AtATM3 is involved in heavy metal resistance in Arabidopsis

AtATM3, an ATP-binding cassette transporter of Arabidopsis (Arabidopsis thaliana), is a mitochondrial protein involved in the biogenesis of iron-sulfur clusters and iron homeostasis in plants. Our gene expression analysis showed that AtATM3 is up-regulated in roots of plants treated with cadmium [Cd(II)] or lead (II); hence, we investigated whether this gene is involved in heavy metal tolerance. We found that AtATM3-overexpressing plants were enhanced in resistance to Cd, whereas atatm3 mutant plants were more sensitive to Cd than their wild-type controls. Moreover, atatm3 mutant plants expressing 35S promoter-driven AtATM3 were more resistant to Cd than wild-type plants. Since previous reports often showed that the cytosolic glutathione level is positively correlated with heavy metal resistance, we measured nonprotein thiols (NPSH) in these mutant plants. Surprisingly, we found that atatm3 contained more NPSH than the wild type under normal conditions. AtATM3-overexpressing plants did not differ under normal conditions, but contained less NPSH than wild-type plants when exposed to Cd(II). These results suggest a role for AtATM3 in regulating cellular NPSH level, a hypothesis that was further supported by our gene expression study. Genetic or pharmacological inhibition of glutathione biosynthesis led to the elevated expression of AtATM3, whereas expression of the glutathione synthase gene GSH1 was increased under Cd(II) stress and in the atatm3 mutant. Because the closest homolog of AtATM3 in fission yeast (Schizosaccharomyces pombe), HMT1, is a vacuolar membrane-localized phytochelatin-Cd transporter, it is tempting to speculate that glutathione-Cd(II) complexes formed in the mitochondria are exported by AtATM3. In conclusion, our data show that AtATM3 contributes to Cd resistance and suggest that it may mediate transport of glutamine synthetase-conjugated Cd(II) across the mitochondrial membrane.     

An Allelic Mutant Series of ATM3 Reveals Its Key Role in the Biogenesis of Cytosolic Iron-Sulfur Proteins in Arabidopsis

The ATP-binding cassette transporters of mitochondria (ATMs) are highly conserved proteins, but their function in plants is poorly defined. Arabidopsis (Arabidopsis thaliana) has three ATM genes, namely ATM1, ATM2, and ATM3. Using a collection of insertional mutants, we show that only ATM3 has an important function for plant growth. Additional atm3 alleles were identified among sirtinol-resistant lines, correlating with decreased activities of aldehyde oxidases, cytosolic enzymes that convert sirtinol into an auxin analog, and depend on iron-sulfur (Fe-S) and molybdenum cofactor (Moco) as prosthetic groups. In the sirtinol-resistant atm3-3 allele, the highly conserved arginine-612 is replaced by a lysine residue, the negative effect of which could be mimicked in the yeast Atm1p ortholog. Arabidopsis atm3 mutants displayed defects in root growth, chlorophyll content, and seedling establishment. Analyses of selected metal enzymes showed that the activity of cytosolic aconitase (Fe-S) was strongly decreased across the range of atm3 alleles, whereas mitochondrial and plastid Fe-S enzymes were unaffected. Nitrate reductase activity (Moco, heme) was decreased by 50% in the strong atm3 alleles, but catalase activity (heme) was similar to that of the wild type. Strikingly, in contrast to mutants in the yeast and mammalian orthologs, Arabidopsis atm3 mutants did not display a dramatic iron homeostasis defect and did not accumulate iron in mitochondria. Our data suggest that Arabidopsis ATM3 may transport (1) at least two distinct compounds or (2) a single compound required for both Fe-S and Moco assembly machineries in the cytosol, but not iron.Plant cells contain more than 50 iron-sulfur (Fe-S) enzymes that carry out important redox and catalytic functions in many aspects of metabolism (Imsande, 1999; Balk and Lobréaux, 2005). The assembly of Fe-S cofactors is mediated by dedicated machinery of ancient evolutionary origin. In plants, mitochondria harbor homologs of the bacterial ISC (for iron sulfur cluster) proteins, while plastids have inherited the sulfur mobilization machinery from their cyanobacterial ancestor (Balk and Lobréaux, 2005; Kessler and Papenbrock, 2005; Pilon et al., 2006). Plant cytosol contains homologs of the cytosolic Fe-S assembly proteins that have recently been identified in yeast (Lill and Mühlenhoff, 2008), including the scaffold protein AtNBP35 (Bych et al., 2008; Kohbushi et al., 2009) and the hydrogenase-like AtNAR1 (Cavazza et al., 2008). It is thought that cytosolic Fe-S cluster assembly is dependent on at least one of the organelles, because the Cys desulfurases that generate sulfur for Fe-S clusters, CpNifS and NFS1, are strictly localized in the plastids and mitochondria, respectively (Kushnir et al., 2001; Frazzon et al., 2007; Van Hoewyk et al., 2007).In yeast, cytosolic and nuclear Fe-S cluster assembly depends on the mitochondrial ISC pathway and on the ATP-binding cassette (ABC) transporter of the mitochondria, Atm1p (Kispal et al., 1999). Atm1p is classified as a “half-transporter” that functions as a homodimer and is localized in the mitochondrial inner membrane with the ATPase domains at the matrix side (Leighton and Schatz, 1995). The orientation indicates that the direction of transport is from the mitochondrial matrix to the intermembrane space and cytosol. In accordance, mutations of yeast ATM1 cause a defect in cytosolic/nuclear Fe-S cluster assembly but not in mitochondrial Fe-S cluster assembly (Kispal et al., 1999). The substrates of Atm1p, however, or of its functional orthologs in other eukaryotes have not been identified thus far.Yeast atm1 mutations also disrupt iron homeostasis: iron uptake transporters are constitutively expressed independent of the iron concentration, and iron accumulates 10- to 30-fold in the mitochondria (Kispal et al., 1997, 1999). Mutations in the human ortholog ABCB7 are the cause of X-linked sideroblastic anemia with ataxia, in which one of the symptoms is mitochondrial iron overload (Rouault and Tong, 2008). Moreover, ATMs are widespread and highly conserved in β-proteobacteria and could be involved in nickel and cobalt resistance (Mikolay and Nies, 2009).The Arabidopsis (Arabidopsis thaliana) genome contains more than 100 ABC transporters, of which the ATMs form a small subfamily in the B group consisting of three genes: ATM1, ATM2, and ATM3 (Rea, 2007). The genes were first identified in Arabidopsis by Kushnir et al. (2001) and were named STA2, STA3, and STA1, respectively. (A proposal for a unified nomenclature [Verrier et al., 2008] has suggested the gene symbols AtABCB24, AtABCB23, and AtABCB25 for AtATM1, AtATM2, and AtATM3, respectively. However, for consistency with previous publications, we have adhered to the ATM gene symbols in this paper.) Expression of GFP fusions showed that all three ATM proteins localized to mitochondria (Kushnir et al., 2001; Chen et al., 2007). Arabidopsis ATM3 could functionally complement the yeast atm1 phenotype (Kushnir et al., 2001; Chen et al., 2007), whereas Arabidopsis ATM1 complemented poorly and ATM2 expression was toxic in yeast (Chen et al., 2007). Until now, functional analysis of the ATM genes in Arabidopsis has been restricted to one atm3 mutant, called starik (Kushnir et al., 2001), in which the protein lacks the C-terminal ATPase domain. The atm3-1 (sta1) mutant is dwarfed and chlorotic, but the biochemical basis of its pleiotropic phenotypes is unknown. In addition, induced expression of ATM3 in response to cadmium and lead, as well as sensitivity of the atm3-1 (sta1) mutant to cadmium, have implicated the ATM3 transporter in heavy metal tolerance (Kim et al., 2006).We have analyzed a comprehensive set of mutants in the Arabidopsis ATM genes and found that ATM3 plays a key role in plant metabolism, while mutants in ATM1 and ATM2 did not display an obvious phenotype. Genetic and biochemical evidence from an atm3 allelic series showed that ATM3 is important for the activity of cytosolic Fe-S and molybdenum cofactor (Moco) enzymes, but it does not play a major role in metal homeostasis.     

A Conserved Mitochondrial ATP-binding Cassette Transporter Exports Glutathione Polysulfide for Cytosolic Metal Cofactor Assembly

An ATP-binding cassette transporter located in the inner mitochondrial membrane is involved in iron-sulfur cluster and molybdenum cofactor assembly in the cytosol, but the transported substrate is unknown. ATM3 (ABCB25) from Arabidopsis thaliana and its functional orthologue Atm1 from Saccharomyces cerevisiae were expressed in Lactococcus lactis and studied in inside-out membrane vesicles and in purified form. Both proteins selectively transported glutathione disulfide (GSSG) but not reduced glutathione in agreement with a 3-fold stimulation of ATPase activity by GSSG. By contrast, Fe²⁺ alone or in combination with glutathione did not stimulate ATPase activity. Arabidopsis atm3 mutants were hypersensitive to an inhibitor of glutathione biosynthesis and accumulated GSSG in the mitochondria. The growth phenotype of atm3-1 was strongly enhanced by depletion of the mitochondrion-localized, GSH-dependent persulfide oxygenase ETHE1, suggesting that the physiological substrate of ATM3 contains persulfide in addition to glutathione. Consistent with this idea, a transportomics approach using mass spectrometry showed that glutathione trisulfide (GS-S-SG) was transported by Atm1. We propose that mitochondria export glutathione polysulfide, containing glutathione and persulfide, for iron-sulfur cluster assembly in the cytosol.     

MTABC3, a novel mitochondrial ATP-binding cassette protein involved in iron homeostasis

Atm1p, a mitochondrial half-type ATP-binding cassette (ABC) protein in Saccharomyces cerevisiae, transports a precursor of the iron-sulfur (Fe/S) cluster from mitochondria to the cytosol. We have identified a novel half-type human ABC protein, designating it MTABC3 (mammalian mitochondrial ABC protein 3). MTABC3 mRNA is ubiquitously expressed in all of the rat and human tissues examined. MTABC3 protein is shown to be present in the mitochondria, as assessed by immunoblot analysis and confocal microscopic analysis of subcellular fractions of Chinese hamster ovary cells stably expressing MTABC3. Accumulation of iron in the mitochondria, mitochondrial DNA damage, and respiratory dysfunction in the yeast ATM1 mutant strain (atm1-1 mutant cells) were almost fully reversed by expressing MTABC3 in these mutant cells. These results indicate that MTABC3 is a novel ortholog of the yeast and suggest an important role in mitochondrial function. Interestingly, the human MTABC3 gene has been mapped to chromosome 2q36, a region within the candidate locus for lethal neonatal metabolic syndrome, a disorder of the mitochondrial function associated with iron metabolism, indicating that MTABC3 is a candidate gene for this disorder.     

The role of the mitochondrion in cellular iron homeostasis

The yeast ATM1 protein is essential for normal mitochondrial iron homeostasis. Deletion of ATM1 results in mitochondrial iron accumulation and oxidative mitochondrial damage. Mutations in ABC7, the human homolog of ATM1, result in X-linked sideroblastic anemia and ataxia. Here we show that a deletion of ATM1 also has effects on extra-mitochondrial iron metabolism. ATM1-deficient cells have an increased iron requirement for growth. When grown in iron-rich medium, mutant cells accumulate excess mitochondrial iron and have increased expression of the genes required for both high and low affinity iron uptake. Thus, ATM1 mutant cells simultaneously demonstrate features of both iron overload and iron starvation. Yfh1p is the yeast homolog of the human frataxin protein, which is deficient in Friedreich's ataxia. As in atm1 cells, a yfh1 deletion results in both mitochondrial iron accumulation and cytosolic iron starvation. In spite of their apparent roles in cellular iron homeostasis, we find that the expression of neither ATM1 nor YFH1 is responsive to cellular iron status. Based on these observations, we propose a model in which cellular iron is prioritized for use by the mitochondrion, and available to the remainder of the cell only after mitochondrial needs have been fulfilled.     

MDL1 is a high copy suppressor of ATM1: evidence for a role in resistance to oxidative stress

The yeast ATM1 gene is essential for normal cellular iron homeostasis. Deletion of ATM1 results in mitochondrial iron accumulation and increased sensitivity to oxidative stress and transition metal toxicity. Atm1p is an ATP-binding cassette (ABC) transporter localized to the mitochondrial inner membrane. The specific function of Atm1p has not been determined, though roles in both mitochondrial iron export and cytosolic Fe-S cluster assembly have been proposed. We undertook a screen for yeast genes capable of suppressing the abnormalities of cellular iron metabolism demonstrated by Deltaatm1 cells. One of the genes we identified was MDL1, which like ATM1, encodes a mitochondrial inner membrane ABC transporter. Mdl1p has previously been shown to function in the export of peptides from the mitochondrial matrix. We demonstrate that over-expression of MDL1 in Deltaatm1 cells results in a reduction of mitochondrial iron content, and decreased sensitivity to H(2)O(2) and transition metal toxicity. Additionally, in studies of the effect of over-expression and deletion of MDL1, we have identified a novel role for Mdl1p in the regulation of cellular resistance to oxidative stress.     

Maturation of cytosolic iron-sulfur proteins requires glutathione

Glutathione is the major protective agent against oxidative stress in Saccharomyces cerevisiae. Deletion of the GSH1 gene (strain Deltagsh1) encoding the enzyme that catalyzes the first step of glutathione biosynthesis leads to growth arrest, which can be relieved by either glutathione or reducing agents such as dithiothreitol. Because defects in the biosynthesis of cellular iron-sulfur (Fe/S) proteins are associated with increases in glutathione levels, we examined the consequences of glutathione depletion on this essential process. No significant defects were detected in the amounts, activities, and maturation of mitochondrial Fe/S proteins in glutathione-depleted Deltagsh1 cells. On the contrary, the maturation of extra-mitochondrial Fe/S proteins was decreased substantially. The defect was rectified neither by addition of dithiothreitol nor under anaerobic conditions excluding oxidative damage of Fe/S clusters. A double mutant in GSH1 and ATM1 encoding a mitochondrial ATP binding cassette (ABC) transporter involved in cytosolic Fe/S protein maturation is nonviable even in the presence of dithiothreitol. Similar to atm1 and other mutants defective in cytosolic Fe/S protein maturation, mitochondria from glutathione-depleted Deltagsh1 cells accumulated high amounts of iron. Together, our data demonstrate that glutathione, in addition to its protective role against oxidative damage, performs a novel and specific function in the maturation of cytosolic Fe/S proteins.     

Mitochondrial and plastidial COG0354 proteins have folate-dependent functions in iron-sulphur cluster metabolism

COG0354 proteins have been implicated in synthesis or repair of iron/sulfur (Fe/S) clusters in all domains of life, and those of bacteria, animals, and protists have been shown to require a tetrahydrofolate to function. Two COG0354 proteins were identified in Arabidopsis and many other plants, one (At4g12130) related to those of α-proteobacteria and predicted to be mitochondrial, the other (At1g60990) related to those of cyanobacteria and predicted to be plastidial. Grasses and poplar appear to lack the latter. The predicted subcellular locations of the Arabidopsis proteins were validated by in vitro import assays with purified pea organelles and by targeting assays in Arabidopsis and tobacco protoplasts using green fluorescent protein fusions. The At4g12130 protein was shown to be expressed mainly in flowers, siliques, and seeds, whereas the At1g60990 protein was expressed mainly in young leaves. The folate dependence of both Arabidopsis proteins was established by functional complementation of an Escherichia coli COG0354 (ygfZ) deletant; both plant genes restored in vivo activity of the Fe/S enzyme MiaB but restoration was abrogated when folates were eliminated by deleting folP. Insertional inactivation of At4g12130 was embryo lethal; this phenotype was reversed by genetic complementation of the mutant. These data establish that COG0354 proteins have a folate-dependent function in mitochondria and plastids, and that the mitochondrial protein is essential. That plants retain mitochondrial and plastidial COG0354 proteins with distinct phylogenetic origins emphasizes how deeply the extant Fe/S cluster assembly machinery still reflects the ancient endosymbioses that gave rise to plants.     

Dual Localized AtHscB Involved in Iron Sulfur Protein Biogenesis in Arabidopsis

Background

Iron-sulfur clusters are ubiquitous structures which act as prosthetic groups for numerous proteins involved in several fundamental biological processes including respiration and photosynthesis. Although simple in structure both the assembly and insertion of clusters into apoproteins requires complex biochemical pathways involving a diverse set of proteins. In yeast, the J-type chaperone Jac1 plays a key role in the biogenesis of iron sulfur clusters in mitochondria.

Methodology/Principal Findings

In this study we demonstrate that AtHscB from Arabidopsis can rescue the Jac1 yeast knockout mutant suggesting a role for AtHscB in iron sulfur protein biogenesis in plants. In contrast to mitochondrial Jac1, AtHscB localizes to both mitochondria and the cytosol. AtHscB interacts with AtIscU1, an Isu-like scaffold protein involved in iron-sulfur cluster biogenesis, and through this interaction AtIscU1 is most probably retained in the cytosol. The chaperone AtHscA can functionally complement the yeast Ssq1knockout mutant and its ATPase activity is enhanced by AtHscB and AtIscU1. Interestingly, AtHscA is also localized in both mitochondria and the cytosol. Furthermore, AtHscB is highly expressed in anthers and trichomes and an AtHscB T-DNA insertion mutant shows reduced seed set, a waxless phenotype and inappropriate trichome development as well as dramatically reduced activities of the iron-sulfur enzymes aconitase and succinate dehydrogenase.

Conclusions

Our data suggest that AtHscB together with AtHscA and AtIscU1 plays an important role in the biogenesis of iron-sulfur proteins in both mitochondria and the cytosol.     

A Novel Role for Arabidopsis Mitochondrial ABC Transporter ATM3 in Molybdenum Cofactor Biosynthesis

The molybdenum cofactor (Moco) is a prosthetic group required by a number of enzymes, such as nitrate reductase, sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Its biosynthesis in eukaryotes can be divided into four steps, of which the last three are proposed to occur in the cytosol. Here, we report that the mitochondrial ABC transporter ATM3, previously implicated in the maturation of extramitochondrial iron-sulfur proteins, has a crucial role also in Moco biosynthesis. In ATM3 insertion mutants of Arabidopsis thaliana, the activities of nitrate reductase and sulfite oxidase were decreased to ∼50%, whereas the activities of xanthine dehydrogenase and aldehyde oxidase, whose activities also depend on iron-sulfur clusters, were virtually undetectable. Moreover, atm3 mutants accumulated cyclic pyranopterin monophosphate, the first intermediate of Moco biosynthesis, but showed decreased amounts of Moco. Specific antibodies against the Moco biosynthesis proteins CNX2 and CNX3 showed that the first step of Moco biosynthesis is localized in the mitochondrial matrix. Together with the observation that cyclic pyranopterin monophosphate accumulated in purified mitochondria, particularly in atm3 mutants, our data suggest that mitochondria and the ABC transporter ATM3 have a novel role in the biosynthesis of Moco.     

A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA

We have isolated a Saccharomyces cerevisiae mutant that shows an increased tendency to form cytoplasmic petites (respiration-deficient ρ⁻ or ρ⁰ mutants) in response to treatment of cells growing on a solid medium with the DNA-damaging agent methyl methanesulfonate or ultraviolet light. The mutation in this strain, atm1-1, was found to cause a single amino acid substitution in ATM1, a nuclear gene that encodes the mitochondrial ATP-binding cassette (ABC) transporter. When the mutant cells were grown in liquid glucose medium, they accumulated free iron within the mitochondria and at the same time gave rise to spontaneous cytoplasmic petite mutants, as seen previously in cells carrying a mutation in a gene homologous to the human gene responsible for Friedreich's ataxia. Analysis of the effects of free iron and malonic acid (an inhibitor of oxidative respiration in mitochondria) on the incidence of petites among the mutant cells indicated that spontaneous induction of petites was a consequence of oxidative stress rather than a direct effect of either a defect in the ATM1 gene or the accumulation of free iron. We observed an increase in the incidence of strand breaks in the mitochondrial DNA of the atm1-1 mutant cells. Furthermore, we found that rates of induction of petites and accumulation of strand breaks in mitochondrial DNA were enhanced in the atm1-1 mutant by the introduction of another mutation, mhr1-1, which results in a deficiency in mitochondrial DNA repair. These observations indicate that spontaneous induction of petites in the atm1-1 mutant is a consequence of oxidative damage to mitochondrial DNA mediated by enhanced accumulation of mitochondrial iron. Received: 26 March 1999 / Accepted: 29 June 1999     

Role of oxidative phosphorylation in Bax toxicity

The Bcl-2-related protein Bax is toxic when expressed either in yeast or in mammalian cells. Although the mechanism of this toxicity is unknown, it appears to be similar in both cell types and dependent on the localization of Bax to the outer mitochondrial membrane. To investigate the role of mitochondrial respiration in Bax-mediated toxicity, a series of yeast mutant strains was created, each carrying a disruption in either a component of the mitochondrial electron transport chain, a component of the mitochondrial ATP synthesis machinery, or a protein involved in mitochondrial adenine nucleotide exchange. Bax toxicity was reduced in strains lacking the ability to perform oxidative phosphorylation. In contrast, a respiratory-competent strain that lacked the outer mitochondrial membrane Por1 protein showed increased sensitivity to Bax expression. Deficiencies in other mitochondrial proteins did not affect Bax toxicity as long as the ability to perform oxidative phosphorylation was maintained. Characterization of Bax-induced toxicity in wild-type yeast demonstrated a growth inhibition that preceded cell death. This growth inhibition was associated with a decreased ability to carry out oxidative phosphorylation following Bax induction. Furthermore, cells recovered following Bax-induced growth arrest were enriched for a petite phenotype and were no longer able to grow on a nonfermentable carbon source. These results suggest that Bax expression leads to an impairment of mitochondrial respiration, inducing toxicity in cells dependent on oxidative phosphorylation for survival. Furthermore, Bax toxicity is enhanced in yeast deficient in the ability to exchange metabolites across the outer mitochondrial membrane.     

Plant mitochondrial rhomboid, AtRBL12, has different substrate specificity from its yeast counterpart

Rhomboid proteins comprise a class of serine proteases that are conserved in all kingdoms of organisms. They contain six or seven transmembrane helices and control a wide range of cellular functions and developmental processes by intramembrane proteolysis. This paper provides experimental evidence for the existence of rhomboid proteases in plant mitochondria and chloroplasts. Among 15 putative rhomboid-like proteins in Arabidopsis thaliana, we selected five predicted as mitochondrially targeted. For these proteins we performed the GFP transient assay, and identified two homologues, AtRBL11 (At5g25752) and AtRBL12 (At1g18600) to be targeted into plastids and mitochondria, respectively. Phylogenetic analysis reveals that AtRBL12 or AtRBL11 have only one clear orthologue in plant species with completely sequenced genomes. Complementation of the yeast lacking a functional copy of mitochondrial rhomboid with AtRBL12 indicates that this plant protease, in contrast to the human orthologue, does not recognize the yeast substrates, cytochrome c peroxidase (Ccp1) or dynamin-like GTPase (Mgm1). In agreement with this, we did not observe processing of Mgm1 when labeled precursor of this protein was incubated in vitro with Arabidopsis mitochondrial extract. Our results imply that plant mitochondrial rhomboids function in a specific manner and thus differ from their yeast and mammal counterparts.     

Targeting capacity and conservation of PreP homologues localization in mitochondria of different species

Mitochondrial presequences and other unstructured peptides are degraded inside mitochondria by presequence proteases (PrePs) identified in Arabidopsis thaliana (AtPreP), humans (hPreP), and yeast (Cym1/Mop112). The presequences of A. thaliana and human PreP are predicted to consist of 85 and 29 amino acids, respectively, whereas the Saccharomyces cerevisiae Cym1/Mop112 presequence contains only 7 residues. These differences may explain the reported targeting of homologous proteins to different mitochondrial subcompartments. Here we have investigated the targeting capacity of the PreP homologues' presequences. We have produced fusion constructs containing N-terminal portions of AtPreP(1-125), hPreP(1-69), and Cym1(1-40) coupled to green fluorescent protein (GFP) and studied their import into isolated plant, mammalian, and yeast mitochondria, followed by mitochondrial subfractionation. Whereas the AtPreP presequence has the capacity to target GFP into the mitochondrial matrix of all three species, the hPreP presequence only targets GFP to the matrix of mammalian and yeast mitochondria. The Cym1/Mop112 presequence has an overall much weaker targeting capacity and only ensures mitochondrial sorting in its host species yeast. Revisiting the submitochondrial localization of Cym1 revealed that endogenous Cym1/Mop112 is localized to the matrix space, as has been previously reported for the plant and human homologues. Moreover, complementation studies in yeast show that native AtPreP restores the growth phenotype of yeast cells lacking Cym1, demonstrating functional conservation.     

NBS1 is involved in DNA repair and plays a synergistic role with ATM in mediating meiotic homologous recombination in plants

The ability of plants to repair DNA double-strand breaks (DSBs) is essential for growth and fertility. The Arabidopsis DSB repair proteins AtRAD50 and AtMRE11 form part of an evolutionarily conserved complex that, in Saccharomyces cerevisiae and mammals, includes a third component termed XRS2 and NBS1, respectively. The MRN complex (MRX in yeast) has a direct role in DSB repair and is also required for DNA damage signaling and checkpoint activation in a pathway mediated by the protein kinase ATM. This study characterizes Arabidopsis and maize NBS1 orthologues that share conserved protein motifs with human NBS1. Both plant NBS1 proteins interact with the corresponding MRE11 orthologues, and deletion analysis of AtNBS1 defines a region towards the C-terminus (amino acids 465-500) that is required for interaction with AtMRE11. Arabidopsis lines homozygous for a T-DNA insertional mutation in AtNBS1 display hypersensitivity to the DNA cross-linking reagent mitomycin C, and this phenotype can be rescued by complementation with the wild-type gene, consistent with a function for AtNBS1 in plant DSB repair. Analysis of atnbs1-1 atatm double mutants revealed a role for AtNBS1 in meiotic recombination. While atatm mutants produce reduced seed numbers, plants deficient in both AtATM and AtNBS1 are completely infertile. Cytological analysis of these double mutants revealed incomplete chromosome pairing and synapsis in meiotic prophase, and extensive chromosome fragmentation in metaphase I and subsequent stages. These results suggest a novel role for AtNBS1 that is independent of AtATM-mediated signaling and functions in the very early stages of meiosis.     

Dre2, a conserved eukaryotic Fe/S cluster protein, functions in cytosolic Fe/S protein biogenesis

In a forward genetic screen for interaction with mitochondrial iron carrier proteins in Saccharomyces cerevisiae, a hypomorphic mutation of the essential DRE2 gene was found to confer lethality when combined with Δmrs3 and Δmrs4. The dre2 mutant or Dre2-depleted cells were deficient in cytosolic Fe/S cluster protein activities while maintaining mitochondrial Fe/S clusters. The Dre2 amino acid sequence was evolutionarily conserved, and cysteine motifs (CX₂CXC and twin CX₂C) in human and yeast proteins were perfectly aligned. The human Dre2 homolog (implicated in blocking apoptosis and called CIAPIN1 or anamorsin) was able to complement the nonviability of a Δdre2 deletion strain. The Dre2 protein with triple hemagglutinin tag was located in the cytoplasm and in the mitochondrial intermembrane space. Yeast Dre2 overexpressed and purified from bacteria was brown and exhibited signature absorption and electron paramagnetic resonance spectra, indicating the presence of both [2Fe-2S] and [4Fe-4S] clusters. Thus, Dre2 is an essential conserved Fe/S cluster protein implicated in extramitochondrial Fe/S cluster assembly, similar to other components of the so-called CIA (cytoplasmic Fe/S cluster assembly) pathway although partially localized to the mitochondrial intermembrane space.     

Requirements of the cytosolic iron–sulfur cluster assembly pathway in Arabidopsis

The assembly of iron–sulfur (Fe–S) clusters requires dedicated protein factors inside the living cell. Striking similarities between prokaryotic and eukaryotic assembly proteins suggest that plant cells inherited two different pathways through endosymbiosis: the ISC pathway in mitochondria and the SUF pathway in plastids. Fe–S proteins are also found in the cytosol and nucleus, but little is known about how they are assembled in plant cells. Here, we show that neither plastid assembly proteins nor the cytosolic cysteine desulfurase ABA3 are required for the activity of cytosolic aconitase, which depends on a [4Fe–4S] cluster. In contrast, cytosolic aconitase activity depended on the mitochondrial cysteine desulfurase NFS1 and the mitochondrial transporter ATM3. In addition, we were able to complement a yeast mutant in the cytosolic Fe–S cluster assembly pathway, dre2, with the Arabidopsis homologue AtDRE2, but only when expressed together with the diflavin reductase AtTAH18. Spectroscopic characterization showed that purified AtDRE2 could bind up to two Fe–S clusters. Purified AtTAH18 bound one flavin per molecule and was able to accept electrons from NAD(P)H. These results suggest that the proteins involved in cytosolic Fe–S cluster assembly are highly conserved, and that dependence on the mitochondria arose before the second endosymbiosis event leading to plastids.     

The ISC [corrected] proteins Isa1 and Isa2 are required for the function but not for the de novo synthesis of the Fe/S clusters of biotin synthase in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is able to use some biotin precursors for biotin biosynthesis. Insertion of a sulfur atom into desthiobiotin, the final step in the biosynthetic pathway, is catalyzed by biotin synthase (Bio2). This mitochondrial protein contains two iron-sulfur (Fe/S) clusters that catalyze the reaction and are thought to act as a sulfur donor. To identify new components of biotin metabolism, we performed a genetic screen and found that Isa2, a mitochondrial protein involved in the formation of Fe/S proteins, is necessary for the conversion of desthiobiotin to biotin. Depletion of Isa2 or the related Isa1, however, did not prevent the de novo synthesis of any of the two Fe/S centers of Bio2. In contrast, Fe/S cluster assembly on Bio2 strongly depended on the Isu1 and Isu2 proteins. Both isa mutants contained low levels of Bio2. This phenotype was also found in other mutants impaired in mitochondrial Fe/S protein assembly and in wild-type cells grown under iron limitation. Low Bio2 levels, however, did not cause the inability of isa mutants to utilize desthiobiotin, since this defect was not cured by overexpression of BIO2. Thus, the Isa proteins are crucial for the in vivo function of biotin synthase but not for the de novo synthesis of its Fe/S clusters. Our data demonstrate that the Isa proteins are essential for the catalytic activity of Bio2 in vivo.