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.

The trace element copper (Cu) is essential for cell growth. In this report we describe the identification of a new component of the high-affinity Cu transport machinery in yeast, encoded by the CTR3 gene. Ctr3p is a small intraceUular cysteine-rich integral membrane protein that restores high-affinity Cu uptake, Cu, Zn superoxide dismutase activity, ferrous iron transport, and respiratory proficiency to strains lacking the CTR1 (Cu transporter 1) gene. In most commonly used Saccharomyces cerevisiae laboratory strains, expression of CTR3 is abolished by a Ty2 transposon insertion that separates the CTR3 promoter from the transcriptional start sites by 6 kb. In strains that do not possess a Ty2 transposon at the CTR3 locus, expression of CTR3 is repressed by copper and activated by copper starvation. In such strains inactivation of both CTRI and CTR3 is required to generate lethal copper-deficient phenotypes. Although Ctrlp and Ctr3p can function independently in copper transport, the expression of both proteins provides maximal copper uptake and growth rate under copper-limiting conditions. These results underscore the importance of mobile DNA elements in the alteration of gene function and phenotypic variation.

Schizosaccharomyces pombe cells acquire iron under high affinity conditions through the action of a cell surface ferric reductase encoded by the frp1 ؉ gene and a two-component iron-transporting complex encoded by the fip1 ؉ and fio1 ؉ genes. When cells are grown in the presence of iron, transcription of all three genes is blocked. A conserved regulatory element, 5-(A/ T)GATAA-3, located upstream of the frp1 ؉ , fip1 ؉ , and fio1 ؉ genes, is necessary for iron repression. We have cloned a novel gene, termed fep1 ؉ , which encodes an iron-sensing transcription factor. Binding studies reveal that the putative DNA binding domain of Fep1 expressed as a fusion protein in Escherichia coli specifically interacts with the 5-(A/T)GATAA-3 sequence in an iron-dependent manner. In a fep1⌬ mutant strain, the fio1 ؉ gene is highly expressed and is unregulated by iron. Furthermore, the fep1⌬ mutation increases activity of the cell surface iron reductase and renders cells hypersensitive to the iron-dependent free radical generator phleomycin. Mutations in the transcriptional corepressors tup11 ؉ and tup12 ؉ are phenocopies to fep1 ؉ . Indeed, strains with both tup11⌬ and tup12⌬ deletions fail to sense iron. This suggests that in the presence of iron and Fep1, the Tup11 and Tup12 proteins may act as co-repressors for down-regulation of genes encoding components of the reductive iron transport machinery.

The fission yeast Schizosaccharomyces pombe responds to the deprivation of iron by inducing the expression of the php4 ؉ gene, which encodes a negative regulatory subunit of the heteromeric CCAAT-binding factor. Once formed, the Php2/3/4/5 transcription complex is required to inactivate a subset of genes encoding iron-using proteins. Here, we used a pan-S. pombe microarray to study the transcriptional response to iron starvation and identified 86 genes that exhibit php4 ؉ -dependent changes on a genome-wide scale. One of these genes encodes the iron-responsive transcriptional repressor Fep1, whose mRNA levels were decreased after treatment with the permeant iron chelator 2,2-dipyridyl. In addition, several genes encoding the components of iron-dependent biochemical pathways, including the tricarboxylic acid cycle, mitochondrial respiration, amino acid biosynthesis, and oxidative stress defense, were downregulated in response to iron deficiency. Furthermore, Php4 repressed transcription when brought to a promoter using a yeast DNA-binding domain, and iron deprivation was required for this repression. On the other hand, Php4 was constitutively active when glutathione levels were depleted within the cell. Based on these and previous results, we propose that iron-dependent inactivation of Php4 is regulated at two distinct levels: first, at the transcriptional level by the iron-responsive GATA factor Fep1 and second, at the posttranscriptional level by a mechanism yet to be identified, which inhibits Php4-mediated repressive function when iron is abundant.

We have identified genes encoding candidate proteins involved in iron storage (pcl1 ؉ ), the tricarboxylic acid cycle (sdh4 ؉ ), and iron-sulfur cluster assembly (isa1 ؉ ) that are negatively regulated in response to iron deprivation. Promoter deletion and site-directed mutagenesis permitted identification of a new cis-regulatory element in the promoter region of the pcl1 ؉ gene. This cis-acting regulatory sequence containing the pentanucleotide sequence CCAAT is responsible for transcriptional repression of pcl1 ؉ under low iron supply conditions. In Schizosaccharomyces pombe, the CCAAT-binding factor is a heteromeric DNA-binding complex that contains three subunits, designated Php2, Php3, and Php5. Inactivation of the php2 ؉ locus negatively affects the transcriptional competency of pcl1 ؉ . A fourth subunit, designated Php4, is not essential for the transcriptional activation of target genes under basal and iron-replete conditions. We demonstrate that, in response to iron-limiting conditions, Php4 is required for down-regulation of pcl1 ؉ , sdh4 ؉ , and isa1 ؉ mRNA levels. In vivo RNase protection studies reveal that the expression of php4 ؉ is negatively regulated by iron and that this regulated expression requires a functional fep1 ؉ gene. The results of these studies reveal that Fep1 represses php4؉ expression in response to iron. In contrast, when iron is scarce, Fep1 becomes inactive and php4؉ is expressed to act as a regulatory subunit of the CCAAT-binding factor that is required to block pcl1 ؉ , sdh4 ؉ , and isa1 ؉ gene transcription.