Copper metallothionein of yeast, structure of the gene, and regulation of expression.

Abstract

Addition of copper to yeast cells leads to the induction of a low molecular weight, cysteine-rich protein that binds copper. This protein, termed copper chelatin or thionein, is related to the metallothionein family of proteins that are induced in response to cadmium and zinc in vertebrate cells. We have determined the structure of the yeast copperbinding protein by DNA sequence analysis of the gene. Although the 6573-dalton yeast protein is substantially divergent from vertebrate metallothioneins, the arrangement of 12 cysteine residues, which is a hallmark of metal-binding proteins, is partially conserved. We analyzed the regulatory DNA sequence of the gene by fusing it with the Escherichia coli galactokinase gene and assaying the levels of enzyme activity in yeast in response to copper. The transcriptional activation has a specific requirement for copper. Zinc, cadmium, and gold were unable to regulate the galactokinase activity. The yeast copper metallothionein regulatory sequences represent a previously unreported class of yeast promoter that is regulated by copper.Methallothioneins (MTs) are thought to play a central role in the protection against heavy metal toxicity and zinc homeostasis (see ref. 1 for a review). Recent interest in the molecular biology of MT stems from the observation that the genes for mammalian MT are regulated by heavy metals as well as by glucocorticoids (2-3). MT genes from several different mammalian systems such as mouse, Chinese hamster, monkey, and human have been cloned and sequenced (4-7).Although the precise mechanism of MT gene activation by heavy metals has not been elucidated, a DNA sequence upstream of the 5' end of the mouse MT I gene, which is responsible for cadmium inducibility, has been identified (8, 9 Recently we cloned a copper-inducible gene from a CUpir strain and showed that it protects a cupP strain from copper toxicity (14). The restriction map of this clone indicated that it is identical to the CUP] gene previously cloned and characterized by Fogel and Welch (12). To provide a basis for understanding the function and regulation of this gene, we determined the primary structure of the coding region and flanking sequences. We show that it encodes a protein with both similarities to and differences from the MTs of higher eukaryotes. We refer to this protein as copper metallothionein (Cu-MT) because it belongs to the metallothionein family of proteins as defined in ref. Fig. 4). Growth 3332The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Mechanisms for activating Cu- and Zn-containing superoxide dismutase in the absence of the CCS Cu chaperone

“…We used a genetic approach to identify the transacting factor(s) responsible for CCS-independent activation of hSOD1. Small Cu-binding proteins were examined, including the Cu carriers encoded by S. cerevisiae ATX1 and COX17 (34), as well as the CUP1 and CRS5 Cu metallothioniens (35,36). However, deletion of these various genes had no effect on CCSindependent activation of hSOD1 (data not shown).…”

Interactions of fungi with toxic metals

“…This protein normally functions to maintain low concentrations of intracellular copper ions and thus prevent futile transcription of the CUPl structural gene (Wright, Hamer & McKenney, 1988). Although yeast metallothionein can also bind Cd and Zn in vitro, it is not transcriptionally induced by these ions and apparently does not protect against them (Butt et al, 1984;Winge et al, 1985).…”

“…The disomic chromosomes exhibit differential patterns of CUPl gene amplification which indicates that the copper resistasnce mechanism involves not only amplification of the CUPl locus on the chromosomes but also disomy or aneuploidy of chromosome VIII (Fogel et al, 1983 ;Butt & Ecker, 1987). The DNA fragments of the CUPl locus that confer copper resistance have been cloned (Fogel & Welch, 1982;Butt et al, 1984;Henderson, Cox & Tubb, 1985) and evidence that the CUPl locus was present as multiple copies in CUPl strains was obtained after restriction enzyme analysis of the cloned DNA (Butt & Ecker, 1987;Fogel et al, 1988). The basic repeating unit 'CUPl locus' is composed of 2 0 kb DNA fragments containing, with respect to restriction enzymes, a unique Xba I site and two sites for Kpn 1 and Sau3A.…”

Structure of the galactokinase gene ofEscherichia coli, the last (?) gene of thegaloperon

“…6 for a review). The use of galK as a selective marker has also been adapted to develop gene fusion vectors to study transcriptional regulatory elements in the gram-positive bacteria Streptomyces (7), yeast (8)(9)(10)(11) and higher cell systems (5,(12)(13)(14)(15).…”