Detection of a [3Fe-4S] cluster intermediate of cytosolic aconitase in yeast expressing iron regulatory protein 1. Insights into the mechanism of Fe-S cluster cycling.

Interconversion of iron regulatory protein 1 (IRP1) with cytosolic aconitase (c-aconitase) occurs via assembly/disassembly of a [4Fe-4S] cluster. Recent evidence implicates oxidants in cluster disassembly. We investigated H(2)O(2)-initiated Fe-S cluster disassembly in c-aconitase expressed in Saccharomyces cerevisiae. A signal for [3Fe-4S] c-aconitase was detected by whole-cell EPR of aerobically grown, aco1 yeast expressing wild-type IRP1 or a S138A-IRP1 mutant (IRP1(S138A)), providing the first direct evidence of a 3Fe intermediate in vivo. Exposure of yeast to H(2)O(2) increased this 3Fe c-aconitase signal up to 5-fold, coincident with inhibition of c-aconitase activity. Untreated yeast expressing IRP1(S138D) or IRP1(S138E), which mimic phosphorylated IRP1, failed to give a 3Fe signal. H(2)O(2) produced a weak 3Fe signal in yeast expressing IRP1(S138D). Yeast expressing IRP1(S138D) or IRP1(S138E) were the most sensitive to inhibition of aconitase-dependent growth by H(2)O(2) and were more responsive to changes in media iron status. Ferricyanide oxidation of anaerobically reconstituted c-aconitase yielded a strong 3Fe EPR signal with wild-type and S138A c-aconitases. Only a weak 3Fe signal was obtained with S138D c-aconitase, and no signal was obtained with S138E c-aconitase. This, paired with loss of c-aconitase activity, strongly argues that the Fe-S clusters of these phosphomimetic c-aconitase mutants undergo more complete disassembly upon oxidation. Our results demonstrate that 3Fe c-aconitase is an intermediate in H(2)O(2)-initiated Fe-S cluster disassembly in vivo and suggest that cluster assembly/disassembly in IRP1 is a dynamic process in aerobically growing yeast. Further, our results support the view that phosphorylation of IRP1 can modulate its response to iron through effects on Fe-S cluster stability and turnover.     

A novel eukaryotic factor for cytosolic Fe-S cluster assembly

Iron regulatory protein 1 (IRP1) is regulated through the assembly/disassembly of a [4Fe-4S] cluster, which interconverts IRP1 with cytosolic aconitase. A genetic screen to isolate Saccharomyces cerevisiae strains bearing mutations in genes required for the conversion of IRP1 to c-aconitase led to the identification of a previously uncharacterized, essential gene, which we call CFD1 (cytosolic Fe-S cluster deficient). CFD1 encodes a highly conserved, putative P-loop ATPase. A non-lethal mutation of CFD1 (cfd1-1) reduced c-aconitase specific activity in IRP1-transformed yeast by >90%, although IRP1 in these cells could be readily converted to c-aconitase in vitro upon incubation with iron alone. IRP1-transformed cfd1-1 yeast lacked EPR-detectable Fe-S clusters in c-aconitase, pointing to a defect in Fe-S cluster assembly. The specific activity of another cytosolic Fe-S protein, Leu1p, was also inhibited by >90% in cfd1-1 yeast, whereas activity of mitochondrial Fe-S proteins was not inhibited. Consistent with a cytosolic site of activity, Cfd1p was localized in the cytoplasm. To our knowledge, Cfd1p is the first cytoplasmic Fe-S cluster assembly factor described in eukaryotes.     

A phosphomimetic mutation at Ser-138 renders iron regulatory protein 1 sensitive to iron-dependent degradation

Iron regulatory protein 1 (IRP1) binds to mRNA iron-responsive elements (IREs) and thereby controls the expression of IRE-containing mRNAs. In iron-replete cells, assembly of a cubane [4Fe-4S] cluster inhibits IRE-binding activity and converts IRP1 to a cytosolic aconitase. Earlier experiments with Saccharomyces cerevisiae suggested that phosphomimetic mutations of Ser-138 negatively affect the stability of the cluster (N. M. Brown, S. A. Anderson, D. W. Steffen, T. B. Carpenter, M. C. Kennedy, W. E. Walden, and R. S. Eisenstein, Proc. Natl. Acad. Sci. USA 95:15235-15240, 1998). Along these lines, we show here that a highly purified preparation of recombinant human IRP1 bearing a phosphomimetic S138E substitution (IRP1(S138E)) lacks aconitase activity, which is a hallmark of [4Fe-4S] cluster integrity. Similarly, IRP1(S138E) expressed in mammalian cells fails to function as aconitase. Furthermore, we demonstrate that the impairment of [4Fe-4S] cluster assembly in mammalian cells sensitizes IRP1(S138E) to iron-dependent degradation. This effect can be completely blocked by the iron chelator desferrioxamine or by the proteasome inhibitors MG132 and lactacystin. As expected, the stability of wild-type or phosphorylation-deficient IRP1(S138A) is not affected by iron manipulations. Ser-138 and flanking sequences appear to be highly conserved in the IRP1s of vertebrates, whereas insect IRP1 orthologues and nonvertebrate IRP1-like molecules contain an S138A substitution. Our data suggest that phosphorylation of Ser-138 may provide a basis for an additional mechanism for the control of vertebrate IRP1 activity at the level of protein stability.     

Recycling of RNA binding iron regulatory protein 1 into an aconitase after nitric oxide removal depends on mitochondrial ATP

Iron regulatory proteins (IRPs) control iron metabolism by specifically interacting with iron-responsive elements (IREs) on mRNAs. Nitric oxide (NO) converts IRP-1 from a [4Fe-4S] aconitase to a trans-regulatory protein through Fe-S cluster disassembly. Here, we have focused on the fate of IRE binding IRP1 from murine macrophages when NO flux stops. We show that virtually all IRP-1 molecules from NO-producing cells dissociated from IRE and recovered aconitase activity after re-assembling a [4Fe-4S] cluster in vitro. The reverse change in IRP-1 activities also occurred in intact cells no longer exposed to NO and did not require de novo protein synthesis. Likewise, inhibition of mitochondrial aconitase via NO-induced Fe-S cluster disassembly was also reversed independently of protein translation after NO removal. Our results provide the first evidence of Fe-S cluster repair of NO-modified aconitases in mammalian cells. Moreover, we show that reverse change in IRP-1 activities and repair of mitochondrial aconitase activity depended on energized mitochondria. Finally, we demonstrate that IRP-1 activation by NO was accompanied by both a drastic decrease in ferritin levels and an increase in transferrin receptor mRNA levels. However, although ferritin expression was recovered upon IRP-1-IRE dissociation, expression of transferrin receptor mRNA continued to rise for several hours after stopping NO flux.     

The essential cytosolic iron-sulfur protein Nbp35 acts without Cfd1 partner in the green lineage

In photosynthetic eukaryotes assembly components of iron-sulfur (Fe-S) cofactors have been studied in plastids and mitochondria, but how cytosolic and nuclear Fe-S cluster proteins are assembled is not known. We have characterized a plant P loop NTPase with sequence similarity to Nbp35 of yeast and mammals, a protein of the cytosolic Cfd1-Nbp35 complex mediating Fe-S cluster assembly. Genome analysis revealed that NBP35 is conserved in the green lineage but that CFD1 is absent. Moreover, plant and algal NBP35 proteins lack the characteristic CXXC motif in the C terminus, thought to be required for Fe-S cluster binding. Nevertheless, chemical reconstitution and spectroscopy showed that Arabidopsis (At) NBP35 bound a [4Fe-4S] cluster in the C terminus as well as a stable [4Fe-4S] cluster in the N terminus. Holo-AtNBP35 was able to transfer an Fe-S cluster to an apoprotein in vitro. When expressed in yeast, AtNBP35 bound 55Fe dependent on the cysteine desulfurase Nfs1 and was able to partially rescue the growth of a cfd1 mutant but not of an nbp35 mutant. The AtNBP35 gene is constitutively expressed in planta, and its disruption was associated with an arrest of embryo development. These results show that despite considerable divergence from the yeast Cfd1-Nbp35 Fe-S scaffold complex, AtNBP35 has retained similar Fe-S cluster binding and transfer properties and performs an essential function.     

Mrs3p, Mrs4p, and frataxin provide iron for Fe-S cluster synthesis in mitochondria

Yeast Mrs3p and Mrs4p are evolutionarily conserved mitochondrial carrier proteins that transport iron into mitochondria under some conditions. Yeast frataxin (Yfh1p), the homolog of the human protein implicated in Friedreich ataxia, is involved in iron homeostasis. However, its precise functions are controversial. Anaerobically grown triple mutant cells (Deltamrs3/4/Deltayfh1) displayed a severe growth defect corrected by in vivo iron supplementation. Because anaerobically grown cells do not synthesize heme, and they do not experience oxidative stress, this growth defect was most likely due to Fe-S cluster deficiency. Fe-S cluster formation was assessed in anaerobically grown cells shifted to air for a brief period. In isolated mitochondria, Fe-S clusters were detected on newly imported yeast ferredoxin precursor and on endogenous aconitase by means of [35S]cysteine labeling and native gel separation. New cluster formation was dependent on iron addition to mitochondria, and the iron concentration dependence was shifted dramatically upward in the Deltamrs3/4 mutant, indicating a role of Mrs3/4p in iron transport. The frataxin mutant strain lacked protein import capacity because of low mitochondrial membrane potential, although this was partially restored by growth in the presence of high iron. Under these conditions, a kinetic defect in new Fe-S cluster formation was still noted. Import of frataxin into frataxin-minus isolated mitochondria promptly corrected the Fe-S cluster assembly defect without further iron addition. These findings show that Mrs3/4p transports iron into mitochondria, whereas frataxin makes iron already within mitochondria available for Fe-S cluster synthesis.     

Human Nbp35 is essential for both cytosolic iron-sulfur protein assembly and iron homeostasis

The maturation of cytosolic iron-sulfur (Fe/S) proteins in mammalian cells requires components of the mitochondrial iron-sulfur cluster assembly and export machineries. Little is known about the cytosolic components that may facilitate the assembly process. Here, we identified the cytosolic soluble P-loop NTPase termed huNbp35 (also known as Nubp1) as an Fe/S protein, and we defined its role in the maturation of Fe/S proteins in HeLa cells. Depletion of huNbp35 by RNA interference decreased cell growth considerably, indicating its essential function. The deficiency in huNbp35 was associated with an impaired maturation of the cytosolic Fe/S proteins glutamine phosphoribosylpyrophosphate amidotransferase and iron regulatory protein 1 (IRP1), while mitochondrial Fe/S proteins remained intact. Consequently, huNbp35 is specifically involved in the formation of extramitochondrial Fe/S proteins. The impaired maturation of IRP1 upon huNbp35 depletion had profound consequences for cellular iron metabolism, leading to decreased cellular H-ferritin, increased transferrin receptor levels, and higher transferrin uptake. These properties clearly distinguished huNbp35 from its yeast counterpart Nbp35, which is essential for cytosolic-nuclear Fe/S protein assembly but plays no role in iron regulation. huNbp35 formed a complex with its close homologue huCfd1 (also known as Nubp2) in vivo, suggesting the existence of a heteromeric P-loop NTPase complex that is required for both cytosolic Fe/S protein assembly and cellular iron homeostasis.     

Metabolic regulation of citrate and iron by aconitases: role of iron–sulfur cluster biogenesis

Iron and citrate are essential for the metabolism of most organisms, and regulation of iron and citrate biology at both the cellular and systemic levels is critical for normal physiology and survival. Mitochondrial and cytosolic aconitases catalyze the interconversion of citrate and isocitrate, and aconitase activities are affected by iron levels, oxidative stress and by the status of the Fe–S cluster biogenesis apparatus. Assembly and disassembly of Fe–S clusters is a key process not only in regulating the enzymatic activity of mitochondrial aconitase in the citric acid cycle, but also in controlling the iron sensing and RNA binding activities of cytosolic aconitase (also known as iron regulatory protein IRP1). This review discusses the central role of aconitases in intermediary metabolism and explores how iron homeostasis and Fe–S cluster biogenesis regulate the Fe–S cluster switch and modulate intracellular citrate flux.     

A bridging [4Fe-4S] cluster and nucleotide binding are essential for function of the Cfd1-Nbp35 complex as a scaffold in iron-sulfur protein maturation

The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo.