Identification of FRA1 and FRA2 as Genes Involved in Regulating the Yeast Iron Regulon in Response to Decreased Mitochondrial Iron-Sulfur Cluster Synthesis

Abstract

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...

Structural and Spectroscopic Insights into BolA-Glutaredoxin Complexes

“…On the other hand, it was reported that Grx-BolA couples from various sources could also form apo-heterodimers (7,9). This is consistent with the observation that an in vivo Grx3/4-Fra2 interaction is found both in iron-replete and iron-depleted yeast cells (5).…”

“…The only characterized physiological role for a GrxBolA holo-heterodimer is in sensing the intracellular iron/Fe-S cluster status in yeast. The current model is that a complex formed by Fra1, Fra2, Grx3, and Grx4 links the mitochondrial Fe-S or iron status to the control of a set of genes involved in iron uptake, transport, and storage known as the iron regulon via the regulation of Aft1 transcription factor (5). Under irondeficient conditions, Aft1 is concentrated in the nucleus and activates the iron regulon, whereas under iron-sufficient conditions it is found predominantly in the cytoplasm.…”

“…BolAs were initially defined as stress-responsive transcriptional regulators whose overexpression in Escherichia coli modified bacterial shape and induced biofilm formation (2,3), whereas glutaredoxins (Grxs) 3 were long thought to uniquely function as glutathione (GSH)-dependent oxidoreductases (4). It was thus surprising that a BolA, referred to as Fra2 (Fe repressor of activation-2), and cytosolic monothiol Grx3/4 contributed to the regulation of iron homeostasis by forming stable [2Fe-2S] cluster-bridged holo-heterodimers controlling the nuclear translocation of the Saccharomyces cerevisiae Aft1/Aft2 transcription factors in response to a mitochondrial signal (5)(6)(7). Hence, BolA may modify monothiol Grx functions by converting Grx homodimers bridging a labile [2Fe-2S] cluster into a heterodimer containing a more stable [2Fe-2S] cluster (7).…”

Both Php4 Function and Subcellular Localization Are Regulated by Iron via a Multistep Mechanism Involving the Glutaredoxin Grx4 and the Exportin Crm1

“…Aft1 is an activator, and in contrast, S. pombe Php4 is a repressor; however, both are active under conditions of iron deficiency and inactive in the presence of iron. Recent studies have provided clues with respect to irondependent inhibition of Aft1 (31)(32)(33)(34)62). One aspect is the requirement of the monothiol glutaredoxins Grx3 and Grx4 to allow Aft1 to sense iron excess (32,33).…”

“…Elegant studies have also determined that S. cerevisiae monothiol glutaredoxins Grx3 and Grx4 are key regulators of Aft1 (32,33). When cells undergo a transition from iron-limiting to iron-sufficient conditions, it has been proposed that Grx3 and Grx4, with the aid of Fra1 and Fra2, transmit an as-yet-unidentified mitochondrial inhibitory signal, which contributes to inactivate Aft1 (32)(33)(34).…”