Transcriptional Activation in Yeast in Response to Copper Deficiency Involves Copper-Zinc Superoxide Dismutase

Dennis J Thiele,L Kent Wood

doi:10.1074/jbc.m807027200

Abstract

Copper is an essential trace element, yet excess copper can lead to membrane damage, protein oxidation, and DNA cleavage. To balance the need for copper with the necessity to prevent accumulation to toxic levels, cells have evolved sophisticated mechanisms to regulate copper acquisition, distribution, and storage. In Saccharomyces cerevisiae, transcriptional responses to copper deficiency are mediated by the copper-responsive transcription factor Mac1. Although Mac1 activates the transcription of genes involved in high affinity copper uptake during periods of deficiency, little is known about the mechanisms by which Mac1 senses or responds to reduced copper availability. Here we show that the copper-dependent enzyme Sod1 (Cu,Zn-superoxide dismutase) and its intracellular copper chaperone Ccs1 function in the activation of Mac1 in response to an external copper deficiency. Genetic ablation of either CCS1 or SOD1 results in a severe defect in the ability of yeast cells to activate the transcription of Mac1 target genes. The catalytic activity of Sod1 is essential for Mac1 activation and promotes a regulated increase in binding of Mac1 to copper response elements in the promoter regions of genomic Mac1 target genes. Although there is precedent for additional roles of Sod1 beyond protection of the cell from oxygen radicals, the involvement of this protein in copper-responsive transcriptional regulation has not previously been observed. Given the presence of both Sod1 and copper-responsive transcription factors in higher eukaryotes, these studies may yield important insights into how copper deficiency is sensed and appropriate cellular responses are coordinated.

Highlights

Organisms have evolved sophisticated homeostatic systems to maintain appropriate intracellular copper levels that are below levels that could lead to cellular damage [4, 5]
The Ccs1 Copper Chaperone Is Required for Robust Activation of Mac1—In S. cerevisiae, expression of the high affinity copper uptake system is regulated by the copper-responsive transcription factor Mac1
We found that the yeasts lacking CCS1 display a severe defect in the activation of Mac1 in response to decreased copper availability induced by supplementation of the growth medium with the copper-specific chelator bathocuproinedisulfonic acid (BCS)

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains and Plasmids—All isogenic S. cerevisiae deletion strains were created by replacement of the endogenous locus with a floxed kanamycin resistance cassette and subsequent removal of this cassette [31]. The ySOD1 plasmid was created by cloning a PCR fragment containing the SOD1 gene and its endogenous promoter and terminator as an XbaI/XhoI fragment into the pRS416 vector. A DNA fragment with the coding sequence for the first 105 amino acids of SCO2 as an in-frame amino-terminal fusion with the SOD1 gene under the control of the SOD1 promoter was created using overlap PCR and cloned by gap repair into pRS415 to create the SCO2-SOD1 plasmid. For the yeast one-hybrid experiment, a PCR product containing codons 42– 417 of the MAC1 gene was cloned by gap repair as an in frame fusion with the GAL4 DNA binding domain of the pGBKT7 plasmid backbone (Clontech). Digital images of ChIP results were quantitated using ImageQuant software and processed using Adobe Photoshop

RESULTS

Hour Growth in Galactose

DISCUSSION