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 transporter Ccc1 imports iron into the vacuole, which is the major site of iron storage in fungi and plants. CCC1 mRNA is destabilized under low-iron conditions by the binding of Cth1 and Cth2 to the 3 untranslated region (S. Puig, E. Askeland, and D. J. Thiele, Cell 120:99-110, 2005). Here, we show that the transcription of CCC1 is stimulated by iron through a Yap consensus site in the CCC1 promoter. We identified YAP5 as being the iron-sensitive transcription factor and show that a yap5⌬ strain is sensitive to high iron. Green fluorescent protein-tagged Yap5 is localized to the nucleus and occupies the CCC1 promoter independent of the iron concentration. Yap5 contains two cysteine-rich domains, and the mutation of the cysteines to alanines in each of the domains affects the transcription of CCC1 but not DNA binding. The fusion of the Yap5 cysteine-containing domains to a GAL4 DNA binding domain results in iron-sensitive GAL1-lacZ expression. Iron affects the sulfhydryl status of Yap5, which is indicative of the generation of intramolecular disulfide bonds. These results show that Yap5 is an iron-sensing transcription factor and that iron regulates transcriptional activation.Iron is an essential nutrient required by all eukaryotes. In high concentrations, however, iron can be toxic, necessitating tight control over its concentration within cells. Multicellular organisms can transfer iron between cell types; however, multicellular and single-cell eukaryotes do not have an excretory mechanism to dispose of iron. Iron homeostasis results from the ability to regulate iron acquisition or to store iron once it is absorbed. The ability to store iron makes it available for future use while preventing toxicity. In fungi and plants, iron is stored in the vacuole, where it can be exported when needed. In the budding yeast Saccharomyces cerevisiae, high-affinity and low-affinity transporters export iron from the vacuole to the cytosol. The high-affinity vacuolar iron transport complex Fet5/ Fth1 is homologous to the high-affinity plasma membrane transport complex Fet3/Ftr1 (22), while the low-affinity vacuolar iron transporter Smf3 is homologous of the cell surface transporter Smf1 (3, 18). The high-affinity vacuolar and cell surface transporters, as well as SMF3, are under the transcriptional control of the iron-sensing transcription factors Aft1 and Aft2 (4,20). Thus, iron transport into the cytosol from the vacuole and across the cell surface into the cytosol is regulated coordinately.Much less is known about the regulation of iron transport from the cytosol to the vacuole. The only identified vacuolar iron importer in plants and fungi is Ccc1 (referred to as VIT1 in plants) (14), which can also transport Mn 2ϩ in yeast (13). The overexpression of Ccc1 leads to profound cytosolic iron depletion, implying that this transporter must be regulated to maintain cytosolic iron homeostasis. Recently, Puig et al. showed that the stability of CCC1 mRNA was regulated by Cth1 and Cth2 and that CTH2 was under the transcriptiona...

Ferritin is a cytosolic molecule comprised of subunits that self‐assemble into a nanocage capable of containing up to 4500 iron atoms. Iron stored within ferritin can be mobilized for use within cells or exported from cells. Expression of ferroportin (Fpn) results in export of cytosolic iron and ferritin degradation. Fpn‐mediated iron loss from ferritin occurs in the cytosol and precedes ferritin degradation by the proteasome. Depletion of ferritin iron induces the monoubiquitination of ferritin subunits. Ubiquitination is not required for iron release but is required for disassembly of ferritin nanocages, which is followed by degradation of ferritin by the proteasome. Specific mammalian machinery is not required to extract iron from ferritin. Iron can be removed from ferritin when ferritin is expressed in Saccharomyces cerevisiae, which does not have endogenous ferritin. Expressed ferritin is monoubiquitinated and degraded by the proteasome. Exposure of ubiquitination defective mammalian cells to the iron chelator desferrioxamine leads to degradation of ferritin in the lysosome, which can be prevented by inhibitors of autophagy. Thus, ferritin degradation can occur through two different mechanisms.

The mechanistic target of rapamycin kinase complex 1 (MTORC1) is a central cellular kinase that integrates major signaling pathways, allowing for regulation of anabolic and catabolic processes including macroautophagy/autophagy and lysosomal biogenesis. Essential to these processes is the regulatory activity of TFEB (transcription factor EB). In a regulatory feedback loop modulating transcriptional levels of RRAG/Rag GTPases, TFEB controls MTORC1 tethering to membranes and induction of anabolic processes upon nutrient replenishment. We now show that TFEB promotes expression of endocytic genes and increases rates of cellular endocytosis during homeostatic baseline and starvation conditions. TFEB-mediated endocytosis drives assembly of the MTORC1-containing nutrient sensing complex through the formation of endosomes that carry the associated proteins RRAGD, the amino acid transporter SLC38A9, and activate AKT/protein kinase B (AKT p-T308). TFEB-induced signaling endosomes en route to lysosomes are induced by amino acid starvation and are required to dissociate TSC2, re-tether and activate MTORC1 on endolysosomal membranes. This study characterizes TFEB-mediated endocytosis as a critical process leading to activation of MTORC1 and autophagic function, thus identifying the importance of the dynamic endolysosomal system in cellular clearance. Abbreviations: CAD: central adrenergic tyrosine hydroxylase-expressing-a-differentiated; ChIP-seq: chromosome immunoprecipitation sequencing; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; EEA1: early endosomal antigen 1; EGF: epidermal growth factor; FBS: fetal bovine serum; GFP: green fluorescent protein; GTPase: guanosine triphosphatase; HEK293T: human embryonic kidney 293 cells expressing a temperature-sensitive mutant of the SV40 large T antigen; LAMP: lysosomal-associated membrane protein; LYNUS: lysosomal nutrient-sensing complex; MAP1LC3/LC3: microtubule associated protein 1 light chain 3 alpha/beta; MTOR: mechanistic target of rapamycin kinase; MTORC: mechanistic target of rapamycin kinase complex; OE: overexpression; PH: pleckstrin homology; PtdIns(3,4,5)P: phosphatidylinositol 3,4,5-trisphosphate; RRAGD: Ras related GTPase binding D; RHEB: Ras homolog enriched in brain; SLC38A9: solute carrier family 38 member 9; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TSC2: tuberous sclerosis 2; TMR: tetramethylrhodamine; ULK1: unc-51 like kinase 1; WT: wild type.

Deletion of two homologous genes, MRS3 and MRS4, that encode mitochondrial iron transporters affects the activity of the vacuolar iron importer Ccc1. Ccc1 levels are decreased in ⌬mrs3⌬mrs4 cells, but the activity of the transporter is increased, resulting is reduced cytosolic iron. Overexpression of CCC1 in ⌬mrs3⌬mrs4 cells results in a severe growth defect due to decreased cytosolic iron, referred to as the mitochondriavacuole signaling (MVS) phenotype. Mutants were identified that suppress the MVS growth defect, and FRA1 was identified as a gene that suppresses the MVS phenotype. Overexpression of FRA1 suppresses altered transition metal metabolism in ⌬mrs3⌬mrs4 cells, whereas deletion of FRA1 is synthetically lethal with ⌬mrs3⌬mrs4. Fra1 binds to Tsa1, which encodes a thioredoxin-dependent peroxidase. Deletion of TSA1 or TRR1 is synthetically lethal in ⌬mrs3⌬mrs4 cells, suggesting that ⌬mrs3⌬mrs4 cells generate reactive oxygen metabolites. The generation of reactive oxygen metabolites in ⌬mrs3⌬mrs4 cells was confirmed by use of the reporter molecule 2,7-dichlorodihydrofluorescein diacetate. These results suggest that mitochondria-induced oxidant damage is responsible for activating Ccc1 and that Fra1 and Tsa1 can reduce oxidant damage.Mitochondria are well recognized as the site for energy production and biosynthetic pathways. Mitochondria are central for a variety of downstream signaling pathways initiated by perturbations in mitochondrial activities. Most notably, in many eukaryotes decreased mitochondrial function results in apoptosis. The budding yeast Saccharomyces cerevisiae has been useful in dissecting the genetics and biochemistry of mitochondrial signaling events. Loss of mitochondrial heme synthesis leads to changes in the transcription of a wide variety of genes mediated by the Hap family of transcription factors (1). Decreased mitochondrial respiration, independent of effects on heme synthesis, affects the transcription of nuclear genes that result in a reconfiguration of energy metabolism, referred to as the retrograde response (2). Finally, alteration in mitochondrial iron-sulfur cluster synthesis can induce expression of genes involved in iron metabolism (3).We showed that deletion of two nuclear genes MRS3 and MRS4, which encode homologous mitochondrial iron transporters, had effects on cellular metal metabolism (4). Deletion of MRS3 or MRS4 individually had no phenotype, but a double deletion showed impaired mitochondrial iron metabolism when cells were grown on low iron medium (4 -6). Mitochondrial iron-consuming processes such as heme and iron-sulfur cluster synthesis appeared normal when ⌬mrs3⌬mrs4 cells were grown in iron-sufficient medium, suggesting that low affinity iron transport systems can supply iron to the mitochondria. In iron-sufficient medium, however, ⌬mrs3⌬mrs4 cells showed abnormalities in iron homeostasis as well as alterations in response to other transition metals (4). We showed that many of the phenotypes in the double deletion could be suppressed by deleting CCC1. The...