The principal iron uptake system of Saccharomyces cerevisiae utilizes a reductase activity that acts on ferric iron chelates external to the cell. The FREI gene product is required for this activity. Iron is an essential nutrient that is required by many enzymes for cellular functions such as DNA synthesis and respiration. Ferric iron forms insoluble ferric hydroxide complexes in the presence of oxygen and water, making iron availability a biological problem under these conditions. Ferrous iron is much more soluble but is rapidly converted to ferric iron in the presence of oxygen (1). Cells have developed two major solutions to the problem of assimilation offerric iron from the environment. In Escherichia coli, specific ferric binding compounds termed siderophores are secreted that bind and solubilize environmental iron and deliver it to specific receptors for transport across the outer and inner membranes (2). In the yeast Saccharomyces cerevisiae, the major iron uptake system under aerobic conditions depends on a transplasma membrane electron transport system (3, 4) that reduces ferric iron external to the cell. Multicellular eukaryotes may also use an externally directed ferric reductase prior to transmembrane movement of ferrous iron (5-8). Our previous results indicated that the FREI gene product in S. cerevisiae is required for external ferric reduction and for ferric iron utilization, thus linking these two processes (9). However, a mutation in FREI did not affect the uptake offerrous iron (9), suggesting that the ferrous transport system is an independent component of the iron utilization apparatus in yeast. Many organisms control their rate of iron assimilation by ascertaining their requirement for iron and expressing a limiting component of the uptake system accordingly (10). Our previous results suggest that during aerobic growth of S. cerevisiae, changes in iron availability are reflected in alterations in the levels of FREI mRNA and of ferric iron uptake (9). In this paper we present the structure of the FREI gene and identify DNA sequences that mediate its regulation by ironA MATERIALS AND METHODSYeast Strains. The following yeast strains were derived from the wild-type strain F113 (MATa, can), inol-13, ura3-52): W103 (MATa, can], inol-13, ura3-52,frel-1) was derived by chemical mutagenesis of F113 and selection for a reductase-negative phenotype (9); W126 was derived by transformation of F113 with the plasmid pWDC20, which carries the FREI gene on the high-copy-number vector YEp24 (11). We used one-step gene disruption (12) to create strain N1 (MATa, can), inol-13, Afrel:: URA3), in which an 800-base-pair (bp) Xho I fragment internal to the FREI coding region was replaced with a URA3 marker gene, and strain N2 (MATa, can, inol-13, Afrel:: URA3), in which a 2.7-kilobase (kb) Cla I fragment containing the entire FREI coding region was similarly replaced. Disruption/deletion strains of the FREI locus were also constructed in the wild-type strain H1085, yielding strain W218 (MATa, leu2-3,112, ...

We have identified a cell surface ferric reductase activity in the fission yeast Schizosaccharomyces pombe. A mutant strain deficient in this activity was also deficient in ferric iron uptake, while ferrous iron uptake was not impaired. Therefore, reduction is a required step in cellular ferric iron acquisition. We have clonedfrpl1, the wild-type allele of the mutant gene.frpl + mRNA levels were repressed by iron addition to the growth medium. Fusion of 138 nucleotides of frpl + promoter sequences to a reporter gene, the bacterial chloramphenicol acetyltransferase gene, conferred iron-dependent regulation upon the latter when introduced into S. pombe. The predicted amino acid sequence of the frp1+ gene exhibits hydrophobic regions compatible with transmembrane domains. It shows similarity to the Saccharomyces cerevisiae FREI gene product and the gp9l-phox protein, a component of the human NADPH phagocyte oxidoreductase that is deficient in X-linked chronic granulomatous disease.

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High-affmity receptors for interleukin 2 (IL-2) are expressed on T cells following activation. These receptors are composed of both a and (3 chains. Expression of a chains and, therefore, expression of high-affinity receptors are critically regulated at the level of transcription initiation. We have further dissected the regulatory elements involved in controlling transcription of the IL-2 receptor a-chain (IL-2Ra) gene. The IL-2Ra promoter contains a KB site and binding sites for additional nuclear factors within a 50-base-pair region (positions -290 to -240 relative to the major transcription start site). These include one upstream of the KB site and one similar to the c-fos serum response element (SRE), which is downstream of the KB site. Mutation ofthe KB site decreases IL-2Ra promoter activity in MT-2 cells (a T-cell line that has been transformed with human T-cell lymphotropic virus type I), but not in Jurkat cells (a T-cell leukemia line) that have been activated by phorbol 12-myristate 13-acetate (PMA). In contrast, mutation of a region upstream of the KB site decreases activity in PMA-induced Jurkat cells but increases activity in MT-2 cells. Mutation of the SRE-llke site decreases activity in both cell types but the effect in PMA-induced Jurkat is more pronounced. Thus, these distinct cis-acting elements play different physiological roles in IL-2Ra gene activation in MT-2 cells and PMA-induced Jurkat T cells. These studies provide direct evidence for a functionally significant SRE-like sequence in a gene other than c-fos and the actin genes and identify other elements that are critical for IL-2Ra gene expression.