The nature of the connection between mitochondrial Fe-S cluster synthesis and the iron-sensitive transcription factor Aft1 in regulating the expression of the iron transport system in Saccharomyces cerevisiae is not known. Using a genetic screen, we identified two novel cytosolic proteins, Fra1 and Fra2, that are part of a complex that interprets the signal derived from mitochondrial Fe-S synthesis. We found that mutations in FRA1 (YLL029W) and FRA2 (YGL220W) led to an increase in transcription of the iron regulon. In cells incubated in high iron medium, deletion of either FRA gene results in the translocation of the low iron-sensing transcription factor Aft1 into the nucleus, where it occupies the FET3 promoter. Deletion of either FRA gene has the same effect on transcription as deletion of both genes and is not additive with activation of the iron regulon due to loss of mitochondrial Fe-S cluster synthesis. These observations suggest that the FRA proteins are in the same signal transduction pathway as Fe-S cluster synthesis. We show that Fra1 and Fra2 interact in the cytosol in an iron-independent fashion. The Fra1-Fra2 complex binds to Grx3 and Grx4, two cytosolic monothiol glutaredoxins, in an iron-independent fashion. These results show that the Fra-Grx complex is an intermediate between the production of mitochondrial Fe-S clusters and transcription of the iron regulon.Iron is an essential element required for all eukaryotes and most prokaryotes. Iron is also potentially dangerous, since it can participate in the generation of toxic oxygen molecules, such as superoxide anion and the hydroxyl radical. Iron transport is highly regulated in all species, and iron transporters are only expressed under conditions of iron need. Transcriptional and post-transcriptional regulation of iron transport systems occurs in all organisms ranging from yeast to humans. Consequently, iron acquisition in all species is tightly controlled and is coordinated with iron use. The budding yeast Saccharomyces cerevisiae expresses two different high affinity iron transport systems. One system is composed of a closely related family of four siderophore transporters. Siderophores are small organic molecules that exhibit an extremely high affinity (K d ϭ 10 Ϫ33 ) for iron (1). Although S. cerevisiae does not synthesize siderophores, it can accumulate siderophores produced by other organisms. The second high affinity iron transport system mediates the acquisition of ionic iron and is composed of a cell surface multicopper oxidase, Fet3, and a transmembrane permease, Ftr1. The multicopper oxidase converts Fe 2ϩ to Fe 3ϩ , which is then transported by the transmembrane permease.The transcriptional activator Aft1 regulates both high affinity iron transport systems (2). Aft1 is cytosolic when cells are iron-replete, but under conditions of iron depletion, Aft1 translocates into the nucleus, where it activates the transcription of ϳ20 genes (3). These genes, referred to as the iron regulon, include the siderophore transporters, the high affini...

The transcription factors Aft1 and Aft2 from Saccharomyces cerevisiae regulate the expression of genes involved in iron homeostasis. These factors induce the expression of iron regulon genes in iron-deficient yeast but are inactivated in iron-replete cells. Iron inhibition of Aft1/Aft2 was previously shown to be dependent on mitochondrial components required for cytosolic iron sulfur protein biogenesis. We presently show that the nuclear monothiol glutaredoxins Grx3 and Grx4 are critical for iron inhibition of Aft1 in yeast cells. Cells lacking both glutaredoxins show constitutive expression of iron regulon genes. Overexpression of Grx4 attenuates wild type Aft1 activity. The thioredoxin-like domain in Grx3 and Grx4 is dispensable in mediating iron inhibition of Aft1 activity, whereas the conserved cysteine that is part of the conserved CGFS motif in monothiol glutaredoxins is essential for this function. Grx3 and Grx4 interact with Aft1 as shown by two-hybrid interactions and co-immunoprecipitation assays. The interaction between glutaredoxins and Aft1 is not modulated by the iron status of cells but is dependent on the conserved glutaredoxin domain Cys residue. Thus, Grx3 and Grx4 are novel components required for Aft1 iron regulation that most likely occurs in the nucleus.

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The high affinity uptake systems for iron and copper ions in Saccharomyces cerevisiae involve metal-specific permeases and two known cell surface Cu(II) and Fe (

X-ray absorption spectroscopy on the minimal copper-regulatory domains of the two copper-regulated transcription factors (Ace1 and Mac1) in Saccharomyces cerevisiae revealed the presence of a remarkably similar polycopper cluster in both proteins. The Cu-regulatory switch motif of Mac1 consisting of the C-terminal first Cys-rich motif, designated the C1 domain, binds four Cu(I) ions as does the Cu-regulatory domain of Ace1. The four Cu(I) ions are bound to each molecule in trigonal geometry. An extended X-ray absorption fine structure (EXAFS) arising from outer-shell Cu...Cu interactions at 2.7 and 2.9 A was apparent in each Cu(I) complex indicative of a polycopper cluster. The intensity of the 2.9 A Cu...Cu backscatter peak, apparently diminished by partial cancellation, dominates the EXAFS. The results suggest that CuAce1 and CuMac1(C1) contain somewhat distorted forms of a known [Cu(4)-S(6)] cage in which a core of Cu atoms forming an approximate tetrahedron is bound by bridging thiolates above each of the six edges. The tetracopper clusters bound by Ace1 and Mac1 differ in that the Ace1 cluster is coordinated entirely by cysteinyl thiolate, whereas the cysteine-deficient Mac1 cluster appears to consist of a Cu(4)(S-Cys)(5)(N-His) cluster with a bridging histidyl-derived nitrogen.