Highlights d Multiple SCLC molecular subtypes arise from a neuroendocrine cell of origin d MYC drives the NEUROD1 + and YAP1 + subtypes of SCLC in a temporal evolution d MYC directly activates NOTCH signaling to reprogram neuroendocrine fate d Multiple SCLC molecular subtypes are present within individual human tumors

The budding yeast Saccharomyces cerevisiae can grow for generations in the absence of exogenous iron, indicating a capacity to store intracellular iron. As cells can accumulate iron by endocytosis we studied iron metabolism in yeast that were defective in endocytosis. We demonstrated that endocytosis-defective yeast (⌬end4) can store iron in the vacuole, indicating a transfer of iron from the cytosol to the vacuole. Using several different criteria we demonstrated that CCC1 encodes a transporter that effects the accumulation of iron and Mn 2؉ in vacuoles. Overexpression of CCC1, which is localized to the vacuole, lowers cytosolic iron and increases vacuolar iron content. Conversely, deletion of CCC1 results in decreased vacuolar iron content and decreased iron stores, which affect cytosolic iron levels and cell growth. Furthermore ⌬ccc1 cells show increased sensitivity to external iron. The sensitivity to iron is exacerbated by ectopic expression of the iron transporter FET4. These results indicate that yeast can store iron in the vacuole and that CCC1 is involved in the transfer of iron from the cytosol to the vacuole.While iron is a required element for all eucaryotes, it is also potentially toxic. Organisms tightly regulate the concentration of cytosolic iron through regulation of iron uptake and storage. In the past few years the mechanisms that mediate plasma membrane iron transport in the budding yeast Saccharomyces cerevisiae have been described in molecular detail (1). Many of the genes required for plasma membrane transport have been cloned. Much less is known, however, about iron storage. Yeast is distinguished from most eucaryotes in not having ferritin as an iron storage molecule. In this regard yeast are more analogous to plants than vertebrates. In plants, ferritin is restricted to the chloroplasts, as opposed to the cytosol in animals. Plants and yeast are thought to store iron in the vacuole, although there is little compelling proof of that supposition. Three lines of evidence have been used to support the view that the vacuole is an iron storage organelle: 1) vacuolar mutants show increased metal sensitivity (2, 3); 2) iron can be found in vacuoles (4); and 3) there are transport systems capable of extracting iron from the vacuole (5, 6). The increased metal sensitivity of vacuolar mutants, however, can result from increased metal uptake rather than decreased storage (7). Uptake of extracellular fluid by endocytosis, a steady state process would lead to vacuolar iron accumulation, which could be extracted by vacuolar iron transporters. There are little data that demonstrate that iron accumulated in the cytosol can be stored in the vacuole.To determine whether S. cerevisiae store iron in the vacuole we studied vacuolar iron storage in yeast strains unable to endocytose. Our studies demonstrated that yeast do store iron in the vacuole. We further demonstrated that CCC1 is an iron/Mn 2ϩ transporter responsible for storing these two metals in the vacuole. EXPERIMENTAL PROCEDURESYeast Strains, Plasm...

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...

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...