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.

When iron repletes, Schizosaccharomyces pombe cells repress transcription of genes encoding components involved in the reductive iron transport system. Fep1 mediates this transcriptional control by interacting specifically with GATA-type cis-acting elements. To further investigate the role that Fep1 plays in iron homeostasis, we searched for additional Fep1-regulated genes. We found that str1+ is subject to negative transcriptional regulation, which is exerted through binding of Fep1 to a single GATA element in the str1+ promoter. Introduction of str1+ into a Saccharomyces cerevisiae fet3Delta arn1-4Delta strain led to assimilation of iron from ferrichrome, revealing that Str1 functions as a siderophore-iron transporter in S.pombe. We also identified two additional target genes of Fep1, named str2+ and str3+. We demonstrate that the str1+, str2+ and str3+ genes share a common promoter element, 5'-(A/T)GATAA-3'. We found that the N-terminal 241 residue segment of Fep1 expressed in Escherichia coli specifically interacts with the 5'-(A/T)GATAA-3' element present in each of these promoters. Consistent with this, constitutive high level str1+, str2+ and str3+ gene expression was observed in a fep1Delta mutant strain. Taken together, these results demonstrate that Fep1 occupies a central role in coordinating transcriptional regulation of genes encoding components of the reductive and non-reductive iron transport systems in fission yeast.

Aerobic organisms possess efficient systems for the transport of copper. This involves transporters that mediate the passage of copper across biological membranes to reach essential intracellular copper-requiring enzymes. In this report, we identify a new copper transporter in Schizosaccharomyces pombe, encoded by the ctr6 ؉ gene. The transcription of ctr6 ؉ is induced under copper-limiting conditions. This regulation is mediated by the cis-acting promoter element CuSE (copper-signaling element) through the copper-sensing transcription factor Cuf1. An S. pombe strain bearing a disrupted ctr6⌬ allele displays a strong reduction of copper,zinc superoxide dismutase activity. When the ctr6؉ gene is overexpressed from the thiamine-inducible nmt1 ؉ promoter, the cells are unable to grow on medium containing exogenous copper. Surprisingly, this copper-sensitive growth phenotype is not due to an increase of copper uptake at the cell surface. Instead, copper delivery across the plasma membrane is reduced. Consistently, this results in repressing ctr4 ؉ gene expression. By using a functional ctr6 ؉ epitope-tagged allele expressed under the control of its own promoter, we localize the Ctr6 protein on the membrane of vacuoles. Furthermore, we demonstrate that Ctr6 is an integral membrane protein that can trimerize. Moreover, we show that Ctr6 harbors a putative copper-binding Met-X-His-Cys-X-Met-X-Met motif in the amino terminus, which is essential for its function. Our findings suggest that under conditions in which copper is scarce, Ctr6 is required as a means to mobilize stored copper from the vacuole to the cytosol.

No abstract

Transcriptional regulation of genes encoding critical components of copper transport is essential for copper homeostasis and growth in yeast. Analysis of regulatory regions in the promoter of the ctr4 ؉ copper transporter gene in fission yeast Schizosaccharomyces pombe reveals the identity of a conserved copper-signaling element (CuSE), which is recognized by the transcription factor Cuf1. We demonstrate that CuSE is necessary for transcriptional activation in response to copper deprivation conditions. Interestingly, the CuSE element bears a strong sequence similarity to the recognition site, denoted MRE (metal regulatory element), which is recognized by a distinct class of copper sensors required for copper detoxification, including Ace1 from Saccharomyces cerevisiae and Amt1 from Candida glabrata. When a consensus MRE from S. cerevisiae is introduced into S. pombe, transcription is induced by copper deprivation in a Cuf1-dependent manner, similar to regulation by Mac1, the nuclear sensor for regulating the expression of genes encoding components involved in copper transport in S. cerevisiae. UV-cross-linking experiments show that the Cuf1 protein directly binds the CuSE. These results demonstrate that the Cuf1 nutritional copper-sensing factor possesses a module that functions similarly to domains found in the Ace1/Amt1 class of metalloregulatory factors, which allows the protein to act through a closely related MRE-like sequence to regulate copper transport gene expression in S. pombe.

Copper uptake in the fission yeast Schizosaccharomyces pombe is carried out by a heteromeric complex formed by two proteins, Ctr4 and Ctr5. In this study, a stable expression system using integrative plasmids was developed to investigate the respective roles of Ctr4 and Ctr5 in copper transport. It was shown that expression of full-length Ctr4 or truncated Ctr4 containing residues 106-289 was required for localization of Ctr5 to the plasma membrane. Likewise, when the full-length Ctr5 or truncated Ctr5 from residues 44-173 was co-expressed with Ctr4, this protein was visualized at the periphery of the cell. To determine the importance of the Mets motifs (consisting of five methionines arranged as Met-X2-Met-X-Met, where X is any amino acid) of Ctr4 and Ctr5 in the heteroprotein complex, we co-expressed Ctr5 lacking the Mets motif and Cys-X-Met-X-Met sequence with wild-type Ctr4 or its mutant derivatives. Conversely, Ctr4 lacking the Mets motif and Met 122 was expressed with wild-type Ctr5 or its mutant derivatives. These experiments revealed that the five Mets motifs of Ctr4 and the Ctr4 residue Met 122 have equally important roles in copper assimilation. Furthermore, the two partially overlapping Mets motifs and the Cys-X-Met-X-Met sequence in Ctr5 have redundant functions in copper transport, with the latter sequence making a greater contribution than the former. Together, the data reveal that co-expression of both Ctr4 and Ctr5 is necessary for the proper function and localization of the heteroprotein complex to the plasma membrane. Once on the cell surface, the N-terminal regions of Ctr4 and Ctr5 can function independently to transport copper; however, the greatest efficiency is achieved when both N termini are present.

In Schizosaccharomyces pombe, the iron sensor Fep1 mediates the transcriptional repression of iron transport genes in response to high concentrations of iron. On the other hand, fep1؉ expression is downregulated under conditions of iron starvation by the CCAAT-binding factor Php4. In this study, we created a fep1⌬ php4⌬ double mutant strain where expression of fep1 ؉ was disengaged from its iron limitation-dependent repression by Php4 to examine the effects of iron on constitutively expressed functional fep1؉ -GFP and TAP-fep1 ؉ alleles and their gene products. In these cells, Fep1-green fluorescent protein was invariably localized in the nucleus under both iron-limiting and iron-replete conditions. Using chromatin immunoprecipitation assays, we found that Fep1 is associated with iron-responsive promoters in vivo. Chromatin binding was iron dependent, with a loss of binding observed in the presence of low iron. Functional dissection of the protein revealed that the N-terminal 241-residue segment that includes two consensus Cys 2 /Cys 2 -type zinc finger motifs and a Cys-rich region is required for optimal promoter occupancy by Fep1. Within this segment, a minimal module encompassing amino acids 60 to 241 is sufficient for iron-dependent chromatin binding. Using yeast one-hybrid analysis, we showed that the replacement of the repression domain of Fep1 by fusing the activation domain of VP16 to the chromatin-binding fragment of amino acids 1 to 241 of Fep1 converts the protein from an iron-dependent repressor into an iron-dependent transcriptional activator. Thus, the repression function of Fep1 can be replaced with that of a transcriptional activation function without the loss of its iron-dependent DNA-binding activity.

In fission yeast, the genes encoding proteins that are components of the copper transporter family are controlled at the transcriptional level by the Cuf1 transcription factor. Under low copper availability, Cuf1 induces expression of the copper transporter genes. In contrast, sufficient levels of copper inactivate Cuf1 and expression of its target genes. Our study reveals that Cuf1 harbors a putative copper-binding motif, Cys-X-Cys-X(3)-Cys-X-Cys-X(2)-Cys-X(2)-His, within its carboxyl-terminal region to sense changing environmental copper levels. Binding studies reveal that the amino-terminal 174-residue segment of Cuf1 expressed as a fusion protein in Escherichia coli specifically interacts with the cis-acting copper transporter promoter element CuSE (copper-signaling element). Within this region, the first 61 amino acids of Cuf1 exhibit more overall homology to the Saccharomyces cerevisiae Ace1 copper-detoxifying factor (from residues 1 to 63) than to Mac1, its functional ortholog. Consistently, we demonstrate that a chimeric Cuf1 protein bearing the amino-terminal 63-residue segment of Ace1 complements cuf1 Delta null phenotypes. Furthermore, we show that Schizosaccharomyces pombe cuf1Delta mutant cells expressing the full-length S. cerevisiae Ace1 protein are hypersensitive to copper ions, with a concomitant up-regulation of CuSE-mediated gene expression in fission yeast. Taken together, these studies reveal that S. cerevisiae Ace1 1-63 is functionally exchangeable with S. pombe Cuf1 1-61, and the nature of the amino acids located downstream of this amino-terminal conserved region may be crucial in dictating the type of regulatory response required to establish and maintain copper homeostasis.