Gyula Kispal

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.

Maturation of cellular Fe-S proteins: An essential function of mitochondria

Iron-sulfur (Fe-S) cluster-containing proteins perform important tasks in catalysis, electron transfer and regulation of gene expression. In eukaryotes, mitochondria are the primary site of cluster formation of most Fe-S proteins. Assembly of the Fe-S clusters is mediated by the iron-sulphate cluster assembly (ISC) machinery consisting of some ten proteins.

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.

The ABC transporter Atm1p is required for mitochondrial iron homeostasis

The function of the ABC transporter Atm1p located in the mitochondrial inner membrane is not yet known. To study its cellular role, we analyzed a mutant in which ATM1 was disrupted. Δatm1 cells are deficient in the holoforms, but not the apoforms of heme‐carrying proteins both within and outside mitochondria, yet both synthesis and transport of heme are functional. Δatm1 cells are hypersensitive for growth in the presence of oxidative reagents, and they contain increased levels of the antioxidant glutathione, in particular of its oxidized form. Mitochondria deficient in Atm1p accumulate 30‐fold higher levels of free iron as compared to wild‐type organelles, i.e. three‐fold more than mitochondria deficient in frataxin, the protein mutated in Friedreichs ataxia. The increased mitochondrial iron content may be causative of the oxidative damage of heme‐containing proteins in Δatm1 cells. Our data assign an important function to Atm1p in mitochondrial iron homeostasis.

A Mutation of the Mitochondrial ABC Transporter Sta1 Leads to Dwarfism and Chlorosis in the Arabidopsis Mutant starik

A mutation in the Arabidopsis gene STARIK leads to dwarfism and chlorosis of plants with an altered morphology of leaf and cell nuclei. We show that the STARIK gene encodes the mitochondrial ABC transporter Sta1 that belongs to a subfamily of Arabidopsis half-ABC transporters. The severity of the starik phenotype is suppressed by the ectopic expression of the STA2 homolog; thus, Sta1 function is partially redundant. Sta1 supports the maturation of cytosolic Fe/S protein in Δatm1 yeast, substituting for the ABC transporter Atm1p. Similar to Atm1p-deficient yeast, mitochondria of the starik mutant accumulated more nonheme, nonprotein iron than did wild-type organelles. We further show that plant mitochondria contain a putative l-cysteine desulfurase. Taken together, our results suggest that plant mitochondria possess an evolutionarily conserved Fe/S cluster biosynthesis pathway, which is linked to the intracellular iron homeostasis by the function of Atm1p-like ABC transporters.

Biogenesis of cytosolic ribosomes requires the essential iron-sulphur protein Rli1p and mitochondria.

Mitochondria perform a central function in the biogenesis of cellular iron–sulphur (Fe/S) proteins. It is unknown to date why this biosynthetic pathway is indispensable for life, the more so as no essential mitochondrial Fe/S proteins are known. Here, we show that the soluble ATP‐binding cassette (ABC) protein Rli1p carries N‐terminal Fe/S clusters that require the mitochondrial and cytosolic Fe/S protein biogenesis machineries for assembly. Mutations in critical cysteine residues of Rli1p abolish association with Fe/S clusters and lead to loss of cell viability. Hence, the essential character of Fe/S clusters in Rli1p explains the indispensable character of mitochondria in eukaryotes. We further report that Rli1p is associated with ribosomes and with Hcr1p, a protein involved in rRNA processing and translation initiation. Depletion of Rli1p causes a nuclear export defect of the small and large ribosomal subunits and subsequently a translational arrest. Thus, ribosome biogenesis and function are intimately linked to the crucial role of mitochondria in the maturation of the essential Fe/S protein Rli1p.

Mitochondrial and cytosolic branched-chain amino acid transaminases from yeast, homologs of the myc oncogene-regulated Eca39 protein

We have isolated a high copy suppressor of a temperature-sensitive mutation in ATM1, which codes for an ABC transporter of Saccharomyces cerevisiae mitochondria. The suppressor, termed BAT1, encodes a protein of 393 amino acid residues with an NH2-terminal extension that directs Bat1p to the mitochondrial matrix. A highly homologous protein, Bat2p, of 376 amino acid residues was found in the cytosol. Both Bat proteins show striking similarity to the mammalian protein Eca39, which is one of the few known targets of the myc oncogene. Deletion of a single BAT gene did not impair growth of yeast cells. In contrast, deletion of both genes resulted in an auxotrophy for branched-chain amino acids (Ile, Leu, and Val) and in a severe growth reduction on glucose-containing media, even after supply of these amino acids. Mitochondria and cytosol isolated from bat1 and bat2 deletion mutants, respectively, contained largely reduced activities for the conversion of branched-chain 2-ketoacids to their corresponding amino acids. Thus, the Bat proteins represent the first known isoforms of yeast branched-chain amino acid transaminases. The severe growth defect of the double deletion mutant observed even in the presence of branched-chain amino acids suggests that the Bat proteins, in addition to the supply of these amino acids, perform another important function in the cell.

Mechanism of Iron Transport to the Site of Heme Synthesis inside Yeast Mitochondria

The import of metals, iron in particular, into mitochondria is poorly understood. Iron in mitochondria is required for the biosynthesis of heme and various iron-sulfur proteins. We have developed an in vitro assay to follow the uptake of iron into isolated yeast mitochondria. By measuring the incorporation of iron into porphyrin by ferrochelatase in the matrix, we were able to define the mechanism of iron import. Iron uptake is driven energetically by a membrane potential across the inner membrane but does not require ATP. Only reduced iron is functional in generating heme. Iron cannot be preloaded in the mitochondrial matrix but rather has to be transported across the inner membrane simultaneously with the synthesis of heme, suggesting that ferrochelatase receives iron directly from the inner membrane. Transport of iron is inhibited by manganese but not by zinc, nickel, and copper ions, explaining whyin vivo these ions are not incorporated into porphyrin. The inner membrane proteins Mmt1p and Mmt2p proposed to be involved in mitochondrial iron movement are not required for the supply of ferrochelatase with iron. Iron transport can be reconstituted efficiently in a membrane potential-dependent fashion in proteoliposomes that were formed from a detergent extract of mitochondria. Our biochemical analysis of iron import into yeast mitochondria provides the basis for the identification of components involved in transport.

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

Abstract 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.

Isa1p is a component of the mitochondrial machinery for maturation of cellular iron-sulfur proteins and requires conserved cysteine residues for function

In eukaryotes, mitochondria execute a central task in the assembly of cellular iron-sulfur (Fe/S) proteins. The organelles synthesize their own set of Fe/S proteins, and they initiate the generation of extramitochondrial Fe/S proteins. In the present study, we identify the mitochondrial matrix protein Isa1p ofSaccharomyces cerevisiae as a new member of the Fe/S cluster biosynthesis machinery. Isa1p belongs to a family of homologous proteins present in prokaryotes and eukaryotes. Deletion of theISA1 gene results in the loss of mitochondrial DNA precluding the use of the Δisa1 strain for functional analysis. Cells in which Isa1p was depleted by regulated gene expression maintained the mitochondrial DNA, yet the cells displayed retarded growth on nonfermentable carbon sources. This finding indicates the importance of Isa1p for mitochondrial function. Deficiency of Isa1p caused a defect in mitochondrial Fe/S protein assembly. Moreover, Isa1p was required for maturation of cytosolic Fe/S proteins. Two cysteine residues in a conserved sequence motif characterizing the Isa1p protein family were found to be essential for Isa1p function in the biogenesis of both intra- and extramitochondrial Fe/S proteins. Our findings suggest a function for Isa1p in the binding of iron or an intermediate of Fe/S cluster assembly.