Yeast Frataxin Homologue Research Articles

Yeast cells depleted of the frataxin homolog Yfh1 redistribute cellular iron: Studies using Mössbauer spectroscopy and mathematical modeling

The neurodegenerative disease Friedreich’s ataxia arises from a deficiency of frataxin, a protein that promotes iron–sulfur cluster (ISC) assembly in mitochondria. Here, primarily using Mössbauer spectroscopy, we investigated the iron content of a yeast strain in which expression of yeast frataxin homolog 1 (Yfh1), oxygenation conditions, iron concentrations, and metabolic modes were varied. We found that aerobic fermenting Yfh1-depleted cells grew slowly and accumulated FeIII nanoparticles, unlike WT cells. Under hypoxic conditions, the same mutant cells grew at rates similar to WT cells, had similar iron content, and were dominated by FeII rather than FeIII nanoparticles. Furthermore, mitochondria from mutant hypoxic cells contained approximately the same levels of ISCs as WT cells, confirming that Yfh1 is not required for ISC assembly. These cells also did not accumulate excessive iron, indicating that iron accumulation into yfh1-deficient mitochondria is stimulated by O2. In addition, in aerobic WT cells, we found that vacuoles stored FeIII, whereas under hypoxic fermenting conditions, vacuolar iron was reduced to FeII. Under respiring conditions, vacuoles of Yfh1-deficient cells contained FeIII, and nanoparticles accumulated only under aerobic conditions. Taken together, these results informed a mathematical model of iron trafficking and regulation in cells that could semiquantitatively simulate the Yfh1-deficiency phenotype. Simulations suggested partially independent regulation in which cellular iron import is regulated by ISC activity in mitochondria, mitochondrial iron import is regulated by a mitochondrial FeII pool, and vacuolar iron import is regulated by cytosolic FeII and mitochondrial ISC activity.

Nitric oxide prevents Aft1 activation and metabolic remodeling in frataxin-deficient yeast

Yeast frataxin homolog (Yfh1) is the orthologue of human frataxin, a mitochondrial protein whose deficiency causes Friedreich Ataxia. Yfh1 deficiency activates Aft1, a transcription factor governing iron homeostasis in yeast cells. Although the mechanisms causing this activation are not completely understood, it is assumed that it may be caused by iron-sulfur deficiency. However, several evidences indicate that activation of Aft1 occurs in the absence of iron-sulfur deficiency. Besides, Yfh1 deficiency also leads to metabolic remodeling (mainly consisting in a shift from respiratory to fermentative metabolism) and to induction of Yhb1, a nitric oxide (NO) detoxifying enzyme. In this work, we have used conditional Yfh1 mutant yeast strains to investigate the relationship between NO, Aft1 activation and metabolic remodeling. We have observed that NO prevents Aft1 activation caused by Yfh1 deficiency. This phenomenon is not observed when Aft1 is activated by iron scarcity or impaired iron-sulfur biogenesis. In addition, analyzing key metabolic proteins by a targeted proteomics approach, we have observed that NO prevents the metabolic remodeling caused by Yfh1 deficiency. We conclude that Aft1 activation in Yfh1-deficient yeasts is not caused by iron-sulfur deficiency or iron scarcity. Our hypothesis is that Yfh1 deficiency leads to the presence of anomalous iron species that can compromise iron bioavailability and activate a signaling cascade that results in Aft1 activation and metabolic remodeling.

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN

The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of ∼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly.

Iron loading site on the Fe-S cluster assembly scaffold protein is distinct from the active site.

Iron-sulfur (Fe-S) cluster containing proteins are utilized in almost every biochemical pathway. The unique redox and coordination chemistry associated with the cofactor allows these proteins to participate in a diverse set of reactions, including electron transfer, enzyme catalysis, DNA synthesis and signaling within several pathways. Due to the high reactivity of the metal, it is not surprising that biological Fe-S cluster assembly is tightly regulated within cells. In yeast, the major assembly pathway for Fe-S clusters is the mitochondrial ISC pathway. Yeast Fe-S cluster assembly is accomplished using the scaffold protein (Isu1) as the molecular foundation, with assistance from the cysteine desulfurase (Nfs1) to provide sulfur, the accessory protein (Isd11) to regulate Nfs1 activity, the yeast frataxin homologue (Yfh1) to regulate Nfs1 activity and participate in Isu1 Fe loading possibly as a chaperone, and the ferredoxin (Yah1) to provide reducing equivalents for assembly. In this report, we utilize calorimetric and spectroscopic methods to provide molecular insight into how wt-Isu1 from S. cerevisiae becomes loaded with iron. Isothermal titration calorimetry and an iron competition binding assay were developed to characterize the energetics of protein Fe(II) binding. Differential scanning calorimetry was used to identify thermodynamic characteristics of the protein in the apo state or under iron loaded conditions. Finally, X-ray absorption spectroscopy was used to characterize the electronic and structural properties of Fe(II) bound to Isu1. Current data are compared to our previous characterization of the D37A Isu1 mutant, and these suggest that when Isu1 binds Fe(II) in a manner not perturbed by the D37A substitution, and that metal binding occurs at a site distinct from the cysteine rich active site in the protein.

Overlapping Binding Sites of the Frataxin Homologue Assembly Factor and the Heat Shock Protein 70 Transfer Factor on the Isu Iron-Sulfur Cluster Scaffold Protein

In mitochondria FeS clusters, prosthetic groups critical for the activity of many proteins, are first assembled on Isu, a 14-kDa scaffold protein, and then transferred to recipient apoproteins. The assembly process involves interaction of Isu with both Nfs1, the cysteine desulfurase serving as a sulfur donor, and the yeast frataxin homolog (Yfh1) serving as a regulator of desulfurase activity and/or iron donor. Here, based on the results of biochemical experiments with purified wild-type and variant proteins, we report that interaction of Yfh1 with both Nfs1 and Isu are required for formation of a stable tripartite assembly complex. Disruption of either Yfh1-Isu or Nfs1-Isu interactions destabilizes the complex. Cluster transfer to recipient apoprotein is known to require the interaction of Isu with the J-protein/Hsp70 molecular chaperone pair, Jac1 and Ssq1. Here we show that the Yfh1 interaction with Isu involves the PVK sequence motif, which is also the site key for the interaction of Isu with Hsp70 Ssq1. Coupled with our previous observation that Nfs1 and Jac1 binding to Isu is mutually exclusive due to partially overlapping binding sites, we propose that such mutual exclusivity of cluster assembly factor (Nfs1/Yfh1) and cluster transfer factor (Jac1/Ssq1) binding to Isu has functional consequences for the transition from the assembly process to the transfer process, and thus regulation of the biogenesis of FeS cluster proteins.

Frataxin Directly Stimulates Mitochondrial Cysteine Desulfurase by Exposing Substrate-binding Sites, and a Mutant Fe-S Cluster Scaffold Protein with Frataxin-bypassing Ability Acts Similarly

For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the "buried" substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation.

Metabolic remodeling in frataxin-deficient yeast is mediated by Cth2 and Adr1

Frataxin is a mitochondrial protein involved in iron metabolism whose deficiency in humans causes Friedreich ataxia. We performed transcriptomic and proteomic analyses of conditional Yeast Frataxin Homologue (Yfh1) mutants (tetO7-YFH1) to investigate metabolic remodeling upon Yfh1 depletion. These studies revealed that Yfh1 depletion leads to downregulation of many glucose-repressed genes. Most of them were Adr1 targets, a key transcription factor required for growth in non-fermentable carbon sources. Using a GFP-tagged Adr1, we observed that Yfh1 depletion promotes the export of Adr1 from the nucleus to the cytosol without affecting its protein levels. This effect was also observed upon H2O2 treatment, but not by iron overload/starvation, indicating the presence of a regulatory pathway involved in Adr1 export and inactivation upon stress conditions. We also observed that CTH2, a gene involved in the mRNA degradation of several iron-containing enzymes, was induced upon Yfh1 depletion. Accordingly, decreased levels of aconitase and succinate dehydrogenase were observed. Nevertheless, their levels were maintained in a Δcth2 mutant even in the absence of Yfh1. From these results we can conclude that, in addition to altering iron homeostasis, frataxin depletion involves drastic metabolic remodeling governed by Adr1 and Cth2 that finally leads to downregulation of iron–sulfur proteins and other proteins involved in respiratory metabolism.

A Dynamic Model of the Proteins that Form the Initial Iron-Sulfur Cluster Biogenesis Machinery in Yeast Mitochondria

The assembly of iron-sulfur clusters (ISCs) in eukaryotes involves the protein Frataxin. Deficits in this protein have been associated with iron inside the mitochondria and impair ISC biogenesis as it is postulated to act as the iron donor for ISCs assembly in this organelle. A pronounced lack of Frataxin causes Friedreich's Ataxia, which is a human neurodegenerative and hereditary disease mainly affecting the equilibrium, coordination, muscles and heart. Moreover, it is the most common autosomal recessive ataxia. High similarities between the human and yeast molecular mechanisms that involve Frataxin have been suggested making yeast a good model to study that process. In yeast, the protein complex that forms the central assembly platform for the initial step of ISC biogenesis is composed by yeast frataxin homolog, Nfs1-Isd11 and Isu. In general, it is commonly accepted that protein function involves interaction with other protein partners, but in this case not enough is known about the structure of the protein complex and, therefore, how it exactly functions. The objective of this work is to model the protein complex in order to gain insight into structural details that end up with its biological function. To achieve this goal several bioinformatics tools, modeling techniques and protein docking programs have been used. As a result, the structure of the protein complex and the dynamic behavior of its components, along with that of the iron and sulfur atoms required for the ISC assembly, have been modeled. This hypothesis will help to better understand the function and molecular properties of Frataxin as well as those of its ISC assembly protein partners.

The Molecular Basis of Iron-induced Oligomerization of Frataxin and the Role of the Ferroxidation Reaction in Oligomerization

The role of the mitochondrial protein frataxin in iron storage and detoxification, iron delivery to iron-sulfur cluster biosynthesis, heme biosynthesis, and aconitase repair has been extensively studied during the last decade. However, still no general consensus exists on the details of the mechanism of frataxin function and oligomerization. Here, using small-angle x-ray scattering and x-ray crystallography, we describe the solution structure of the oligomers formed during the iron-dependent assembly of yeast (Yfh1) and Escherichia coli (CyaY) frataxin. At an iron-to-protein ratio of 2, the initially monomeric Yfh1 is converted to a trimeric form in solution. The trimer in turn serves as the assembly unit for higher order oligomers induced at higher iron-to-protein ratios. The x-ray crystallographic structure obtained from iron-soaked crystals demonstrates that iron binds at the trimer-trimer interaction sites, presumably contributing to oligomer stabilization. For the ferroxidation-deficient D79A/D82A variant of Yfh1, iron-dependent oligomerization may still take place, although >50% of the protein is found in the monomeric state at the highest iron-to-protein ratio used. This demonstrates that the ferroxidation reaction controls frataxin assembly and presumably the iron chaperone function of frataxin and its interactions with target proteins. For E. coli CyaY, the assembly unit of higher order oligomers is a tetramer, which could be an effect of the much shorter N-terminal region of this protein. The results show that understanding of the mechanistic features of frataxin function requires detailed knowledge of the interplay between the ferroxidation reaction, iron-induced oligomerization, and the structure of oligomers formed during assembly.

Rim2, a pyrimidine nucleotide exchanger, is needed for iron utilization in mitochondria

Mitochondria transport and utilize iron for the synthesis of haem and Fe-S clusters. Although many proteins are known to be involved in these processes, additional proteins are likely to participate. To test this hypothesis, in the present study we used a genetic screen looking for yeast mutants that are synthetically lethal with the mitochondrial iron carriers Mrs3 and Mrs4. Several genes were identified, including an isolate mutated for Yfh1, the yeast frataxin homologue. All such triple mutants were complemented by increased expression of Rim2, another mitochondrial carrier protein. Rim2 overexpression was able to enhance haem and Fe-S cluster synthesis in wild-type or Δmrs3/Δmrs4 backgrounds. Conversely Rim2 depletion impaired haem and Fe-S cluster synthesis in wild-type or Δmrs3/Δmrs4 backgrounds, indicating a unique requirement for this mitochondrial transporter for these processes. Rim2 was previously shown to mediate pyrimidine exchange in and out of vesicles. In the present study we found that isolated mitochondria lacking Rim2 exhibited concordant iron defects and pyrimidine transport defects, although the connection between these two functions is not explained. When organellar membranes were ruptured to bypass iron transport, haem synthesis from added iron and porphyrin was still markedly deficient in Rim2-depleted mitochondrial lysate. The results indicate that Rim2 is a pyrimidine exchanger with an additional unique function in promoting mitochondrial iron utilization.

Co-precipitation of Phosphate and Iron Limits Mitochondrial Phosphate Availability in Saccharomyces cerevisiae Lacking the Yeast Frataxin Homologue (YFH1)

Saccharomyces cerevisiae cells lacking the yeast frataxin homologue (Δyfh1) accumulate iron in the mitochondria in the form of nanoparticles of ferric phosphate. The phosphate content of Δyfh1 mitochondria was higher than that of wild-type mitochondria, but the proportion of mitochondrial phosphate that was soluble was much lower in Δyfh1 cells. The rates of phosphate and iron uptake in vitro by isolated mitochondria were higher for Δyfh1 than wild-type mitochondria, and a significant proportion of the phosphate and iron rapidly became insoluble in the mitochondrial matrix, suggesting co-precipitation of these species after oxidation of iron by oxygen. Increasing the amount of phosphate in the medium decreased the amount of iron accumulated by Δyfh1 cells and improved their growth in an iron-dependent manner, and this effect was mostly transcriptional. Overexpressing the major mitochondrial phosphate carrier, MIR1, slightly increased the concentration of soluble mitochondrial phosphate and significantly improved various mitochondrial functions (cytochromes, [Fe-S] clusters, and respiration) in Δyfh1 cells. We conclude that in Δyfh1 cells, soluble phosphate is limiting, due to its co-precipitation with iron.

Evidence that yeast frataxin is not an iron storage protein in vivo

Yeast cells deficient in the yeast frataxin homolog (Yfh1p) accumulate iron in their mitochondria. Whether this iron is toxic, however, remains unclear. We showed that large excesses of iron in the growth medium did not inhibit growth and did not decrease cell viability. Increasing the ratio of mitochondrial iron-to-Yfh1p by decreasing the steady-state level of Yfh1p to less than 100 molecules per cell had very few deleterious effects on cell physiology, even though the mitochondrial iron concentration greatly exceeded the iron-binding capacity of Yfh1p in these conditions. Mössbauer spectroscopy and FPLC analyses of whole mitochondria or of isolated mitochondrial matrices showed that the chemical and biochemical forms of the accumulated iron in mitochondria of mutant yeast strains (Δ yfh1, Δ ggc1 and Δ ssq1) displayed a nearly identical distribution. This was also the case for Δ ggc1 cells, in which Yfh1p was overproduced. In these mitochondria, most of the iron was insoluble, and the ratio of soluble-to-insoluble iron did not change when the amount of Yfh1p was increased up to 4500 molecules per cell. Our results do not privilege the hypothesis of Yfh1p being an iron storage protein in vivo.

Yeast frataxin mutants display decreased superoxide dismutase activity crucial to promote protein oxidative damage

Iron overload is involved in several pathological conditions, including Friedreich ataxia, a disease caused by decreased expression of the mitochondrial protein frataxin. In a previous study, we identified 14 proteins selectively oxidized in yeast cells lacking Yfh1, the yeast frataxin homolog. Most of these were magnesium-binding proteins. Decreased Mn-SOD activity, oxidative damage to CuZn-SOD, and increased levels of chelatable iron were also observed in this model. This study explores the relationship between low SOD activity, the presence of chelatable iron, and protein damage. We observed that addition of copper and manganese to the culture medium restored SOD activity and prevented both oxidative damage and inactivation of magnesium-binding proteins. This protection was compartment specific: recovery of mitochondrial enzymes required the addition of manganese, whereas cytosolic enzymes were recovered by adding copper. Copper treatment also decreased Δ yfh1 sensitivity to menadione. Finally, a Δ sod1 mutant showed high levels of chelatable iron and inactivation of magnesium-binding enzymes. These results suggest that reduced superoxide dismutase activity contributes to the toxic effects of iron overloading. This would also apply to pathologies involving iron accumulation.

Frataxin interacts with Isu1 through a conserved tryptophan in its β-sheet

Friedreich's ataxia is a neurodegenerative disease caused by the low expression of frataxin, a mitochondrial iron-binding protein which plays an important, but non-essential, role in the formation of iron-sulfur (Fe/S) clusters. It has been shown that Yfh1, the yeast frataxin homologue, interacts functionally and physically with Isu1, the scaffold protein on which the Fe/S clusters are assembled. The large beta-sheet platform of frataxin is a good ligand candidate for this interaction. We have generated 12 yeast mutants in conserved residues of the beta-sheet protruding at the surface or buried in the protein core. The Q129A, I130A, W131A(F) and R141A mutations, which reside in surface exposed residues of the fourth and fifth beta-strands, result in severe cell growth inhibition on high-iron media and low aconitase activity, indicating that Fe/S cluster biosynthesis is impaired. The null phenotype of the I130A mutant results from the high instability of the protein, pointing that this buried residue is essential for folding. In contrast, Gln-129, Trp-131 and Arg-141 residues which are spatially closely clustered define a patch important for protein function. Co-immunoprecipitation experiments using cell extracts show that W131A, unlike W131F, is the sole mutation that strongly decreases the interaction with Isu1. Therefore, Trp-131, which is the only strictly conserved frataxin residue in all sequenced species, appears as a major contributor to the interaction with Isu1 through its surface-exposed aromatic side chain.

Deficiency in frataxin homologue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation

Pichia guilliermondii is a representative of yeast species that overproduce riboflavin (vitamin B2) in response to iron deprivation. P. guilliermondii YFH1 gene coding for frataxin homologue, eukaryotic mitochondrial protein involved in iron trafficking and storage, was identified and deleted. Constructed P. guilliermondii Δyfh1 mutant grew very poorly in a sucrose-containing synthetic medium supplemented with sulfate or sulfite as a sole sulfur source. Addition of sodium sulfide, glutathione, cysteine, methionine, N-acetyl-L-cysteine partially restored growth rate of the mutant suggesting that it is impaired in sulfate assimilation. Cellular iron content in Δyfh1 mutant was ~3-3.5 times higher as compared to the parental strain. It produced 50-70 times more riboflavin in iron sufficient synthetic media relative to the parental wildtype strain. Biomass yield of the mutant in the synthetic glutathione containing medium supplemented with glycerol as a sole carbon source was 1.4- and 2.6-fold increased as compared to sucrose and succinate containing media, respectively. Oxygen uptake of the Δyfh1 mutant on sucrose, glycerol or succinate, when compared to the parental strain, was decreased 5.5-, 1.7- and 1.5-fold, respectively. Substitution of sucrose or glycerol in the synthetic iron sufficient medium with succinate completely abolished riboflavin overproduction by the mutants. Deletion of the YFH1 gene caused hypersensitivity to hydrogen peroxide and exogenously added riboflavin and led to alterations in superoxide dismutase activities. Thus, deletion of the gene coding for yeast frataxin homologue has pleiotropic effect on metabolism in P. guilliermondii.

Overexpression of the yeast frataxin homolog (Yfh1): Contrasting effects on iron–sulfur cluster assembly, heme synthesis and resistance to oxidative stress

Friedreich’s ataxia is generally associated with defects in [Fe–S] cluster assembly/stability and heme synthesis and strong susceptibility to oxidative stress. We used the yeast ( Saccharomyces cerevisiae) model of Friedreich’s ataxia to study the physiological consequences of modulating the expression of the frataxin gene ( YFH1). We show that the number of frataxin molecules per wild-type cell varies from less than 200 to 1500 according to the iron concentration in the medium. Cells overexpressing YFH1 on a plasmid (2μYFH1; about 3500 molecules Yfh1/cell) took up more iron than wild-type cells and displayed defective [Fe–S] cluster assembly/stability in vivo. By contrast, endogenous mitochondrial iron was more available to ferrochelatase in 2μYFH1 cells than in wild-type cells, resulting in higher levels of heme synthesis in vitro. Frataxin overproduction resulted in a shift from frataxin trimers to frataxin oligomers of higher molecular mass in the mitochondrial matrix. Much fewer carbonylated proteins were present in 2μYFH1 cells, and these cells were more resistant to oxidizing agents than wild-type cells, which probably resulted from the lower production of hydrogen peroxide by the mitochondria of 2μYFH1 cells compared to wild-type cells. To our knowledge, this work is the first description where major frataxin-related phenotypes ([Fe–S] cluster assembly and heme synthesis) can be split in vivo, suggesting that frataxin has independent roles in both processes, and that the optimal conditions for these independent roles are different.

Frataxin and Mitochondrial Carrier Proteins, Mrs3p and Mrs4p, Cooperate in Providing Iron for Heme Synthesis

Frataxin is a conserved mitochondrial protein implicated in cellular iron metabolism. Deletion of the yeast frataxin homolog (YFH1) was combined with deletions of MRS3 and MRS4, mitochondrial carrier proteins implicated in iron homeostasis. As previously reported, the Deltayfh1 mutant accumulated iron in mitochondria, whereas the triple mutant (DeltaDeltaDelta) did not. When wild-type, Deltamrs3/4, Deltayfh1, and DeltaDeltaDelta strains were incubated anaerobically, all strains were devoid of heme and protected from iron and oxygen toxicity. The cultures were then shifted to air for a short time (4-5 h) or a longer time (15 h), and the evolving mutant phenotypes were analyzed (heme-dependent growth, total heme, cytochromes, heme proteins, and iron levels). A picture emerges from these data of defective heme formation in the mutants, with a markedly more severe defect in the DeltaDeltaDelta than in the individual Deltamrs3/4 or Deltayfh1 mutants (a "synthetic" defect in the genetic sense). The defect(s) in heme formation could be traced to lack of iron. Using a real time assay of heme biosynthesis, porphyrin precursor and iron were presented to permeabilized cells, and the appearance and disappearance of fluorescent porphyrins were followed. The Mrs3/4p carriers were required for rapid iron transport into mitochondria for heme synthesis, whereas there was also evidence for an alternative slower system. A different role for Yfh1p was observed under conditions of low mitochondrial iron and aerobic growth (revealed in the DeltaDeltaDelta), acting to protect bioavailable iron within mitochondria and to facilitate its use for heme synthesis.

Functional frataxin homologue in hydrogenosomes of Trichomonas vaginalis

The cellular structure of Trichomonas vaginalis substantially differs from other eukaryotes. Instead of mitochondria it possesses hydrogenosomes where key metabolic reactions are mediated by FeS proteins. The gene expression and the activity of FeS proteins is fully dependent on the iron availability. Although hydrogenosomes contain a high amount of iron, there is no information about hydrogenosomal iron homeostasis and FeS cluster formation. In mitochondria, frataxin plays an unclear role in iron metabolism. The deficiency of frataxin in human causes Friedreich's ataxia, which is associated with an increased level of mitochondrial iron. It has been suggested that frataxin is involved in various functions such as FeS cluster formation or iron storage. We cloned T. vaginalis frataxin homologue (TvFTX) coding for a protein of 121 amino acids. Although lacking transcription initiator element in 5′UTR region, the expression of tvftx was verified by a nuclear run‐on assay. Moreover, the gene upregulation in iron deficiency was shown. The N‐terminal part of TvFTX contains an extension similar to presequences targeting proteins to hydrogenosomes and mitochondria. To identify its cellular localization we transfected T. vaginalis with TvFTX fused to HA tag. The immunodetection of HA tag specifically labeled hydrogenosomes and the corresponding cellular fraction. The hydrogenosomal targeting sequence however does not support the translocation into yeast mitochondria. Thus for the complementation of yeast frataxin homologue (YFH1) a mitochondrial targeting sequence is required. When expressed in mitochondria of δYFH1 cells TvFTX complements the mutant growth defect and restores the activity of aconitase (iron‐sulfur protein). Together with recent characterization of T. vaginalis cystein desulfurase these data indicate the conserved role of hydrogenosomes and mitochondria in FeS cluster assembly.

Mitochondrial functioning of constitutive iron uptake mutations in Cryptococcus neoformans

Randomly obtained, constitutive plasma membrane ferric reductase/ferrous uptake mutants of Cryptococcus neoformans were mapped to four distinct loci by meiotic analysis. One of those loci, FRR1 , was previously found homologous to MRS3 and MRS4 of Saccharomyces cerevisiae , which determine proteins involved in mitochondrial transport of iron. We were able to complement, clone, sequence and thereby identify two of the three remaining constitutive uptake loci. FRR3 was found to be homologous to ISU1 and ISU2 of S. cerevisiae, which form mitochondrial iron-sulfur complexes; FRR4 was found to be homologous to YFH1, the yeast frataxin homologue, which also participates in iron-sulfur cluster biogenesis. Because of the constitutive iron uptake seen in these mutants, mitochondria appear to have a central role in the cellular iron economy; moreover, as judged by our mutational statistics, the genetic machinery for mitochondrial iron accumulation may be more complex than that of the cytoplasm.

Iron-induced oligomerization of yeast frataxin homologue Yfh1 is dispensable in vivo.

The neurodegenerative disease Friedreich's ataxia is caused by reduced levels of frataxin, a mitochondrial matrix protein. The in vivo role of frataxin is under debate. Frataxin, as well as its yeast homologue Yfh1, binds multiple iron atoms as an oligomer and has been proposed to function as a crucial iron-storage protein. We identified a mutant Yfh1 defective in iron-induced oligomerization. This mutant protein was able to replace functionally wild-type Yfh1, even when expressed at low levels, when mitochondrial iron levels were high and in mutant strains having deletions of genes that had synthetic growth defects with a YFH1 deletion. The ability of an oligomerization-deficient Yfh1 to function in vivo suggests that oligomerization, and thus oligomerization-induced iron storage, is not a critical function of Yfh1. Rather, the capacity of this oligomerization-deficient mutant to interact with the Isu protein suggests a more direct role of Yfh1 in iron-sulphur cluster biogenesis.