Eukaryotic cells contain dozens, perhaps hundreds, of iron-dependent proteins, which perform critical functions in nearly every major cellular process. Nutritional iron is frequently available to cells in only limited amounts; thus, unicellular and higher eukaryotes have evolved mechanisms to cope with iron scarcity. These mechanisms have been studied at the molecular level in the model eukaryotes Saccharomyces cerevisiae and Schizosaccharomyces pombe, as well as in some pathogenic fungi. Each of these fungal species exhibits metabolic adaptations to iron deficiency that serve to reduce the cell’s reliance on iron. However, the regulatory mechanisms that accomplish these adaptations differ greatly between fungal species.
Although cells express hundreds of metalloenzymes, the mechanisms by which apoenzymes receive their metal cofactors are largely unknown. Poly(rC)-binding proteins PCBP1 and PCBP2 are multifunctional adaptor proteins that bind iron and deliver it to ferritin for storage or to prolyl and asparagyl hydroxylases to metallate the mononuclear iron center. Here, we show that PCBP1 and PCBP2 also deliver iron to deoxyhypusine hydroxylase (DOHH), the dinuclear iron enzyme required for hypusine modification of the translation factor eukaryotic initiation factor 5A. Cells depleted of PCBP1 or PCBP2 exhibited loss of DOHH activity and loss of the holo form of the enzyme in cells, particularly when cells were made mildly iron-deficient. Lysates containing PCBP1 and PCBP2 converted apo-DOHH to holo-DOHH in vitro with greater efficiency than lysates lacking PCBP1 or PCBP2. PCBP1 bound to DOHH in iron-treated cells but not in control or iron-deficient cells. Depletion of PCBP1 or PCBP2 had no effect on the cytosolic Fe-S cluster enzyme xanthine oxidase but led to loss of cytosolic aconitase activity. Loss of aconitase activity was not accompanied by gain of RNAbinding activity, a pattern suggesting the incomplete disassembly of the [4Fe-4S] cluster. PCBP depletions had minimal effects on total cellular iron, mitochondrial iron levels, and heme synthesis. Thus, PCBP1 and PCBP2 may serve as iron chaperones to multiple classes of cytosolic nonheme iron enzymes and may have a particular role in restoring metal cofactors that are spontaneously lost in iron deficient cells.diiron enzyme | iron regulatory protein T ransition metal ions, especially zinc, iron, copper, and manganese, serve as cofactors for hundreds of cellular proteins and play key roles in most biological processes (1). Coordination of metal centers within proteins typically involves a small number of amino acid side chains, and yet the identity and configuration of these side chains are frequently not sufficient to account for the incorporation of the native metal cofactor and the exclusion of nonnative cofactors. In vitro and in heterologous cells, many metalloproteins will bind nonnative metal cofactors with equal or greater affinity than the native metal. In both instances, misincorporation is likely to inactivate the enzyme. It is clear that cellular factors, particularly the subcellular compartment and the state of the cytosolic metal pools, may determine the species incorporated into a specific metal center. The pool of "free" metal ions in the cytosol is exceedingly low (2), because most metals are either tightly bound to proteins or coordinated by small molecules, such as glutathione (3). For most metalloproteins, the specific cellular factors contributing to metallation are unknown, but some proteins rely on metal chaperones: proteins that specifically bind metal ions and deliver them to target enzymes through direct protein-protein interactions (4).Iron is one of the most abundant metals in eukaryotic cells and cofactors in the form of heme, Fe-S clusters, a...
Cells contain hundreds of proteins that require iron cofactors for activity. Iron cofactors are synthesized in the cell, but the pathways involved in distributing heme, iron-sulfur clusters, and ferrous/ferric ions to apoproteins remain incompletely defined. In particular, cytosolic monothiol glutaredoxins and BolA-like proteins have been identified as [2Fe-2S]-coordinating complexes in vitro and iron-regulatory proteins in fungi, but it is not clear how these proteins function in mammalian systems or how this complex might affect Fe-S proteins or the cytosolic Fe-S assembly machinery. To explore these questions, we use quantitative immunoprecipitation and live cell proximitydependent biotinylation to monitor interactions between Glrx3, BolA2, and components of the cytosolic iron-sulfur cluster assembly system. We characterize cytosolic Glrx3⅐BolA2 as a Although hundreds of cellular proteins require iron-containing cofactors for activity (1), the machinery responsible for distributing these cofactors remains relatively obscure. Separate systems must exist for both the mitochondrial and the cytosolic/nuclear compartments and systems must selectively distribute ferrous iron ions, iron-sulfur (Fe-S) centers, and heme.Recent studies indicate that the poly(rC)-binding proteins are involved in the distribution of ferrous iron to ferritin, the principal iron storage protein, and to mono-and dinuclear iron enzymes in the cytosol of mammalian cells (2-4). Studies in both bakers' yeast and vertebrates indicate that many proteins are involved in the assembly and distribution of Fe-S clusters in the cytosol (5-7). These proteins are structurally and functionally conserved across many species. One protein class, the monothiol glutaredoxins, has been functionally implicated in the trafficking of both ionic iron and Fe-S clusters (8 -12).Monothiol glutaredoxins are members of the thioredoxin (Trx) 3 -fold family of proteins. Most members of the Trx family utilize a dithiol active site to catalyze the oxido-reduction of thiol-disulfide residues. In contrast, monothiol glutaredoxins contain a Cys-Gly-Phe-Ser active site that lacks catalytic activity and instead coordinates a [2Fe-2S] cluster via the active site cysteine and the sulfhydryl residue of a molecule of glutathione, which is non-covalently bound adjacent to the glutaredoxin active site (10,(13)(14)(15). In vitro analysis of this Fe-S-containing species indicates that two glutathione-bound glutaredoxin proteins can coordinate a single [2Fe-2S] cluster as a bridging complex. In eukaryotes, distinct monothiol glutaredoxins are expressed in the mitochondria and cytosol. Genetic evidence suggests that mitochondrial glutaredoxins are involved in the transfer of newly assembled Fe-S clusters to recipient apoproteins (8,9,16,17). Cytosolic monothiol glutaredoxins differ from their mitochondrial paralogs in that they contain an amino-terminal Trx-like domain followed by one or more glutaredoxin domains. Studies in fungi suggest these proteins are involved in iron homeostasis.Th...
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