Copper-specific Transcriptional Repression of Yeast Genes Encoding Critical Components in the Copper Transport Pathway

Abstract

Copper is an essential micronutrient that is toxic in excess. To maintain an adequate yet non-toxic concentration of copper, cells possess several modes of control. One involves copper uptake mediated by genes encoding proteins that play key roles in high affinity copper transport. These include the FRE1-encoded Cu 2؉ /Fe 3؉ reductase and the CTR1 and CTR3-encoded membraneassociated copper transport proteins. Each of these genes is transcriptionally regulated as a function of copper availability: repressed when cells are grown in the presence of copper and highly activated during copper starvation. Our data demonstrate that repression of CTR3 transcription is exquisitely copper-sensitive and specific. Although copper represses CTR3 gene expression at picomolar metal concentrations, cadmium and mercury down-regulate CTR3 expression only at concentrations 3 orders magnitude greater. Furthermore, copper-starvation rapidly and potently induces CTR3 gene expression. We demonstrate that the CTR1, CTR3, and FRE1 genes involved in high affinity copper uptake share a common promoter element, TTTGCTC, which is necessary for both copper repression and copper-starvation activation of gene expression. Furthermore, the Mac1p is essential for down-or up-regulation of the copper-transport genes. In vivo footprinting studies reveal that the cis-acting element, termed CuRE (copperresponse element), is occupied under copper-starvation and accessible to DNA modifying agents in response to copper repression, and that this regulated occupancy requires a functional MAC1 gene. Therefore, yeast cells coordinately express genes involved in high affinity copper transport through the action of a common signaling pathway.

Dynamic Regulation of Copper Uptake and Detoxification Genes in Saccharomyces cerevisiae

“…The high-affinity Cu(I) transport genes CTR1, CTR3, and FRE1 are transcriptionally downregulated by Cu ions and induced by Cu ion starvation. This Cu ion-dependent regulation requires a wild-type Mac1p Cu metalloregulatory transcription factor (CuMRTF) (15,24,29,48). Regulation of these genes by Mac1p is highly specific for and exquisitely sensitive to Cu ions (29).…”

“…This Cu ion-dependent regulation requires a wild-type Mac1p Cu metalloregulatory transcription factor (CuMRTF) (15,24,29,48). Regulation of these genes by Mac1p is highly specific for and exquisitely sensitive to Cu ions (29). Deletion of the MAC1 gene results in Cu ion starvation phenotypes similar to those associated with deletions in the CTR1 and CTR3 genes, which can be corrected by added Cu ions.…”

“…In mac1⌬ strains, transcription of CTR1 and CTR3 is undetectable and the FRE1 gene is transcribed at low levels. In addition, yeast strains which possess a dominant MAC1 up1 allele exhibit high basal levels of CTR1, CTR3, and FRE1 mRNAs, a lack of Cu-dependent repression of CTR1 and CTR3 (29), and hypersensitivity to Cu ions (24). The mutations in Mac1p that lead to a dominant MAC1 up1 allele all map to the first of two cysteine clusters found in the carboxyl-terminal half of Mac1p (16,24,48,56).…”

Stalking metal-linked dimers

“…Eukaryotic Cu(I)-responsive transcription factors include AceI and MacI of the organism Saccharomyces cerevisiae. Ace1 activates the transcription of copper sequestration and detoxification genes under conditions of copper excess [3][4][5], while Mac1 activates the Ctr1 and Ctr3 high-affinity copper uptake transporters under conditions of copper depletion [6,7]. In a similar vein, copper(I) has been shown to regulate the CopY repressors in bacteria.…”

“…1. CXCX (4)(5)(6) CXC motif in CopY homologs. Phylogenetic tree of amino acid sequences similar to E. hirae CopY, obtained by BLASTing (cutoff evalue of 0.00001) CopY to 496 complete bacterial genome sequences.…”