An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins

Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria. Furthermore, Erv1p was found to be important for cellular iron homeostasis. The homologous mammalian protein ALR (‘augmenter of liver regeneration’), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p. Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor. Both Erv1p and full-length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins. It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process.     

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 mitochondrial proteins Atm1p and Nfs1p are essential for biogenesis of cytosolic Fe/S proteins.

Iron-sulfur (Fe/S) cluster-containing proteins catalyse a number of electron transfer and metabolic reactions. Little is known about the biogenesis of Fe/S clusters in the eukaryotic cell. Here, we demonstrate that mitochondria perform an essential role in the synthesis of both intra- and extra-mitochondrial Fe/S proteins. Nfs1p represents the yeast orthologue of the bacterial cysteine desulfurase NifS that initiates biogenesis by producing elemental sulfur. The matrix-localized protein is required for synthesis of both mitochondrial and cytosolic Fe/S proteins. The ATP-binding cassette (ABC) transporter Atm1p of the mitochondrial inner membrane performs an essential function only in the generation of cytosolic Fe/S proteins by mediating export of Fe/S cluster precursors synthesized by Nfs1p and other mitochondrial proteins. Assembly of cellular Fe/S clusters constitutes an indispensable biosynthetic task of mitochondria with potential relevance for an iron-storage disease and the control of cellular iron uptake.     

The essential role of mitochondria in the biogenesis of cellular iron-sulfur proteins

Iron-sulfur (Fe/S) proteins play an important role in electron transfer processes and in various enzymatic reactions. In eukaryotic cells, known Fe/S proteins are localised in mitochondria, the cytosol and the nucleus. The biogenesis of these proteins has only recently become the focus of investigations. Mitochondria are the major site of Fe/S cluster biosynthesis in the cell. The organelles contain an Fe/S cluster biosynthesis apparatus that resembles that of prokaryotic cells. This apparatus consists of some ten proteins including a cysteine desulfurase producing elemental sulfur for biogenesis, a ferredoxin involved in reduction, and two chaperones. The mitochondrial Fe/S cluster synthesis apparatus not only assembles mitochondrial Fe/S proteins, but also initiates formation of extra-mitochondrial Fe/S proteins. This involves the export of sulfur and possibly iron from mitochondria to the cytosol, a reaction performed by the ABC transporter Atm1p of the mitochondrial inner membrane. A possible substrate of Atm1p is an Fe/S cluster that may be stabilised for transport. Constituents of the cytosol involved in the incorporation of the Fe/S cluster into apoproteins have not been described yet. Many of the mitochondrial proteins involved in Fe/S cluster formation are essential, illustrating the central importance of Fe/S proteins for life. Defects in Fe/S protein biogenesis are associated with the abnormal accumulation of iron within mitochondria and are the cause of an iron storage disease.     

Stimulation of the ATPase activity of the yeast mitochondrial ABC transporter Atm1p by thiol compounds

The ATP binding cassette (ABC) transporter Atm1p of the mitochondrial inner membrane performs crucial roles in both the biogenesis of cytosolic/nuclear iron-sulfur proteins and cellular iron homeostasis. Since the function of the mitochondrial iron-sulfur cluster (ISC) assembly machinery is also required for these two processes, Atm1p is thought to translocate a still unknown product of this pathway to the cytosol. Here, we provide a detailed in vitro characterization of Atm1p in order to better understand its function. Atm1p was purified using an expression system in E. coli. The detergent-solubilised protein exhibits a stable ATPase activity. Reconstitution of Atm1p into proteoliposomes allowed us to determine the biochemical characteristics of the ATPase such as: (i) the strong inhibition by the transition state analogue vanadate, (ii) a Km value of 0.1 mM, and (iii) a turnover number of 127 min-1. The ATPase activity of ABC transporters is generally stimulated by their specific substrate. We used this property to define the chemical properties of the substrate transported by Atm1p. ATPase hydrolysis by Atm1p-containing proteoliposomes was specifically increased 3-5-fold by thiol-containing compounds, in particular by micromolar concentrations of cysteine thiol groups in peptides, even though Atm1p is not a general peptide transporter such as yeast Mdl1p or mammalian TAP which share sequence similarity with Atm1p. We speculate that the physiological substrate of Atm1p may contain multiple sulfhydryl groups in a peptidic environment.     

Mitochondrial ABC transporter Atm1p is required for protection against oxidative stress and vacuolar functions in Schizosaccharomyces pombe

A potential correlation between mitochondrial and vacuolar functions is known to exit in yeast. Fission yeast atm1(+), SPAC15A10.01, encodes a putative half-type ABC transporter with an N-terminal mitochondrial-targeting signal. In an attempt to evaluate the possible involvement of mitochondrion in vacuole function, a functional analysis of atm1(+) was performed by gene disruption. Growth of the atm1 mutant was inhibited in the presence of oxidizing agents, and S. cerevisiae Atm1p was found to complement this growth defect. atm1Delta cells exhibited defects in fluid-phase endocytosis and vacuolar fusion under hypotonic stress. GFP-tagged Atm1p was observed to be localized in the mitochondria. These data strongly suggest that fission yeast Atm1p was not only involved in protection against oxidative stress, but also played a role in vacuolar functions.     

Functional characterization of AtATM1, AtATM2, and AtATM3, a subfamily of Arabidopsis half-molecule ATP-binding cassette transporters implicated in iron homeostasis

The functional capabilities of one of the smallest subfamilies of ATP-binding cassette transporters from Arabidopsis thaliana, the AtATMs, are described. Designated AtATM1, AtAATM2, and AtATM3, these half-molecule ABC proteins are homologous to the yeast mitochondrial membrane protein ATM1 (ScATM1), which is clearly implicated in the export of mitochondrially synthesized iron/sulfur clusters. Yeast ATM1-deficient (atm1) mutants grow very slowly (have a petite phenotype), are respiration-deficient, accumulate toxic levels of iron in their mitochondria, and show enhanced compensatory high affinity iron uptake. Of the three Arabidopsis ATMs, AtATM3 bears the closest functional resemblance to ScATM1. Heterologously expressed AtATM3 is not only able to complement the yeast atm1 petite phenotype but is also able to suppress the constitutively high capacity for high affinity iron uptake associated with loss of the chromosomal copy of ScATM1, abrogate intra-mitochondrial iron hyperaccumulation, and restore mitochondrial respiratory function and cytochrome c levels. By comparison, AtATM1 only weakly suppresses the atm1 phenotype, and AtATM2 exerts little or no suppressive action but instead is toxic when expressed in this system. The differences between AtATM3 and AtATM1 are maintained after exchanging their target peptides, and these proteins as well as AtATM2 colocalize with the mitochondrial fluor MitoTracker Red when expressed in yeast as GFP fusions. Although its toxicity when heterologously expressed in yeast, except when fused with GFP, precludes the functional analysis of native AtATM2, a common function, mitochondrial export of Fe/S clusters or their precursors for the assembly of cytosolic Fe/S proteins, is inferred for AtATM3 and AtATM1.     

The hydrogenase-like Nar1p is essential for maturation of cytosolic and nuclear iron-sulphur proteins

The genome of the yeast Saccharomyces cerevisiae encodes the essential protein Nar1p that is conserved in virtually all eukaryotes and exhibits striking sequence similarity to bacterial iron-only hydrogenases. A human homologue of Nar1p was shown previously to bind prenylated prelamin A in the nucleus. However, yeast neither exhibits hydrogenase activity nor contains nuclear lamins. Here, we demonstrate that Nar1p is predominantly located in the cytosol and contains two adjacent iron-sulphur (Fe/S) clusters. Assembly of its Fe/S clusters crucially depends on components of the mitochondrial Fe/S cluster biosynthesis apparatus such as the cysteine desulphurase Nfs1p, the ferredoxin Yah1p and the ABC transporter Atm1p. Using functional studies in vivo, we show that Nar1p is required for maturation of cytosolic and nuclear, but not of mitochondrial, Fe/S proteins. Nar1p-depleted cells do not accumulate iron in mitochondria, distinguishing these cells from mutants in components of the mitochondrial Fe/S cluster biosynthesis apparatus. In conclusion, Nar1p represents a crucial, novel component of the emerging cytosolic Fe/S protein assembly machinery that catalyses an essential and ancient process in eukaryotes.     

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.     

An ABC transporter in the mitochondrial inner membrane is required for normal growth of yeast.

In an attempt to identify a mitochondrial ATP binding cassette (ABC) transporter, we have used the polymerase chain reaction to amplify 10 DNA fragments homologous to members of the ABC family from the yeast Saccharomyces cerevisiae. We disrupted five of the corresponding genes and found that one of the resulting null mutants barely grew on rich medium and failed to grow on minimal medium. This gene, termed ATM1, encodes a putative 'half-transporter' of 694 amino acids. Atm1p is synthesized with an N-terminal mitochondrial matrix-targeting signal and is located in the mitochondrial inner membrane, with its C-terminal ATPase domain exposed to the matrix. Cells lacking a functional ATM1 gene have an unstable mitochondrial genome and have white mitochondria that completely lack cytochromes. Atm1p is the first mitochondrial member of the ABC family to be identified and the only eukaryotic ABC transporter that has been shown to be necessary for normal cellular growth.     

A mitochondrial half-size ABC transporter is involved in cadmium tolerance in Chlamydomonas reinhardtii

Five cadmium-sensitive insertional mutants, all affected at the CDS1 (' cadmium-sensitive 1 ') locus, have been previously isolated in the unicellular green alga Chlamydomonas reinhardtii . We here describe the cloning of the Cds1 gene (8314 bp with 26 introns) and the corresponding cDNA. The Cds1 gene, strongly induced by cadmium, encodes a putative protein (CrCds1) of 1062 amino acid residues that belongs to the ATM/HMT subfamily of half-size ABC transporters. This subfamily includes both vacuolar HMT-type proteins transporting phytochelatin-cadmium complexes from the cytoplasm to the vacuole and mitochondrial ATM-type proteins involved in the maturation of cytosolic Fe/S proteins. Unlike the Δsphmt1 cadmium-sensitive mutant of Schizosaccharomyces pombe that lacks a vacuolar HMT-type transporter, the cds1 mutant accumulates a high amount of phytochelatin–cadmium complexes. By epitope tagging, the CrCds1 protein was localized in the mitochondria. Even though mitochondria of cds1 do not accumulate important amounts of 'free' iron, the mutant cells are hypersensitive to high iron concentrations . Our data show for the first time that a mitochondrial ATM-like transporter plays a major role in tolerance to cadmium.     

Arabidopsis IRT2 gene encodes a root-periphery iron transporter

Iron uptake from the soil is a tightly controlled process in plant roots, involving specialized transporters. One such transporter, IRT1, was identified in Arabidopsis thaliana and shown to function as a broad-range metal ion transporter in yeast. Here we report the cloning and characterization of the IRT2 cDNA, a member of the ZIP family of metal transporters, highly similar to IRT1 at the amino-acid level. IRT2 expression in yeast suppresses the growth defect of iron and zinc transport yeast mutants and enhances iron uptake and accumulation. However, unlike IRT1, IRT2 does not transport manganese or cadmium in yeast. IRT2 expression is detected only in roots of A. thaliana plants, and is upregulated by iron deficiency. By fusing the IRT2 promoter to the uidA reporter gene, we show that the IRT2 promoter is mainly active in the external cell layers of the root subapical zone, and therefore provide the first tissue localization of a plant metal transporter. Altogether, these data support a role for the IRT2 transporter in iron and zinc uptake from the soil in response to iron-limited conditions.     

Biogenesis of iron-sulfur proteins in eukaryotes: a novel task of mitochondria that is inherited from bacteria

Fe/S clusters are co-factors of numerous proteins with important functions in metabolism, electron transport and regulation of gene expression. Presumably, Fe/S proteins have occurred early in evolution and are present in cells of virtually all species. Biosynthesis of these proteins is a complex process involving numerous components. In mitochondria, this process is accomplished by the so-called ISC (iron-sulfur cluster assembly) machinery which is derived from the bacterial ancestor of the organelles and is conserved from lower to higher eukaryotes. The mitochondrial ISC machinery is responsible for biogenesis iron-sulfur proteins both within and outside the organelle. Maturation of the latter proteins involves the ABC transporter Atm1p which presumably exports iron-sulfur clusters from the organelle. This review summarizes recent developments in our understanding of the biogenesis of iron-sulfur proteins both within bacteria and eukaryotes.     

In yeast, the 3' untranslated region or the presequence of ATM1 is required for the exclusive localization of its mRNA to the vicinity of mitochondria

We isolated mitochondria from Saccharomyces cerevisiae to selectively study polysomes bound to the mitochondrial surface. The distribution of several mRNAs coding for mitochondrial proteins was examined in free and mitochondrion-bound polysomes. Some mRNAs exclusively localize to mitochondrion-bound polysomes, such as the ones coding for Atm1p, Cox10p, Tim44p, Atp2p, and Cot1p. In contrast, mRNAs encoding Cox6p, Cox5a, Aac1p, and Mir1p are found enriched in free cytoplasmic polysome fractions. Aac1p and Mir1p are transporters that lack cleavable presequences. Sequences required for mRNA asymmetric subcellular distribution were determined by analyzing the localization of reporter mRNAs containing the presequence coding region and/or the 3'-untranslated region (3'UTR) of ATM1, a gene encoding an ABC transporter of the mitochondrial inner membrane. Biochemical analyses of mitochondrion-bound polysomes and direct visualization of RNA localization in living yeast cells allowed us to demonstrate that either the presequence coding region or the 3'UTR of ATM1 is sufficient to allow the reporter mRNA to localize to the vicinity of the mitochondrion, independently of its translation. These data demonstrate that mRNA localization is one of the mechanisms used, in yeast, for segregating mitochondrial proteins.     

AtCCMA interacts with AtCcmB to form a novel mitochondrial ABC transporter involved in cytochrome c maturation in Arabidopsis

ABC transporters make a large and diverse family of proteins found in all phylae. AtCCMA is the nucleotide binding domain of a novel Arabidopsis mitochondrial ABC transporter. It is encoded in the nucleus and imported into mitochondria. Sub-organellar and topology studies find AtCCMA bound to the mitochondrial inner membrane, facing the matrix. AtCCMA exhibits an ATPase activity, and ATP/Mg(2+) can facilitate its dissociation from membranes. Blue Native PAGE shows that it is part of a 480-kDa complex. Yeast two-hybrid assays reveal interactions between AtCCMA and domains of CcmB, the mitochondria-encoded transmembrane protein of a conserved ABC transporter. All these properties designate the protein as the ortholog in plant mitochondria of the bacterial CcmA required for cytochrome c maturation. The transporter that involves AtCCMA defines a new category of eukaryotic ABC proteins because its transmembrane and nucleotide binding domains are encoded by separate genomes.     

Differential regulation of nramp and irt metal transporter genes in wild type and iron uptake mutants of tomato

Metal transporters regulated by iron can transport a variety of divalent metals, suggesting that iron regulation is important for specificity of iron transport. In plants, the iron-regulated broad-range metal transporter IRT1 is required for uptake of iron into the root epidermis. Functions of other iron-regulated plant metal transporters are not yet established. To deduce novel plant iron transport functions we studied the regulation of four tomato metal transporter genes belonging to the nramp and irt families with respect to environmental and genetic factors influencing iron uptake. We isolated Lenramp1 and Lenramp3 from tomato and demonstrate that these genes encode functional NRAMP metal transporters in yeast, where they were iron-regulated and localized mainly to intracellular vesicles. Lenramp1 and Leirt1 revealed both root-specific expression and up-regulation by iron deficiency, respectively, in contrast to Leirt2 and Lenramp3. Lenramp1 and Leirt1, but not Lenramp3 and Leirt2, were down-regulated in the roots of fer mutant plants deficient in a bHLH gene regulating iron uptake. In chloronerva mutant plants lacking the functional enzyme for synthesis of the plant-specific metal chelator nicotianamine Leirt1 and Lenramp1 were up-regulated despite sufficient iron supply independent of a functional fer gene. Lenramp1 was expressed in the vascular root parenchyma in a similar cellular pattern as the fer gene. However, the fer gene was not sufficient for inducing Lenramp1 and Leirt1 when ectopically expressed. Based on our results, we suggest a novel function for NRAMP1 in mobilizing iron in the vascular parenchyma upon iron deficiency in plants. We discuss fer/nicotianamine synthase-dependent and -independent regulatory pathways for metal transporter gene regulation.