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.     

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.     

An interaction between frataxin and Isu1/Nfs1 that is crucial for Fe/S cluster synthesis on Isu1

Depletion of the mitochondrial matrix protein frataxin is the molecular cause of the neurodegenerative disease Friedreich ataxia. The function of frataxin is unclear, although recent studies have suggested a function of frataxin (yeast Yfh1) in iron/sulphur (Fe/S) protein biogenesis. Here, we show that Yfh1 specifically binds to the central Fe/S-cluster (ISC)-assembly complex, which is composed of the scaffold protein Isu1 and the cysteine desulphurase Nfs1. Association between Yfh1 and Isu1/Nfs1 was markedly increased by ferrous iron, but did not depend on ISCs on Isu1. Functional analyses in vivo showed an involvement of Yfh1 in de novo ISC synthesis on Isu1. Our data demonstrate a crucial function of Yfh1 in Fe/S protein biogenesis by defining its function in an early step of this essential process. The iron-dependent binding of Yfh1 to Isu1/Nfs1 suggests a role of frataxin/Yfh1 in iron loading of the Isu scaffold proteins.     

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.     

The cytosolic factor required for import of precursors of mitochondrial proteins into mitochondria

The mechanism of import of proteins into mitochondria was studied by using the peptide of the presequence of ornithine aminotransferase (the extrapeptide), which was chemically synthesized and is composed of 34 amino acids. When the extrapeptide was incubated with isolated mitochondria in the presence of a rabbit reticulocyte lysate at 25 degrees C, it was imported into the mitochondrial matrix, and the import depended on the inner membrane potential, but not added ATP. The import of several precursors of mitochondrial proteins was competitively inhibited by the presence of excess extrapeptide in the reaction system, indicating that the extrapeptide and mitochondrial proteins were imported by the same machinery. Import of the extrapeptide was significantly stimulated by addition of a rabbit reticulocyte lysate, and a component of the lysate (the cytosolic factor) stimulating import of the extrapeptide was purified about 20,000 times by successive column chromatography on DEAE-cellulose and aminopentyl-Sepharose 4B. The binding of the extrapeptide to liposomes composed of egg lecithin and partially purified receptor of the precursor of mitochondrial protein (Ono, H., and Tuboi, S., (1985) Biochem. Int. 10, 351-357) required the cytosolic factor when the concentration of the peptide was less than 1.5 X 10(-8) M, suggesting that the physiological binding of the precursors of mitochondrial proteins to the receptor is dependent on the cytosolic factor. The extrapeptide and the cytosolic factor were shown to form a complex. From these results, the mechanism of binding of the extrapeptide to the receptor of the mitochondrial outer membrane is suggested to be as follows: the peptide (the precursor of mitochondrial protein) and the cytosolic factor form a complex, and then the complex is recognized by and bound to the receptor.     

J-domain protein, Jac1p, of yeast mitochondria required for iron homeostasis and activity of Fe-S cluster proteins

J-proteins are molecular chaperones with a characteristic domain predicted to mediate interaction with Hsp70 proteins. We have previously isolated yeast mutants of the mitochondrial Hsp70, Ssq1p, in a genetic screen for mutants with altered iron homeostasis. Here we describe the isolation of mutants of the J-domain protein, Jac1p, using the same screen. Mutant jac1 alleles predicted to encode severely truncated proteins (lacking 70 or 152 amino acids) were associated with phenotypes strikingly similar to the phenotypes of ssq1 mutants. These phenotypes include activation of the high affinity cellular iron uptake system and iron accumulation in mitochondria. In contrast to iron accumulation, Fe-S proteins of mitochondria were specifically deficient. In jac1 mutants, like in ssq1 mutants, processing of the Yfh1p precursor protein from intermediate to mature forms was delayed. In the genetic backgrounds used in this study, jac1 null mutants were found to be viable, permitting analysis of genetic interactions. The Deltajac1 Deltassq1 double mutant was more severely compromised for growth than either single mutant, suggesting a synthetic or additive effect of these mutations. Overexpression of Jac1p partially suppressed ssq1 slow growth and vice versa. Similar mitochondrial localization and similar mutant phenotypes suggest that Ssq1p and Jac1p are functional partners in iron homeostasis.     

The Hsp70 chaperone Ssq1p is dispensable for iron-sulfur cluster formation on the scaffold protein Isu1p

The specialized yeast mitochondrial chaperone system, composed of the Hsp70 Ssq1p, its co-chaperone J-protein Jac1p, and the nucleotide release factor Mge1p, perform a critical function in the biogenesis of iron-sulfur (Fe/S) proteins. Using a spectroscopic assay, we have analyzed the potential role of the chaperones in Fe/S cluster assembly on the scaffold protein Isu1p in vitro in the presence of the cysteine desulfurase Nfs1p. In the absence of chaperones, the kinetics of Fe/S cluster formation on Isu1p were compatible with a chemical reconstitution pathway with Nfs1p functioning as a sulfide donor. Addition of Ssq1p improved the rates of Fe/S cluster assembly 3-fold. However, this stimulatory effect of Ssq1p required neither ATP nor Jac1p and could be fully attributed to the activation of the Nfs1p desulfurase activity by Ssq1p. Furthermore, chaperone-stimulated Fe/S cluster assembly did not involve the specific interaction between Isu1p and Ssq1p, since the effect was observed with Isu1p mutant proteins defective in this interaction, suggesting that nonspecific binding of Ssq1p to Nfs1p helped to prevent its unfolding. Consistent with this idea, these Isu1p mutants were capable of binding an Fe/S cluster in vivo but failed to restore the growth and Fe/S cluster assembly defects of a Isu1p/Isu2p-deficient yeast strain. Taken together, these data suggest that Ssq1p/Jac1p/Mge1p are not important for Fe/S cluster synthesis on Isu1p. Hence, consistent with previous in vivo data, these chaperones likely function in steps subsequent to the de novo synthesis of the Fe/S cluster on Isu1p.     

The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation

Members of the bacterial and mitochondrial iron-sulfur cluster (ISC) assembly machinery include the so-called A-type ISC proteins, which support the assembly of a subset of Fe/S apoproteins. The human genome encodes two A-type proteins, termed ISCA1 and ISCA2, which are related to Saccharomyces cerevisiae Isa1 and Isa2, respectively. An additional protein, Iba57, physically interacts with Isa1 and Isa2 in yeast. To test the cellular role of human ISCA1, ISCA2, and IBA57, HeLa cells were depleted for any of these proteins by RNA interference technology. Depleted cells contained massively swollen and enlarged mitochondria that were virtually devoid of cristae membranes, demonstrating the importance of these proteins for mitochondrial biogenesis. The activities of mitochondrial [4Fe-4S] proteins, including aconitase, respiratory complex I, and lipoic acid synthase, were diminished following depletion of the three proteins. In contrast, the mitochondrial [2Fe-2S] enzyme ferrochelatase and cellular heme content were unaffected. We further provide evidence against a localization and direct Fe/S protein maturation function of ISCA1 and ISCA2 in the cytosol. Taken together, our data suggest that ISCA1, ISCA2, and IBA57 are specifically involved in the maturation of mitochondrial [4Fe-4S] proteins functioning late in the ISC assembly pathway.     

Nop53p is required for late 60S ribosome subunit maturation and nuclear export in yeast

We report that Ypl146cp/Nop53p is associated with pre-60S ribosomal complexes and localized to the nucleolus and nucleoplasm. In cells depleted of Nop53p synthesis of the rRNA components of the 60S ribosomal subunit is severely inhibited, with strikingly strong accumulation of the 7S pre-rRNA and a 5' extended form of the 25S rRNA. In cells depleted of Nop53p pre-60S subunits accumulate in the nucleus. However, a heterokaryon assay demonstrated that Nop53p is not transferred between nuclei, indicating that it is not released into the cytoplasm. We conclude that Nop53p is a late-acting factor in the nuclear maturation of 60S ribosomal subunits, which is required for normal acquisition of export competence. The strong accumulation of preribosomes in the Nop53p-depleted strain further suggests that it may participate in targeting aberrant preribosomes to surveillance and degradation pathways.     

Characterization of the interaction between the J-protein Jac1p and the scaffold for Fe-S cluster biogenesis, Isu1p

Jac1p is a conserved, specialized J-protein that functions with Hsp70 in Fe-S cluster biogenesis in mitochondria of the yeast Saccharomyces cerevisiae. Although Jac1p as well as its specialized Hsp70 partner, Ssq1p, binds directly to the Fe-S cluster scaffold protein Isu, the Jac1p-Isu1p interaction is not well understood. Here we report that a C-terminal fragment of Jac1p lacking its J-domain is sufficient for interaction with Isu1p, and amino acid alterations in this domain affect interaction with Isu1p but not Ssq1p. In vivo, such JAC1 mutations had no obvious phenotypic effect. However, when present in combination with a mutation in SSQ1 that causes an alteration in the substrate binding cleft, growth was significantly compromised. Wild type Jac1p and Isu1p cooperatively stimulate the ATPase activity of Ssq1p. Jac1p mutant protein is only slightly compromised in this regard. Our in vivo and in vitro results indicate that independent interaction of Jac1p and the Isu client protein with Hsp70 is sufficient for robust growth under standard laboratory conditions. However, our results also support the idea that Isu protein can be "targeted" to Ssq1p after forming a complex with Jac1p. We propose that Isu protein targeting may be particularly important when environmental conditions place high demands on Fe-S cluster biogenesis or in organisms lacking specialized Hsp70s for Fe-S cluster biogenesis.     

Adrenodoxin reductase homolog (Arh1p) of yeast mitochondria required for iron homeostasis

Arh1p is an essential mitochondrial protein of yeast with reductase activity. Here we show that this protein is involved in iron metabolism. A yeast strain was constructed in which the open reading frame was placed under the control of a galactose-regulated promoter. Protein expression was induced by galactose and repressed to undetectable levels in the absence of galactose, although cells grew quite well in the absence of inducer. Under noninducing conditions, cellular iron uptake was dysregulated, exhibiting a failure to repress in response to medium iron. Iron trafficking within the cell was also disturbed. Exposure of Arh1p-depleted cells to increasing iron concentrations during growth led to drastic increases in mitochondrial iron, indicating a loss of homeostatic control. Activity of aconitase, a prototype Fe-S protein, was deficient at all concentrations of mitochondrial iron, although the protein level was unaltered. Heme protein deficiencies were exacerbated in the iron-loaded mitochondria, suggesting a toxic side effect of accumulated iron. Finally, a time course correlated the cellular depletion of Arh1p with the coordinated appearance of various mutant phenotypes including dysregulated cellular iron uptake, deficiency of Fe-S protein activities in mitochondria and cytoplasm, and deficiency of hemoproteins. Thus, Arh1p is required for control of cellular and mitochondrial iron levels and for the activities of Fe-S cluster proteins.     

The mitochondrial proteins Ssq1 and Jac1 are required for the assembly of iron sulfur clusters in mitochondria

Mitochondria of the yeast Saccharomyces cerevisiae contain three different Hsp70 chaperones, Ssc1, Ecm10 and Ssq1. Ssc1 is an essential protein that mediates the import of nuclear-encoded proteins into the organelle and their subsequent folding. The nucleotide state of Ssc1 is thereby regulated by the nucleotide exchange factor Mge1. Here, we show that Mge1 interacts with Ssq1 in an ATP-dependent manner, suggesting that Mge1 also regulates Ssq1 function. In contrast to Ssc1, Ssq1 does not associate with the Tim44 subunit of the protein translocating complex, indicating a different function of both chaperones. Mutants in Ssq1 were reported to have low levels of iron sulfur (FeS) cluster-containing enzymes. Employing an assay that allowed us to monitor the conversion of the apoform of mitochondrial ferredoxin into its FeS-containing holoform, Ssq1 was demonstrated to be required for the FeS cluster assembly in mitochondria. The mitochondrial DnaJ homolog Jac1 is crucial for this process, whereas Mdj1 function is dispensable. Furthermore, the presence of frataxin is necessary for FeS cluster assembly into ferredoxin suggesting a role for frataxin at the level of the formation of holo-ferredoxin.     

S. cerevisiae Tel1p and Mre11p are required for normal levels of Est1p and Est2p telomere association

In diverse organisms, the Mre11 complex and phosphoinositide 3-kinase-related kinases (PIKKs), such as Tel1p and Mec1p from S. cerevisiae, are key mediators of DNA repair and DNA damage checkpoints that also function at telomeres. Here, we use chromatin immunoprecipitation (ChIP) to determine if Mre11p, Tel1p, or Mec1p affects telomere maintenance by promoting recruitment of telomerase subunits to S. cerevisiae telomeres. We find that recruitment of Est2p, the catalytic subunit of telomerase, and Est1p, a telomerase accessory protein, was severely reduced in mre11Delta and tel1Delta cells. In contrast, the levels of Est2p and Est1p binding in late S/G2 phase, the period in the cell cycle when yeast telomerase lengthens telomeres, were indistinguishable in wild-type (WT) and mec1Delta cells. These data argue that Mre11p and Tel1p affect telomere length by promoting telomerase recruitment to telomeres, whereas Mec1p has only a minor role in telomerase recruitment in a TEL1 cell.     

Glycosylphosphatidylinositol-anchored proteins are required for the transport of detergent-resistant microdomain-associated membrane proteins Tat2p and Fur4p

In eukaryotic cells many cell surface proteins are attached to the membrane via the glycosylphosphatidylinositol (GPI) moiety. In yeast, GPI also plays important roles in the production of mannoprotein in the cell wall. We previously isolated gwt1 mutants and found that GWT1 is required for inositol acylation in the GPI biosynthetic pathway. In this study we isolated a new gwt1 mutant allele, gwt1-10, that shows not only high temperature sensitivity but also low temperature sensitivity. The gwt1-10 cells show impaired acyltransferase activity and attachment of GPI to proteins even at the permissive temperature. We identified TAT2, which encodes a high affinity tryptophan permease, as a multicopy suppressor of cold sensitivity in gwt1-10 cells. The gwt1-10 cells were also defective in the import of tryptophan, and a lack of tryptophan caused low temperature sensitivity. Microscopic observation revealed that Tat2p is not transported to the plasma membrane but is retained in the endoplasmic reticulum in gwt1-10 cells grown under tryptophan-poor conditions. We found that Tat2p was not associated with detergent-resistant membranes (DRMs), which are required for the recruitment of Tat2p to the plasma membrane. A similar result was obtained for Fur4p, a uracil permease localized in the DRMs of the plasma membrane. These results indicate that GPI-anchored proteins are required for the recruitment of membrane proteins Tat2p and Fur4p to the plasma membrane via DRMs, suggesting that some membrane proteins are redistributed in the cell in response to environmental and nutritional conditions due to an association with DRMs that is dependent on GPI-anchored proteins.     

Components involved in assembly and dislocation of iron-sulfur clusters on the scaffold protein Isu1p

The mitochondrial proteins Isu1p and Isu2p play an essential role in the maturation of cellular iron-sulfur (Fe/S) proteins in eukaryotes. By radiolabelling of yeast cells with 55Fe we demonstrate that Isu1p binds an oxygen-resistant non-chelatable Fe/S cluster providing in vivo evidence for a scaffolding function of Isu1p during Fe/S cluster assembly. Depletion of the cysteine desulfurase Nfs1p, the ferredoxin Yah1p or the yeast frataxin homologue Yfh1p by regulated gene expression causes a strong decrease in the de novo synthesis of Fe/S clusters on Isu1p. In contrast, depletion of the Hsp70 chaperone Ssq1p, its co-chaperone Jac1p or the glutaredoxin Grx5p markedly increased the amount of Fe/S clusters bound to Isu1p, even though these mitochondrial proteins are crucial for maturation of Fe/S proteins. Hence Ssq1p/Jac1p and Grx5p are required in a step after Fe/S cluster synthesis on Isu1p, for instance in dissociation of preassembled Fe/S clusters from Isu1p and/or their insertion into apoproteins. We propose a model that dissects Fe/S cluster biogenesis into two major steps and assigns its central components to one of these two steps.     

The LYR protein Mzm1 functions in the insertion of the Rieske Fe/S protein in yeast mitochondria

The assembly of the cytochrome bc(1) complex in Saccharomyces cerevisiae is shown to be conditionally dependent on a novel factor, Mzm1. Cells lacking Mzm1 exhibit a modest bc(1) defect at 30°C, but the defect is exacerbated at elevated temperatures. Formation of bc(1) is stalled in mzm1Δ cells at a late assembly intermediate lacking the Rieske iron-sulfur protein Rip1. Rip1 levels are markedly attenuated in mzm1Δ cells at elevated temperatures. Respiratory growth can be restored in the mutant cells by the overexpression of the Rip1 subunit. Elevated levels of Mzm1 enhance the stabilization of Rip1 through physical interaction, suggesting that Mzm1 may be an important Rip1 chaperone especially under heat stress. Mzm1 may function primarily to stabilize Rip1 prior to inner membrane (IM) insertion or alternatively to aid in the presentation of Rip1 to the inner membrane translocation complex for extrusion of the folded domain containing the iron-sulfur center.     

Sue1p is required for degradation of labile forms of altered cytochromes C in yeast mitochondria

Previous studies on certain altered holo-isocytochromes c revealed a rho(-)-dependent degradation (RDD) phenotype, in which certain altered holo-iso-1-cytochromes c are at normal or nearly normal levels in rho+ strains, but are at low levels or absent in rho- strains, although wild-type holo-iso-1-cytochrome c is present at normal levels in both rho+ and related rho- strains. The diminished levels of altered holo-iso-1-cytochrome c are due to the rapid degradation that is carried out by a novel proteolytic pathway in the IMS of mitochondria. SUE1, a nuclear gene that encodes a mitochondrial protein, was identified with a genetic screen for mutants that diminish RDD. The levels of RDD and certain other types of altered holo-iso-1-cytochrome c were elevated in rho- sue1 strains. Also, rho+ sue1 strains containing certain altered holo-iso-1-cytochromes c grew better on non-fermentable carbon sources than the corresponding rho+ SUE1 strains. These results indicate that Sue1p may play an important role in the degradation of abnormal holo-iso-1-cytochrome c in the mitochondria.     

Factors beyond enolase 2 and mitochondrial lysyl-tRNA synthetase precursor are required for tRNA import into yeast mitochondria

In yeast, the import of tRNA^Lys with CUU anticodon (tRK1) relies on a complex mechanism where interaction with enolase 2 (Eno2p) dictates a deep conformational change of the tRNA. This event is believed to mask the tRNA from the cytosolic translational machinery to re-direct it towards the mitochondria. Once near the mitochondrial outer membrane, the precursor of the mitochondrial lysyl-tRNA synthetase (preMsk1p) takes over enolase to carry the tRNA within the mitochondrial matrix, where it is supposed to participate in translation following correct refolding. Biochemical data presented in this report focus on the role of enolase. They show that despite the inability of Eno2p alone to form a complex with tRK1, mitochondrial import can be recapitulated in vitro using fractions of yeast extracts sharing either recombinant or endogenous yeast Eno2p as one of the main components. Taken together, our data suggest the existence of a protein complex containing Eno2p that is involved in RNA mitochondrial import.     

DNA resection proteins Sgs1 and Exo1 are required for G1 checkpoint activation in budding yeast

Double-strand breaks (DSBs) in budding yeast trigger activation of DNA damage checkpoints, allowing repair to occur. Although resection is necessary for initiating damage-induced cell cycle arrest in G2, no role has been assigned to it in the activation of G1 checkpoint. Here we demonstrate for the first time that the resection proteins Sgs1 and Exo1 are required for efficient G1 checkpoint activation. We find in G1 arrested cells that histone H2A phosphorylation in response to ionizing radiation is independent of Sgs1 and Exo1. In contrast, these proteins are required for damage-induced recruitment of Rfa1 to the DSB sites, phosphorylation of the Rad53 effector kinase, cell cycle arrest and RNR3 expression. Checkpoint activation in G1 requires the catalytic activity of Sgs1, suggesting that it is DNA resection mediated by Sgs1 that stimulates the damage response pathway rather than protein–protein interactions with other DDR proteins. Together, these results implicate DNA resection, which is thought to be minimal in G1, as necessary for activation of the G1 checkpoint.