The YFH1 gene is the yeast homologue of the human FRDA gene, which encodes the frataxin protein. Saccharomyces cerevisiae cells lacking the YFH1 gene showed very low cytochrome content. In Deltayfh1 strains, the level of ferrochelatase (Hem15p) was very low, as a result of transcriptional repression of HEM15. However, the low amount of Hem15p was not the cause of haeme deficiency in Deltayfh1 cells. Ferrochelatase, a mitochondrial protein, able to mediate insertion of iron or zinc into the porphyrin precursor, made primarily the zinc protoporphyrin product. Zinc protoporphyrin instead of haeme accumulated during growth of Deltayfh1 mutant cells and, furthermore, preferential formation of zinc protoporphyrin was observed in real time. The method for these studies involved direct presentation of porphyrin to mitochondria and to ferrochelatase of permeabilized cells with intact architecture, thereby specifically testing the iron delivery portion of the haeme biosynthetic pathway. The studies showed that Deltayfh1 mutant cells are defective in iron use by ferrochelatase. Mössbauer spectroscopic analysis showed that iron was present as amorphous nano-particles of ferric phosphate in Deltayfh1 mitochondria, which could explain the unavailability of iron for haeme synthesis. A high frequency of suppressor mutations was observed, and the phenotype of such mutants was characterized by restoration of haeme synthesis in the absence of Yfh1p. Suppressor strains showed a normal cytochrome content, normal respiration, but remained defective in Fe-S proteins and still accumulated iron into mitochondria although to a lesser extent. Yfh1p and Hem15p were shown to interact in vitro by Biacore studies. Our results suggest that Yfh1 mediates iron use by ferrochelatase.

The trace element copper (Cu) is essential for cell growth. In this report we describe the identification of a new component of the high-affinity Cu transport machinery in yeast, encoded by the CTR3 gene. Ctr3p is a small intraceUular cysteine-rich integral membrane protein that restores high-affinity Cu uptake, Cu, Zn superoxide dismutase activity, ferrous iron transport, and respiratory proficiency to strains lacking the CTR1 (Cu transporter 1) gene. In most commonly used Saccharomyces cerevisiae laboratory strains, expression of CTR3 is abolished by a Ty2 transposon insertion that separates the CTR3 promoter from the transcriptional start sites by 6 kb. In strains that do not possess a Ty2 transposon at the CTR3 locus, expression of CTR3 is repressed by copper and activated by copper starvation. In such strains inactivation of both CTRI and CTR3 is required to generate lethal copper-deficient phenotypes. Although Ctrlp and Ctr3p can function independently in copper transport, the expression of both proteins provides maximal copper uptake and growth rate under copper-limiting conditions. These results underscore the importance of mobile DNA elements in the alteration of gene function and phenotypic variation.

Nfs1p is the yeast homolog of the bacterial proteins NifS and IscS, enzymes that release sulfur from cysteine for iron-sulfur cluster assembly. Here we show that the yeast mitochondrial protein Nfs1p regulates cellular and mitochondrial iron homeostasis. A strain of Saccharomyces cerevisiae, MA14, with a missense NFS1 allele (I191S) was isolated in a screen for altered iron-dependent gene regulation. This mutant exhibited constitutive up-regulation of the genes of the cellular iron uptake system, mediated through effects on the Aft1p iron-regulatory protein. Iron accumulating in the mutant cells was retained in the mitochondrial matrix while, at the same time, iron-sulfur proteins were deficient. In this work, the yeast protein was localized to mitochondria, and the gene was shown to be essential for viability. Furthermore, Nfs1p in the MA14 mutant was found to be markedly decreased, suggesting that this low protein level produced the observed regulatory effects. This hypothesis was confirmed by experiments in which expression of wild-type Nfs1p from a regulated galactose-induced promoter was turned off, leading to recapitulation of the iron regulatory phenotypes characteristic of the MA14 mutant. These phenotypes include decreases in iron-sulfur protein activities coordinated with increases in cellular iron uptake and iron distribution to mitochondria.Iron-sulfur (Fe-S) clusters are cofactors of proteins involved in oxidation-reduction, electron transport, metabolic conversions, and regulatory functions (1). The iron and sulfur are assembled in fixed stoichiometries (e.g. 2Fe-2S, 4Fe-4S) characteristic of the particular protein and coordinated to critical cysteines in the primary peptide backbone (2). Within cells, iron availability for synthesis of iron-sulfur proteins and other biological functions must be tightly regulated, because excess iron is toxic (3). Excess iron leads to free radical reactions that damage membranes, proteins, and DNA (4). Here we describe a regulatory control mechanism that coordinates iron uptake, iron distribution, and the levels of iron-sulfur cluster proteins in the eukaryote Saccharomyces cerevisiae. The regulator responsible for these effects is Nfs1p.Examination of the S. cerevisiae genome data base reveals that Nfs1p is the single yeast homolog of bacterial IscS (5, 6) and NifS (7). There is strong evidence, both biochemical and genetic, showing that the bacterial protein NifS mobilizes sulfur from cysteine and mediates Fe-S cluster assembly. Bacterial mutants of NifS were found to be deficient in the assembly of both Fe protein and MoFe protein subunits of nitrogenase (8, 9). NifS through its enzymatic activity was found to reactivate the apo form of nitrogenase in which the Fe-S cluster was removed by chelation (10). Elegant biochemical work has elucidated this catalytic process: NifS was shown to be a pyridoxal phosphate-containing homodimer that catalyzes the formation of elemental sulfur from L-cysteine (7). A conserved lysine residue in the bacterial NifS protein (equiva...

High-affinity iron uptake by a ferrous permease in the opportunistic pathogen Candida albicans is required for virulence. Here this iron uptake system has been characterized by investigating three distinct activities : an externally directed surface ferric reductase, a membrane-associated PPD ( pphenylenediamine) oxidase and a cellular ferrous iron transport activity. Copper was required for the PPD oxidase and ferrous transport activities. In contrast, copper was not required for iron uptake from siderophores. Addition of iron to the growth medium repressed ferric reductase and ferrous transport, indicating homeostatic regulation. To identify the genes involved, orthologous mutants of Saccharomyces cerevisiae were transformed with a genomic library of C. albicans. CFL95, a gene with sequence similarity to ferric reductases, restored reductase activity to the orthologous S. cerevisiae mutant. CaFTR2 and CaFTR1, genes with homology to ferrous permeases, conferred ferrous transport activity to the orthologous S. cerevisiae mutant. However, neither a genomic library nor CaFET99, a multicopper oxidase homologue and candidate gene for the PPD oxidase, complemented the S. cerevisiae mutant, possibly because of problems with targeting or assembly. Transcripts for CFL95, CaFTR1 and CaFET99 were strongly repressed by iron, whereas the CaFTR2 transcript was induced by iron. Deletion of the TUP1 regulator perturbed the homeostatic control of reductive iron uptake. Incidentally, iron starvation was noted to induce flavin production and this was misregulated in the absence of TUP1 control. The opposite regulation of two iron permease genes and the role of TUP1 indicate that the process of iron acquisition by C. albicans may be more complex and potentially more adaptable than by S. cerevisiae.

Here we show that the yeast mitochondrial chaperone Ssc2p, a homolog of mt-Hsp70, plays a critical role in mitochondrial iron homeostasis. Yeast with ssc2-1 mutations were identified by a screen for altered iron-dependent gene regulation and mitochondrial dysfunction. These mutants exhibit increased cellular iron uptake, and the iron accumulates exclusively within mitochondria. Yfh1p is homologous to frataxin, the human protein implicated in the neurodegenerative disease, Friedreich's ataxia. Like mutants of yfh1, ssc2-1 mutants accumulate vast quantities of iron in mitochondria. Furthermore, using import studies with isolated mitochondria, we demonstrate a specific role for Ssc2p in the maturation of Yfh1p within this organelle. This function for a mitochondrial Hsp70 chaperone is likely to be conserved, implying that a human homolog of Ssc2p may be involved in iron homeostasis and in neurodegenerative disease.