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* This work was supported by grants from the Sonderforschungsbereich 286 of the Deutsche Forschungsgemeinschaft, the Volkswagen-Stiftung, the Fonds der Chemischen Industrie, and the Alexander von Humboldt-Stiftung and by Hungarian Funds OKTA Grants T6378, T020079, and T022581.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Present address: Inst. of Biochemistry, University Medical School of Pecs, Szigeti 12, 7624 Pecs, Hungary.
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.
polymerase chain reaction
Investigations during the past few years have demonstrated the complexity of the assembly of iron-sulfur (Fe/S)1 proteins in a living cell (reviewed by Refs.
). In some bacteria, these genes are clustered in operons termed isc (iron sulfurcluster assembly). The encoded proteins were suggested to perform crucial tasks in the biogenesis of the normal equipment of Fe/S proteins within a prokaryotic cell (
). However, only a few proteins have been functionally investigated in detail. The best studied examples are the cysteine desulfurases NifS and IscS that produce elemental sulfur from cysteine for biogenesis (
Recently, eukaryotic homologues of some of the Nif and Isc proteins have been identified. All of these factors have been localized to mitochondria, and an involvement in the biosynthesis of mitochondrial Fe/S proteins has been demonstrated in yeast. Nfs1p has been shown to represent the functional orthologue of the cysteine desulfurase NifS/IscS (
At least some of these factors appear to perform an additional role in the generation of Fe/S proteins outside the organelle. Direct evidence for a function in the production of Fe/S clusters for incorporation into cytosolic apoproteins has been reported for Nfs1p and Yah1p (
). Apparently, mitochondria produce a component required for the maturation of Fe/S proteins in the cytosol. Export of this component, presumably an Fe/S cluster, to the cytosol is mediated by the ATP-binding cassette transporter Atm1p of the mitochondrial inner membrane (
). This expectation is based on sequence similarity to bacterial proteins encoded by thenif and isc operons. One of these proteins is Isa1p of the yeast Saccharomyces cerevisiae. It displays sequence homology to bacterial proteins termed IscA (also coined HesB; Refs.
) and to a few eukaryotic proteins including Isa2p of yeast. Recently, E. coli IscA was shown to improve the efficiency of the maturation of an overexpressed [2Fe-2S] ferredoxin, suggesting a function of this protein in Fe/S cluster biosynthesis (
Here, we present a study on the subcellular localization and functional characterization of Isa1p. The protein was shown to reside in the mitochondrial matrix, where it performs a crucial role in the generation of Fe/S clusters for both mitochondrial and cytosolic apoproteins. Two conserved cysteine residues located in a sequence motif characteristic for the Isa/IscA proteins were shown to be essential for Isa1p function. Our study adds Isa1p, and by extrapolation the eukaryotic and bacterial homologues, to the growing list of components involved in cellular Fe/S cluster biogenesis.
Yeast Strains and Cell Growth
The following strains ofS. cerevisiae were used: strain W303 (MATα,ura3-1, ade2-1, trp1-1, his3-11, 15, leu2-3, 112) in diploid and haploid form served as wild type. Strain W303ρ0 is derived from W303 and lacks mitochondrial DNA (mtDNA). Deletion of the ISA1 gene in W303 diploid cells was performed by a PCR-based method using the kanamycin resistance marker (
). The haploid Δisa1 strain was obtained after sporulation and tetrad dissection. Strain Δisa1/ISA1 was generated by transforming Δisa1 cells with the yeast expression plasmid pRS416MET25 carrying the ISA1 gene (
). PCR fragments corresponding to the coding region (nucleotides −6 to 871) and the 5′-upstream region (nucleotides −601 to −4) of ISA1 were cloned into theBamHI/HindIII andHindIII/HpaI restriction sites, respectively, of the Yep51 vector carrying the GAL10 promoter. Cells were grown as detailed previously using rich (YP) or minimal media (
). The standard error of the determination of enzyme activities varied between 5 and 15%. The labeling of yeast cells with radioactive iron (55Fe) and the measurement of the incorporation of 55Fe into the cytosolic protein Leu1p by immunoprecipitation and liquid scintillation counting was described earlier (
). The standard error for detection of 55Fe associated with cytosolic Leu1p in a cell lysate was less than 15%, the cellular uptake of 55Fe varied by 30%.
Mutagenesis of ISA1
The exchange of the two conserved cysteine residues 242 and 244 of Isa1p to serines was accomplished by PCR-based mutagenesis of the ISA1 gene using appropriate degenerate primers. Single mutations (C242S and C244S) and double mutations (C242S, C244S) were created. PCR fragments encompassing the mutated ISA1 genes were cloned into the BamHI andHindIII restriction sites of the yeast expression vector pRS416MET25 (
). These plasmids and the vector lacking a DNA insert were transformed into the Gal-ISA1 cells. DNA sequencing was used to verify the desired sequences of all mutant ISA1 genes.
Yeast Isa1p Is a Member of a Large Protein Family
The protein Isa1p of S. cerevisiae (open reading frameYLL027w) contains 250 amino acid residues corresponding to a molecular mass of 27.7 kDa. A data bank search identified homologous proteins in yeasts, man, plants, and many prokaryotes, but not in archaebacteria (Fig. 1). In some organisms such as S. cerevisiae and E. coli, two members of the Isa/IscA protein family have been described. However, we were unable to classify the family members into two distinct subgroups by sequence criteria. The Isa/IscA proteins show relatively low overall sequence conservation except for two regions in the center and at the extreme C terminus. These segments coined “IsaI” and “IsaII” motifs contain one or two, respectively, conserved cysteine residues (Fig. 1). Isa2p of S. cerevisiae harbors an extra sequence block of 20 mostly charged residues that are not present in other Isa/IscA proteins. The fungal and human Isa proteins carry N-terminal extensions that are predicted to function as mitochondrial targeting signals (
). Even though the plant Isa proteins carry only small extensions as compared with the bacterial members, their N termini also resemble mitochondrial presequences.
Isa1p Is Localized in the Mitochondrial Matrix
The cellular localization of Isa1p was determined by immunostaining analysis using specific antibodies. Isa1p was detected exclusively in isolated mitochondria and was absent in the post-mitochondrial supernatant (Fig.2A). To verify that the putative N-terminal presequence of Isa1p can direct the protein into mitochondria and to determine its submitochondrial localization, Isa1p was translated in reticulocyte lysate and used for in vitroimport into isolated mitochondria. The radioactively labeled translation product migrated at a molecular mass of 32 kDa in polyacrylamide gels. Upon incubation with mitochondria, the protein was processed to a shorter form of 29 kDa (Fig. 2B, top panel). This mature protein was protected against degradation by added proteinase K in both intact mitochondria and in mitoplasts that were generated by a hypotonic swelling procedure. Upon lysis of the organelles by addition of detergent, Isa1p was degraded by the protease. An identical behavior was observed for the endogenous Isa1p and for marker proteins of the mitochondrial matrix (Fig.2B, bottom panel). Thus, both imported and endogenous Isa1p are located in the matrix space. Both processing and protease protection of Isa1p were not observed, when the import reaction was performed in the presence of the uncoupler CCCP (Fig.2B, top panel). Hence, the import of Isa1p was dependent on the presence of a membrane potential (ΔΨ). This is characteristic for proteins of the mitochondrial matrix. We conclude from these observations that Isa1p is a constituent of the mitochondrial matrix and uses a typical N-terminal targeting sequence for import into the organelles.
Genetic Inactivation of the ISA1 Gene
To initiate the functional investigation of Isa1p, the ISA1 gene was deleted. The resulting yeast strain Δisa1 grew slowly on glucose-containing medium and did not give rise to colonies on nonfermentable carbon sources such as glycerol (not shown). The growth phenotype was not reverted by reintroducing the ISA1 gene into Δisa1 cells on a yeast expression plasmid (Δisa1/ISA1 cells; not shown), suggesting that the deletion of ISA1 was accompanied by the loss of mtDNA. This expectation was confirmed by DAPI staining, which detected no mtDNA in Δisa1 cells (Fig.3A). Apparently, Isa1p, like numerous other mitochondrial proteins (
The rho0 phenotype of Δisa1 cells hindered the analysis of Isa1p function in these cells (see below) and made it necessary to construct a strain in which ISA1 gene expression could be turned off. To this end, the ISA1 promoter was exchanged for a galactose-inducible promoter. In the resulting Gal-ISA1 cells the concentration of Isa1p was decreased strongly after 16 h of growth in the absence of galactose, whereas the levels of other mitochondrial proteins (Tom70p, CC1HL, Mge1p) were not affected (Fig.3B). When Gal-ISA1 cells depleted in Isa1p were analyzed by DAPI staining, no significant loss of mtDNA was apparent, even after growth for several days in the absence of galactose (Fig.3A). It appears from these data that the depletion of Isa1p in Gal-ISA1 cells did not bring about the severe pleiotropic consequences that were observed in the deletion strain Δisa1. The rapid and specific depletion of Isa1p allowed the functional investigation of this protein.
First, the growth phenotype of Gal-ISA1 cells was analyzed. The cells grew normally on the nonfermentable carbon source glycerol in the presence of galactose but displayed strongly retarded growth in the absence of this sugar (Fig. 3C). On glucose-containing medium, hardly any difference to wild-type cells was observed. The growth defect of Gal-ISA1 cells on nonfermentable carbon sources indicates an important role of Isa1p in the generation of functional mitochondria.
Isa1p Is Necessary for the Maturation of Mitochondrial Fe/S Proteins
To analyze the potential role of Isa1p in Fe/S cluster formation, we first used Δisa1 cells and measured the activities of the Fe/S cluster-containing enzymes aconitase and succinate dehydrogenase of isolated mitochondria. Both activities were undetectable in mitochondria of Δisa1 cells, indicating a severe defect in these Fe/S proteins (Fig.4A). However, this result cannot be taken as evidence for a function of Isa1p in the maturation of mitochondrial Fe/S proteins. We found a strong (5-fold compared with wild-type cells) decrease in the activities of the Fe/S proteins of ρ0 (wild-type background) and Δisa1/ISA1 cells that both lack mtDNA (see above). Thus, to a large extent the ρ0 character of Δisa1 cells accounts for the dramatic defect observed in mitochondrial Fe/S proteins. Citrate synthase, a non-Fe/S protein, displayed wild-type activity in these cells (Fig.4A). Taken together, the ρ0 character of Δisa1 cells precludes the examination of the role of Isa1p in the maturation of cellular Fe/S proteins.
To circumvent these problems, mitochondria were isolated from Gal-ISA1 cells that did not lose mtDNA upon growth in the absence of galactose (Fig. 3A). Depletion of Isa1p caused an up to 4-fold decrease in the activities of succinate dehydrogenase and a 2-fold decline of aconitase (Fig. 4B). In comparison, the activity of citrate synthase slightly increased upon depletion of Isa1p. The amounts of both Fe/S cluster-containing enzymes were hardly affected by the reduction of Isa1p levels as determined by immunostaining (Fig.3B). We conclude from these data that Isa1p is required for efficient maturation of mitochondrial Fe/S proteins.
Isa1p Participates in the Generation of the Cytosolic Fe/S Protein Leu1p
Mitochondria have a primary function in the maturation of Fe/S proteins located in the cytosol (
). Does Isa1p participate in the generation of extramitochondrial Fe/S proteins? The consequences of Isa1p depletion on the enzymatic activity of the cytosolic Fe/S cluster-containing enzyme isopropyl malate isomerase (Leu1p) was measured. Lysates were prepared from wild-type cells and from Gal-ISA1 cells before and after depletion of Isa1p and used for detection of Leu1p activity. In comparison with extracts derived from wild-type cells and from Gal-ISA1 cells grown in the presence of galactose, cells depleted in Isa1p displayed an up to 8-fold reduction in Leu1p activity (Fig. 5A). This effect was similar to the results obtained for cells deficient in the proteins Atm1p, Nfs1p, and Yah1p that are essential for the generation of cytosolic Fe/S proteins (
). As estimated by immunostaining, the levels of Leu1p were only slightly reduced (less than twofold; Fig.3B), suggesting that the reason for the lack of Leu1p activity is not the defective synthesis of the Leu1p polypeptide chain.
To test the requirement of Isa1p for the de novo assembly of the Fe/S cluster in Leu1p, Gal-ISA1 cells were depleted in Isa1p and radiolabeled for 1 h with (55Fe) iron chloride. A cell extract was prepared, and the de novo synthesis of the Fe/S cluster in Leu1p was estimated from the amount of radioactive55Fe that could be immunoprecipitated with antibodies against Leu1p (cf. Ref.
). Depletion of Isa1p resulted in a 4-fold reduction in the assembly of 55Fe with Leu1p as compared with wild-type cells (Fig. 5B, top panel) or with Gal-ISA1 cells grown in the presence of galactose (not shown). Immunoprecipitation performed with preimmune serum did not yield significant amounts of radioactive iron associated with the immuno-beads (Fig. 5B and Ref.
). Further, the uptake of55Fe into the cells was independent of the cellular concentration of Isa1p, illustrating that Isa1p-deficient cells were not impaired in iron uptake (Fig. 5B, bottom panel). Together, these data demonstrate a crucial function of the mitochondrial protein Isa1p in the de novo assembly of a cytosolic Fe/S protein.
Conserved Cysteine Residues Are Essential for Isa1p Function
The functional importance of two conserved cysteine residues in the IsaII sequence motif of Isa1p was investigated (Fig.1). First, we generated cells expressing mutant Isa1p proteins in which the cysteine residues at positions 242 and 244 were changed either individually or simultaneously to serine residues. To this end, mutantISA1 genes were synthesized by PCR and inserted into the yeast expression plasmid pRS416MET25. Gal-ISA1 cells were transformed with these plasmids or with plasmids carrying either the wild-typeISA1 gene or no DNA insert. We then tested the functionality of the mutant Isa1p proteins by turning off the expression of the nucleus-encoded wild-type Isa1p by cultivation of the cells in the absence of galactose. The three cells containing the mutant proteins Isa1p(C242S), Isa1p(C244S), and Isa1p(C242S, C244S) displayed a growth phenotype indistinguishable from Gal-ISA1 cells lacking Isa1p (not shown). On the contrary, expression of wild-type ISA1restored wild-type growth of Gal-ISA1 cells in the absence of galactose. This suggested an important role of the conserved cysteine residues for Isa1p function.
To more thoroughly analyze the function of the mutated Isa1p proteins in Fe/S cluster biogenesis, the enzyme activities of the mitochondrial Fe/S proteins aconitase and succinate dehydrogenase and the cytosolic Leu1p were measured. Depletion of the wild type and expression of the mutant Isa1p proteins (Fig.6C, middle panel) caused a specific decrease in the enzymatic activities of both the mitochondrial Fe/S proteins (Fig. 6A) and the cytosolic Leu1p (Fig. 6B). Expression of the ISA1 gene restored wild-type activities of these proteins. Likewise, the de novo incorporation of Fe/S clusters into cytosolic Leu1p (Fig.5B) was strongly impaired in cells exclusively synthesizing the mutated Isa1p proteins (Fig. 6C, top panel). In all cases, the defects in Fe/S protein maturation were similar to those observed in Gal-ISA1 cells lacking Isa1p. These results amply demonstrate the essential contribution of each of the cysteine residues of the IsaII motif for Isa1p function. Without an active sulfhydryl group at these positions, Isa1p appears to be nonfunctional.
Our investigation provides evidence for a function of the protein Isa1p of S. cerevisiae mitochondria in Fe/S cluster formation. Isa1p is a new component of the mitochondrial Fe/S cluster biosynthesis machinery and is required for the maturation of both mitochondrial and cytosolic Fe/S proteins. Thus, the protein seems to participate in the generation of most, if not all, cellular Fe/S proteins similar to what this has been reported for the cysteine desulfurase Nfs1p and the ferredoxin Yah1p (
). However, in contrast to the latter components, the deficiency in Isa1p has a somewhat weaker effect on the biogenesis of the mitochondrial and cytosolic Fe/S proteins. For instance, aconitase was only slightly (2-fold) decreased in the absence of functional Isa1p, whereas succinate dehydrogenase was reduced 4-fold. These biochemical observations fit well to the observed growth phenotype of cells depleted in Isa1p. These cells grew slowly on nonfermentable carbon sources, whereas cells defective in Nfs1p or Yah1p are unable to maintain growth (
). A possible reason for the comparatively mild consequences of the Isa1p deficiency on cellular Fe/S proteins may be the presence of a homologue, termed Isa2p, in yeast. However, simultaneous deletion of the ISA1 and ISA2 genes does not exacerbate the growth defects of single mutants (
This suggests that the Isa proteins perform nonoverlapping functions. The nonessential character of the Isa proteins points to an auxiliary function in the maturation of cellular Fe/S proteins distinguishing them from Nfs1p, the Isu proteins, Yah1p and Jac1p that perform essential functions.
All members of the Isa/IscA protein family are characterized by two sequence motifs termed IsaI and IsaII in the middle and at the C terminus, respectively, of these proteins (Fig. 1). The motifs contain either one or two conserved cysteine residues. Our study demonstrates that each of the cysteine residues of the IsaII motif is essential for function of the protein. Mutation of the cysteines to serine residues yielded mutant proteins that did not revert the growth phenotype of cells depleted in wild-type Isa1p. Moreover, these proteins were inactive in Fe/S cluster biosynthesis for apoproteins both inside and outside mitochondria. Thus, these amino acid residues are central structural elements that might be involved in the binding of iron or an intermediate of Fe/S cluster formation to the Isa proteins. In general, cysteines appear to be important residues of proteins involved in Fe/S cluster biosynthesis. For instance, NifS contains a conserved cysteine residue that transiently carries the sulfur liberated from free cysteine as an enzyme-bound persulfide (
). Similarly, bacterial NifU protein contains conserved cysteine residues at its N terminus that have been shown to be essential for the binding of the iron that is used for Fe/S cluster synthesis by NifS (
The accumulation of iron within mitochondria has emerged to be a rather common consequence of defects in components with a function in Fe/S cluster assembly. 10–30-fold elevated levels of free (i.e.non-heme, non-Fe/S) iron within mitochondria have been observed for mutants defective in ATM1, NFS1, SSQ1, and YAH1 genes (
). Gal-ISA1 cells deficient in Isa1p contained only 2-fold higher amounts of mitochondrial iron as compared with wild-type organelles (not shown). This result is compatible with the auxiliary function of Isa1p in Fe/S cluster biosynthesis.
Our study is of importance for the proposed role of mitochondria in initiating the maturation of cytosolic Fe/S proteins (
and this study). The requirement of these proteins for maturation of Fe/S proteins outside the mitochondria strongly suggests that the entire mitochondrial Fe/S cluster biosynthesis machinery participates in this pathway. This makes it likely that an Fe/S cluster is preassembled in the matrix and, after proper stabilization, becomes exported from the mitochondria, presumably by the transport function of Atm1p. It should be noted that the requirement of Isa1p for Fe/S protein maturation in the cytosol does not necessarily indicate a direct function in the production of these Fe/S clusters. Because the mitochondrial ferredoxin Yah1p itself is an Fe/S protein, the rather strong defect in the maturation of cytosolic Leu1p upon depletion of Isa1p may be due to defective assembly of functional Yah1p, which then causes the defects in the incorporation of Fe/S clusters into cytosolic apoproteins (
Currently available protein sequence data suggest the existence of a universal apparatus for the biosynthesis of Fe/S clusters in eukaryotes, prokaryotes, and archaea. Many of the central components of Fe/S cluster biosynthesis are conserved in organisms representing these evolutionary kingdoms. These proteins include homologues of NifS, NifU, and the [2Fe-2S] ferredoxins. Interestingly, Isa/IscA homologues are present in higher and lower eukaryotes and in prokaryotes but have not been identified yet in archaebacteria. Presumably, the prokaryotic and eukaryotic proteins use similar molecular mechanisms for the assembly of the Fe/S clusters. Nevertheless, some variance in the pathways in different species might be expected, e.g. from the absence of IscU homologues in some bacteria.
Mitochondria have been shown to represent the primary site of Fe/S cluster synthesis in a eukaryotic cell and may be involved in the assembly of virtually all cellular Fe/S proteins (Refs.
). Yeast provides an excellent experimental system to study this fundamental process and to dissect its molecular mechanisms by facilitating the combined use of genetic, cell biological, and biochemical methodologies.