Abstract
Protection from copper toxicity in the bakers' yeast Saccharomyces cerevisiae involves the action of a copper binding metallothionein encoded by the CUP1 locus. To identify additional factors contributing to copper ion homeostasis and detoxification, we screened a genomic library for genes that confer high levels of copper resistance to yeast strains lacking CUP1. This screen led to the identification of the CRS5 (copper-resistant suppressor) gene. By sequence analyses, CRS5 encodes a small molecular weight cysteine-rich protein with an amino acid sequence bearing all the features of a eukaryotic metallothionein. The CRS5 polypeptide exhibits a striking similarity to a number of mammalian and invertebrate metallothioneins, yet shares surprisingly little homology with CUP1. In yeast, CRS5 is expressed as a 0.5-kilobase mRNA that is regulated both by copper ions and by oxidative stress, and expression is dependent upon ACE1, a copper and DNA binding transcription factor also known to regulate CUP1. Deletion of the chromosomal CRS5 locus was found to increase cellular sensitivity to copper, but not cadmium, toxicity. These studies support an important role for the CRS5 metallothionein-like protein in copper homeostasis and detoxification.
Highlights
From the Division of Toxicological Sciences, Department of Environmental Health Sciences, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205
CRS5 encodes a small sitivity to cadmium toxicity has been associated with MT gene molecular weight cysteine-rich protein with an amino deletions in transgenic mice [8, 9]
The budding yeast, S. cereuisiae, has provided a valuable tool for genetically probing MT gene function and untilnow, this organism has been associated with just a single MT locus, CUPl [13, 14]
Summary
The nucleotide sequence(s1 reportedin this paper has been submitted to the GenBankTMIEMBLData Bank with accession number(s) L29056. $ To whom correspondence should be addressed. Strains DTY22 [17],DTY6O [27],and EG103 and EG133 [28] were constructed as described. VC-sp is a MATa Met’ Leu- derivative of 19.10b,a cupl”strain containing a single. The -abbreviations used are: MT, metallothioneins;kb, kilobase cuplA::URA3 strain; Ref. 16) to VC-sp and by isolating a Zeu2-.
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