William E. Walden

Structure of dual function iron regulatory protein 1 complexed with ferritin IRE-RNA.

Iron regulatory protein 1 (IRP1) binds iron-responsive elements (IREs) in messenger RNAs (mRNAs), to repress translation or degradation, or binds an iron-sulfur cluster, to become a cytosolic aconitase enzyme. The 2.8 angstrom resolution crystal structure of the IRP1:ferritin H IRE complex shows an open protein conformation compared with that of cytosolic aconitase. The extended, L-shaped IRP1 molecule embraces the IRE stem-loop through interactions at two sites separated by ∼30 angstroms, each involving about a dozen protein:RNA bonds. Extensive conformational changes related to binding the IRE or an iron-sulfur cluster explain the alternate functions of IRP1 as an mRNA regulator or enzyme.

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.

The Aconitase Function of Iron Regulatory Protein 1 GENETIC STUDIES IN YEAST IMPLICATE ITS ROLE IN IRON-MEDIATED REDOX REGULATION

Iron regulatory proteins (IRP) are sequence-specific RNA-binding proteins that mediate iron-responsive gene regulation in animals. IRP1 is also the cytosolic isoform of aconitase (c-aconitase). This latter activity could complement a mitochondrial aconitase mutation (aco1) in Saccharomyces cerevisiae to restore glutamate prototrophy. In yeast, the c-aconitase activity of IRP1 was responsive to iron availability in the growth medium. Although IRP1 expression rescuedaco1 yeast from glutamate auxotrophy, cells remained growth-limited by glutamate, displaying a slow-growth phenotype on glutamate-free media. Second site mutations conferringenhanced cytosolicaconitase-dependent (ECA) growth were recovered. Relative c-aconitase activity was increased in extracts of strains harboring these mutations. One of the ECA mutations was found to be in the gene encoding cytosolic NADP+-dependent isocitrate dehydrogenase (IDP2). This mutation, an insertion of a Ty delta element into the 5′ region of IDP2, markedly elevates expression of Idp2p in glucose media. Our results demonstrate the physiological significance of the aconitase activity of IRP1 and provide insight into the role of c-aconitase with respect to iron and cytoplasmic redox regulation.

From bacteria to mitochondria: Aconitase yields surprises

More than a century of biochemical investigation has brought a thorough understanding of metabolism in a wide variety of organisms in the biosphere. Organisms inhabiting diverse niches ranging from anaerobic to highly aerobic environments share a surprisingly similar spectrum of enzyme activities, which are organized into pathways that provide the products for growth and development. Although this great body of information provides details of metabolic pathways, relatively little is understood regarding the forces that drove the evolution of metabolic pathways or the evolution of the specific enzymes involved. Nowhere is this more evident than in the evolution of mitochondria. Mitochondria participate in a wide range of activities in eukaryotic cells, from energy metabolism to apoptotic signaling in multicellular organisms. This is quite remarkable considering that mitochondria are believed to have evolved from a bacterial endosymbiont (1). Current views on the origins of mitochondria look to a α-proteobacterium endosymbiont as the ancestor (1). The α-proteobacteriaciae includes present-day intracellular parasites such as Rickettsia prowazekii. It was the completion of the R. prowazekii genome sequence in 1998 that revealed the close relationship of mitochondria to the α-proteobacteriaciae (2). In this issue of PNAS, Baughn and Malamy (3) report the identification and characterization of genes encoding the enzymes of the oxidative branch of the Krebs cycle in Bacteroides flagilis. Based on phylogenetic analyses, these authors suggest a common origin of the enzymes of the oxidative branch of the Krebs cycle in Bacteroides sp. and mitochondria. This surprising finding supports a polyphyletc origin of the mitochondrial Krebs cycle, and raises the question of what forces drove the evolution of the mitochondrial proteome. Perhaps a consortium of bacterial endosymbionts contributed to the evolution of mitochondria.

Complex Formation of the Elongation Factor Tu from Pseudomonas aeruginosa with Nucleoside Diphosphate Kinase Modulates Ribosomal GTP Synthesis and Peptide Chain Elongation

The elongation factor Tu (EF-Tu) fromPseudomonas aeruginosa was purified as a 45-kDa polypeptide that forms a complex with both the 12- and 16-kDa forms of nucleoside-diphosphate kinase (Ndk) and predominantly synthesizes GTP. 70 S ribosomes of P. aeruginosa predominantly synthesize GTP, which is inhibited in presence of anti-Ndk antibodies. Anti-EF-Tu antibodies change the specificity of ribosomal GTP synthesis to all nucleoside triphosphate synthesis. Ndk has been shown to be a part of 30 S ribosomes, whereas EF-Tu is found to be associated with the 50 S ribosomal subunit. These data indicate that GTP synthesis in the ribosome is modulated both by Ndk and by EF-Tu. Peptide chain elongation as measured by polymerization of Phe-tRNA on a poly(U) template in presence of GDP can be inhibited by anti-Ndk antibodies and restored by the addition of GTP. Anti-EF-Tu antibodies similarly inhibit peptide chain elongation by P. aeruginosa ribosomes in the in vitro translation assay; however, this inhibition cannot be overcome by adding back GTP. Because the purified EF-Tu·16-kDa Ndk complex predominantly synthesizes GTP, it seems likely that this complex is a significant source of GTP for translational elongation in protein biosynthesis.

Characteristics of the interaction of the ferritin repressor protein with the iron-responsive element

SummaryThe iron-responsive regulation of ferritin mRNA translation is mediated by the specific interaction of the ferritin repressor protein (FRP) with the iron-responsive element (IRE), a highly conserved 28-nucleotide sequence located in the 5′ untranslated region of ferritin mRNAs. The IRE alone is necessary and sufficient to confer repression of translation by FRP upon a heterologous message, chloramphenicol acetyltransferase, in an in vitro translation system. The activity of FRP is sensitive to iron in vivo. Cytoplasmic extracts of rabbit kidney cells show reduction of FRP activity when grown in the presence of iron, as detected by RNA band shift assay. Using a nitrocellulose filter binding assay to examine the interaction of FRP with the IRE in more detail, we find that purified FRP has a single high-affinity binding site for the IRE with aKd of 20–50 pM. Hemin pretreatment decreases the total amount of FRP which can bind to the IRE. This effect is dependent on hemin concentration. Interestingly, the FRP which remains active at a given hemin concentration binds to the IRE with the same high affinity as untreated FRP. A variety of hemin concentrations were examined for their effect on preformed FRP/IRE complexes. All hemin concentrations tested resulted in rapid complex breakdown. The final amount of complex breakdown corresponds to the concentration of hemin present in the reaction. The effect of hemin on FRP activity suggests that a specific hemin binding site exists on FRP.

Crystallization and preliminary X-ray diffraction analysis of iron regulatory protein 1 in complex with ferritin IRE RNA

Iron regulatory protein 1 (IRP1) is a bifunctional protein with activity as an RNA-binding protein or as a cytoplasmic aconitase. Interconversion of IRP1 between these mutually exclusive states is central to cellular iron regulation and is accomplished through iron-responsive assembly and disassembly of a [4Fe-4S] cluster. When in its apo form, IRP1 binds to iron responsive elements (IREs) found in mRNAs encoding proteins of iron storage and transport and either prevents translation or degradation of the bound mRNA. Excess cellular iron stimulates the assembly of a [4Fe-4S] cluster in IRP1, inhibiting its IRE-binding ability and converting it to an aconitase. The three-dimensional structure of IRP1 in its different active forms will provide details of the interconversion process and clarify the selective recognition of mRNA, Fe-S sites and catalytic activity. To this end, the apo form of IRP1 bound to a ferritin IRE was crystallized. Crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 109.6, b = 80.9, c = 142.9 A, beta = 92.0 degrees. Native data sets have been collected from several crystals with resolution extending to 2.8 A and the structure has been solved by molecular replacement.

Specificity of the induction of ferritin synthesis by hemin

We have previously reported that hemin derepresses ferritin mRNA translation in vitro. As noted earlier, pre-incubation of a 90 kDa ferritin repressor protein (FRP) with hemin prevented subsequent repression of ferritin synthesis in a wheat germ extract. The significance of this observation has been investigated further. Evidence is presented here that this inactivation of FRP is temperature dependent. Neither FeCl3, Fe3+ chelated with EDTA, nor protoporphyrin IX caused significant inactivation of FRP under comparable conditions, whereas Zn2(+)-protoporphyrin IX produced an intermediate degree of inhibition. The presence of a glutathione redox buffer (GSB), which was previously shown to minimize non-specific side-effects of hemin, was not necessary for the derepression reaction. Inclusion of mannitol, a free radical scavenger, did not alter the inactivation caused by hemin. Calculation of the expected ratio of hemin monomers to dimers suggests that the active species is the monomer.

Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions

Metal ion binding was previously shown to destabilize IRE-RNA/IRP1 equilibria and enhanced IRE-RNA/eIF4F equilibria. In order to understand the relative importance of kinetics and stability, we now report rapid rates of protein/RNA complex assembly and dissociation for two IRE-RNAs with IRP1, and quantitatively different metal ion response kinetics that coincide with the different iron responses in vivo. kon, for FRT IRE-RNA binding to IRP1 was eight times faster than ACO2 IRE-RNA. Mn2+ decreased kon and increased koff for IRP1 binding to both FRT and ACO2 IRE-RNA, with a larger effect for FRT IRE-RNA. In order to further understand IRE-mRNA regulation in terms of kinetics and stability, eIF4F kinetics with FRT IRE-RNA were determined. kon for eIF4F binding to FRT IRE-RNA in the absence of metal ions was 5-times slower than the IRP1 binding to FRT IRE-RNA. Mn2+ increased the association rate for eIF4F binding to FRT IRE-RNA, so that at 50 µM Mn2+ eIF4F bound more than 3-times faster than IRP1. IRP1/IRE-RNA complex has a much shorter life-time than the eIF4F/IRE-RNA complex, which suggests that both rate of assembly and stability of the complexes are important, and that allows this regulatory system to respond rapidly to change in cellular iron.

Interaction with Cfd1 Increases the Kinetic Lability of FeS on the Nbp35 Scaffold

Background: A Cfd1 and Nbp35 heterocomplex serves as scaffold for cytosolic iron-sulfur cluster assembly. Results: Deficiency in Cfd1-Nbp35 interaction impaired iron turnover on Nbp35. Conclusion: Cfd1 promotes binding and transfer of labile iron-sulfur cluster on the Nbp35 scaffold. Significance: This is the first insight into the unique roles of these P-loop ATPases in cytosolic iron-sulfur cluster assembly. P-loop NTPases of the ApbC/Nbp35 family are involved in FeS protein maturation in nearly all organisms and are proposed to function as scaffolds for initial FeS cluster assembly. In yeast and animals, Cfd1 and Nbp35 are homologous P-loop NTPases that form a heterotetrameric complex essential for FeS protein maturation through the cytosolic FeS cluster assembly (CIA) pathway. Cfd1 is conserved in animals, fungi, and several archaeal species, but in many organisms, only Nbp35 is present, raising the question of the unique roles played by Cfd1 and Nbp35. To begin to investigate this issue, we examined Cfd1 and Nbp35 function in budding yeast. About half of each protein was detected in a heterocomplex in logarithmically growing yeast. Nbp35 readily bound 55Fe when fed to cells, whereas 55Fe binding by free Cfd1 could not be detected. Rapid 55Fe binding to and release from Nbp35 was impaired by Cfd1 deficiency. A Cfd1 mutation that caused a defect in heterocomplex stability supported iron binding to Nbp35 but impaired iron release. Our results suggest a model in which Cfd1-Nbp35 interaction increases the lability of assembled FeS on the Nbp35 scaffold for transfer to target apo-FeS proteins.