional Ace1 binding site was found to be necessary for the aerobic repression ofCRS5. Moreover, the aerobic down-regulation of the metallothioneins was abolished when cells were treated with elevated levels of copper. Our studies show that anaerobic cultures accumulate higher levels of copper than do aerobic cells and that this copper is rapidly lost when cells are exposed to air. In fact, the kinetics of this copper loss closely parallels the kinetics ofCUP1 andCRS5 gene repression. The yeast metallothionein genes, therefore, serve as excellent markers for variations in copper accumulation and homeostasis that occur in response to changes in the oxidative status of the cell.     

Studies on metal resistance system inKluyveromyces marxianus

Through preliminary plate tests,Kluyveromyces marxianus was found to be much more resistant to toxic heavy metals compared to aCUP1 ^R strain ofSaccharomyces cerevisiae. Specific growth rate and maximum dry weights affected by increasing metal concentrations were determined to obtain precise patterns of resistance. Metal biosorption was also monitored during the course of growth in synthetic media containing respective metals at 0.5 mM final concentration. Although Zn- and Co-binding was negligible, as much as 90% of silver, 60% of copper, and 65% of cadmium were found to be absorbed by the end of active growth. Analysis of the protein profiles ofS. cerevisiae andK. marxianus on metal exposure suggested constitutive production of metallothionein inK. marxianus. Furthermore, a smaller protein synthesized byK. marxianus on induction by silver or cadmium accounts for the high resistance of the organism to these metals.     

Detoxification of cadmium Ultrastructural study and electron-probe microanalysis of the midgut in a cadmium-resistant strain ofDrosophila melanogaster

Summary The midgut of a cadmium-resistant strain ofDrosophila melanogaster has been studied at the ultrastructural level and by electronprobe microanalysis (EPMA). Chronic exposure to cadmium leads to a concentration of the metal in a lysosomal system developed in both anterior and posterior segments of the midgut, where it coexists with copper and sulfur. This mechanism apparently ensures a permanent cadmium detoxification and prevents cellular injury. Wild-type flies fed on a cadmium-contaminated medium manifest the same detoxification process. As a result of contamination, copper is stored along the entire length of the midgut, including a part of the middle-midgut previously named copper-accuumulating region. Our data demonstrate that the midgut, particularly the posterior segment, is an accumulative organ for both cadmium and copper. The involvement of the metallothionein system in the detoxification process is discussed.     

Copper ions and the regulation ofSaccharomyces cerevisiae metallothionein genes under aerobic and anaerobic conditions

We have previously reported that theSaccharomyces cerevisiae CRS5 metallothionein gene is negatively regulated by oxygen. The mechanism of this repression was the focus of the current study. We observed that the aerobic repression ofCRS5 is rapid and occurs within minutes of exposing anaerobic cultures to air. Furthermore, theCUP1 metallothionein gene ofS. cerevisiae was found to be subject to a similar down-regulation of gene expression. We provide evidence that the aerobic repression of yeast metallothioneins involves copper ions and Ace1, the coppertrans-activator ofCUP1 andCRS5 gene expression. A functional Ace1 binding site was found to be necessary for the aerobic repression ofCRS5. Moreover, the aerobic down-regulation of the metallothioneins was abolished when cells were treated with elevated levels of copper. Our studies show that anaerobic cultures accumulate higher levels of copper than do aerobic cells and that this copper is rapidly lost when cells are exposed to air. In fact, the kinetics of this copper loss closely parallels the kinetics ofCUP1 andCRS5 gene repression. The yeast metallothionein genes, therefore, serve as excellent markers for variations in copper accumulation and homeostasis that occur in response to changes in the oxidative status of the cell.     

Amplification of a gene for metallothionein by tandem repeat in a strain of cadmium-resistant yeast cells

Abstract In a cadmium-resistant strain of Saccharomyces cerevisiae , cells are protected against cadmium toxicity by the production of large amounts of cadmium-binding metallothionein, as occurs similarly in a copper-resistant strain. The apoprotein of the metallothionein is encoded by the CUP1 gene on chromosome VIII. The CUP1 gene is present as 8–10 copies in the cadmium-resistant strain as a result of tandem repeat of a 2.0-kb fragment of DNA that includes CUP1 , while the wild-type strain contains only a single copy of CUP1 . In the cadmium-resistant strain, some evidence for elongation of chromosome VIII with variations in length (maximum to 200 kb) was obtained. However, the elongation was not due to the tandem repeats of the CUP1 -containing region.     

The role of zinc transporters in cadmium and manganese transport in mammalian cells

To understand the mechanism of cadmium accumulation, it is important to know the precise mechanisms of transport systems for other metals. Recently, utilization of genomics and metallomics has clarified the involvement of specific metal transporter(s) in cadmium uptake. Studies with metallothionein (MT)-null cadmium-resistant cells have revealed the involvement of the manganese/zinc transport system in cadmium uptake. Genomic studies of strain differences in sensitivity to cadmium-induced testicular hemorrhage revealed that a zinc transporter, Zrt-, Irt-related protein (ZIP) 8 encoded by slc39a8, is responsible for the strain difference. Ectopic expression of ZIP8 in various cells enhanced the uptake of cadmium, manganese, and zinc. ZIP8-transgenic mice showed high expression of ZIP8 in the vasculature of testis and apical membrane of proximal tubules in kidney, and exhibited enhanced cadmium accumulation and toxicity when treated with cadmium. The expression of ZIP8 was found to be down-regulated in MT-null cadmium-resistant cells, in which the uptake rates of both cadmium and manganese were decreased. These data suggest that ZIP8 plays an important role in the uptake of both cadmium and manganese in mammalian cells. The role of ZIP14 in the uptake of cadmium and manganese is also 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.     

不同因素诱导豚鼠肝中金属硫蛋白的研究

通过皮下注射的方法诱导豚鼠产生金属硫蛋白（ＭＴ）,研究了重金属元素（Ｃｄ）、微量元素（Ｃｕ,Ｚｎ）及有机试剂（ＣＣｌ４,在体内可产生自由基）等因素的诱导与豚鼠肝脏中ＭＴ不同亚型的含量及金属结合状态的变化关系．实验结果表明,微量元素及有机试剂的诱导可使豚鼠肝脏中ＭＴ１的产量明显高于ＭＴ２,说明在体内ＭＴ１在参与微量元素的储存及清除自由基功能方面比ＭＴ２强．在重金属元素诱导下体内ＭＴ１对重金属元素的结合量远远大于ＭＴ２．表明ＭＴ１的重金属解毒能力比ＭＴ２强．上述实验结果与对不同亚型ＭＴ生物学功能差异的体外研究结果相吻合．此外,无论采用上述何种因素诱导,所得ＭＴ中均结合有Ｃｕ．对Ｃｕ在ＭＴ形成过程中的作用也进行了初步探讨．     

The binding of gold(I) to metallothionein

The binding of gold(I) to metallothionein, MT, has been unambiguously established by the reaction of Na₂AuTM with purified horse kidney MT. Zinc was displaced more readily than cadmium although the latter could be displaced using large Au/Cd ratios. The metal exchange reactions were complete within 2 hr of mixing. Further evidence that such reactions might be physiologically significant were obtained by studying in vitro metal displacements in the liver cytosol of in vivo metal treated rats: When Na₂AuTM was added to the cytosol of rats administered CdCl₂ in vivo, zinc, copper and cadmium were displaced in 2/1/1 ratios from the metallothionein fraction. The zinc and cadmium displacement provide direct evidence that the gold was binding to MT. Addition of Cd⁺² to liver cytosol of gold-treated rats resulted in displacement of copper and zinc, but not gold, from the MT fractions. When liver MT is prepared from rats exposed to Au or Cd, the Cd/protein ratio increased during the preparation, but the Au/protein ratio decreased. The Mt-bound metals account for 95% of the cytosolic Cd but only 15%–30% of the cytosolic gold in these studies. Thus, the nonspecific binding of gold to MT in vivo should be considered as one aspect in its equilibration among protein binding sites, which include, inter alia, metallothionein. Gold was found to coelute with zinc and cadmium in the MT fraction of rat kidney cytosol, when both Cd and Na₂AuTM were administered to the rats. The possible significance of gold binding to MT in the treatment of rheumatoid arthritis-chrysotherapy-is briefly discussed.     

The many highways for intracellular trafficking of metals

Metal ions such as copper and manganese represent a unique problem to living cells in that these ions are not only essential co-factors for metalloproteins, but are also potentially toxic. To aid in the homeostatic balance of essential but toxic metals, cells have evolved with a complex network of metal trafficking pathways. The object of such pathways is two-fold: to prevent accumulation of the metal in the freely reactive form (metal detoxification pathways) and to ensure proper delivery of the ion to target metalloproteins (metal utilization pathways). Much of what we currently know regarding these complex pathways of metal trafficking has emerged from molecular genetic studies in baker's yeast, Saccharomyces cerevisiae. In this review, we shall briefly highlight the current understanding of factors that function in the trafficking and handling of copper, including copper detoxification factors, copper transporters and copper chaperones. In addition, very recent findings on the players involved in manganese trafficking will be presented. The goal is to provide a paradigm for the intracellular handling of metals that may be applied in a more general sense to metals that serve essential functions in biology.Electronic Supplementary Material Supplementary material is available in the online version of this article Abbreviations CTR cell surface transporter - GSH glutathione - MCF mitochondrial carrier family - mito mitochondria - MT metallothionein - SOD superoxide dismutase