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

Simon Labbé,Zhiwu Zhu,Dennis J Thiele

doi:10.1074/jbc.272.25.15951

Simon Labbé, Zhiwu Zhu + Show 1 more

Open Access

https://doi.org/10.1074/jbc.272.25.15951

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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 Cu2+/Fe3+ reductase and the CTR1 and CTR3-encoded membrane-associated 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 (copper-response 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.

Highlights

Copper is an essential trace element that is required for a number of cellular enzymes including cytochrome c oxidase, Cu/Zn-superoxide dismutase, lysyl oxidase, and dopamine-␤monooxygenase [1]
S. cerevisiae cells acquire copper as Cu(I) under high affinity conditions through the action of a plasma membrane-associated Cu(II)-Fe(III) reductase activity encoded by the FRE1 gene [24, 25] and two high affinity copper transport proteins encoded by the CTR1 [26] and CTR3 [27] genes
Metal Ion Specificity and Sensitivity of CTR3 mRNA Downregulation—Previous investigations have demonstrated that all three yeast genes known to be involved in high affinity copper transport, CTR1, CTR3, and FRE1, are repressed by copper at the level of steady-state mRNA [10, 27]

Summary

This study

Consistent with the involvement of the FRE1encoded Cu(II)-Fe(III) reductase in both high affinity copper and iron transport, the expression of this gene is repressed by both copper and iron [24, 25]. A dominant mutation in a duplicated region rich in cysteine exhibits high levels of Cu(II) reductase activity and FRE1 mRNA that are virtually not repressed by copper [25] These characteristics of Mac1p, as well as the nuclear localization of a Mac1-␤-galactosidase fusion protein [32], suggests that Mac plays a key role in copper homeostasis. In vivo footprinting studies reveal that the cis-acting element, termed CuRE (copper-response element), is occupied under copper-starvation and accessible to DNA modifying agents in response to copper repression, and that this copper-responsive occupation of the CuREs requires a functional MAC1 gene Taken together these results show that yeast cells coordinately express genes involved in high affinity copper transport through the action of a common signaling pathway to acquire this micronutrient from the environment

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DISCUSSION