Transcription of the Yeast Iron Regulon Does Not Respond Directly to Iron but Rather to Iron-Sulfur Cluster Biosynthesis

Abstract

Saccharomyces cerevisiae responds to iron deprivation by increased transcription of the iron regulon, including the high affinity cell-surface transport system encoded by FET3 and FTR1. Here we demonstrate that transcription of these genes does not respond directly to cytosolic iron but rather to the mitochondrial utilization of iron for the synthesis of iron-sulfur (Fe-S) clusters. We took advantage of a mutant form of an iron-dependent enzyme in the sterol pathway (Erg25-2p) to assess cytosolic iron levels. We showed that disruption of mitochondrial Fe-S biosynthesis, which results in excessive mitochondrial iron accumulation, leads to transcription of the iron transport system independent of the cytosolic iron level. There is an inverse correlation between the activity of the mitochondrial Fe-S-containing enzyme aconitase and the induction of FET3. Regulation of transcription by Fe-S biosynthesis represents a mechanism by which cellular iron acquisition is integrated with mitochondrial iron metabolism.High affinity iron uptake in Saccharomyces cerevisiae is mediated by a transport system comprising the multicopper oxidase Fet3p and the transmembrane permease Ftr1p (1). Genes that encode these proteins are part of an iron regulon in which transcription is activated by Aft1p. Aft1p is cytosolic when iron is replete and translocates to the nucleus when iron is limited (2). It has been speculated that Aft1p responds to cytosolic levels of elemental iron, as conditions that lower cytosolic iron result in increased transcription of the iron regulon. Binding of iron to Aft1p, however, has not been demonstrated.Mutations that affect mitochondrial Fe-S biosynthesis or export result in increased cellular and mitochondrial iron as a result of decreased mitochondrial iron efflux (3). It has been thought that the increase in mitochondrial iron occurs at the expense of cytosolic iron, and the decrease in cytosolic iron is responsible for the increase in Aft1p-mediated transcription of the iron regulon (3,4). Herein, we demonstrate that activation of Aft1p does not directly respond to cytosolic iron. Using a novel measure of cytosolic iron, we showed that activation of the iron regulon can occur when cytosolic iron levels are high. We further demonstrated that activation of the iron regulon is controlled by the synthesis of Fe-S clusters, which in yeast is localized within mitochondria. Our studies revealed that cellular iron acquisition is coordinated with the mitochondrial use of iron. MATERIALS AND METHODSYeast Strains-All yeast strains used in this study were derived from a W303 background strain. The following yeast strains have been described previously: DY150 (Mata ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 can1-100(oc)), METYFH1 (Mat␣ ade2-1 his3-11 ⌬yfh1::HIS3 leu2-3,112 trp1-1 ura3-52 can1-100(oc), pMET3YFH1 [URA3]) (5), METNFS1 (Mat␣ ade2-1 his3-11 ⌬nfs1::HIS3 leu2-3,112 trp1-1 ura3-52 can1-100(oc), pMET3NFS1[URA3]) (3), and erg25-2 (Mat␣ erg25-2 ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 can1-100(oc)) (6). DNA trans...

Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis

“…Third, the severe defect in COX activity in Yah1-depleted Gal-YAH1 cells (15) was fully rescued by synthesis of Fdx2, indicating that human Fdx2 can participate not only in yeast mitochondrial Fe/S cluster synthesis but also replace Yah1 function in the hydroxylation of heme O to heme A. Finally, we tested whether expression of Fdx2 can normalize the activation of the yeast iron regulon observed upon functional defects of the mitochondrial iron-sulfur cluster (ISC) assembly and export machineries (31,32). As a reporter we used the promoter of FET3, which encodes the multi-copper ferroxidase of the high affinity iron uptake system (33), fused to GFP.…”

Metal Ion availability in mitochondria

“…Although the identity of the ligand remains unresolved, the matrix CuL pool is accessible to a heterologous cuproenzyme (Cobine et al 2004). The mechanism by which Aft1 (and Aft2) sense cellular iron levels involves the mitochondrion (Chen et al 2004;Chen et al 2002). Cells defective for Fe-S cluster biogenesis within the mitochondrial matrix exhibit constitutive expression of the iron regulon genes (Chen et al 2002).…”

“…Cells defective for Fe-S cluster biogenesis within the mitochondrial matrix exhibit constitutive expression of the iron regulon genes (Chen et al 2002). The constitutive activity of Aft1 is due to an impairment of a signal arising from the mitochondrial Fe-S biosynthetic machinery and not due to Fe-deficiency within the cytoplasm (Chen et al 2004).…”

A Mitochondrial-Vacuolar Signaling Pathway in Yeast That Affects Iron and Copper Metabolism

“…Storage of iron would make it unavailable to the iron-sensing transcription factor Aft1p. This could occur through either through a direct reduction in cytosolic iron or by affecting the synthesis of iron-sulfur clusters in mitochondria, as the level of iron-sulfur clusters appears to regulate transcription of the iron-regulon (16). Our previous studies demonstrated that yeast store iron in the vacuole and that Ccc1p is a vacuolar protein that transports iron from the cytosol to the vacuole (15).…”