Abstract

Article15 January 2004free access Genetic ablations of iron regulatory proteins 1 and 2 reveal why iron regulatory protein 2 dominates iron homeostasis Esther G Meyron-Holtz Esther G Meyron-Holtz Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Manik C Ghosh Manik C Ghosh Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Kazuhiro Iwai Kazuhiro Iwai Cell Biology and Metabolism Branch, Bethesda, MD, USAPresent address: Osaka City University, Osaka, Japan Search for more papers by this author Timothy LaVaute Timothy LaVaute Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Xavier Brazzolotto Xavier Brazzolotto Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Urs V Berger Urs V Berger UB-In Situ, Natick, MA, USA Search for more papers by this author William Land William Land Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Hayden Ollivierre-Wilson Hayden Ollivierre-Wilson Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Alex Grinberg Alex Grinberg Laboratory of Mammalian Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Paul Love Paul Love Laboratory of Mammalian Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Tracey A Rouault Corresponding Author Tracey A Rouault Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Esther G Meyron-Holtz Esther G Meyron-Holtz Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Manik C Ghosh Manik C Ghosh Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Kazuhiro Iwai Kazuhiro Iwai Cell Biology and Metabolism Branch, Bethesda, MD, USAPresent address: Osaka City University, Osaka, Japan Search for more papers by this author Timothy LaVaute Timothy LaVaute Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Xavier Brazzolotto Xavier Brazzolotto Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Urs V Berger Urs V Berger UB-In Situ, Natick, MA, USA Search for more papers by this author William Land William Land Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Hayden Ollivierre-Wilson Hayden Ollivierre-Wilson Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Alex Grinberg Alex Grinberg Laboratory of Mammalian Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Paul Love Paul Love Laboratory of Mammalian Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Tracey A Rouault Corresponding Author Tracey A Rouault Cell Biology and Metabolism Branch, Bethesda, MD, USA Search for more papers by this author Author Information Esther G Meyron-Holtz1, Manik C Ghosh1, Kazuhiro Iwai1, Timothy LaVaute1, Xavier Brazzolotto1, Urs V Berger3, William Land1, Hayden Ollivierre-Wilson1, Alex Grinberg2, Paul Love2 and Tracey A Rouault 1 1Cell Biology and Metabolism Branch, Bethesda, MD, USA 2Laboratory of Mammalian Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, MD, USA 3UB-In Situ, Natick, MA, USA *Corresponding author. Section on Human Iron Metabolism, NIH/NICHD/CBMB, Building 18T/Room 101, Bethesda, MD 20892, USA. Tel.: +1 301 496 6368/402; Fax: +1 301 402 0078; E-mail: [email protected] The EMBO Journal (2004)23:386-395https://doi.org/10.1038/sj.emboj.7600041 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The two iron regulatory proteins IRP1 and IRP2 bind to transcripts of ferritin, transferrin receptor and other target genes to control the expression of iron metabolism proteins at the post-transcriptional level. Here we compare the effects of genetic ablation of IRP1 to IRP2 in mice. IRP1−/− mice misregulate iron metabolism only in the kidney and brown fat, two tissues in which the endogenous expression level of IRP1 greatly exceeds that of IRP2, whereas IRP2−/− mice misregulate the expression of target proteins in all tissues. Surprisingly, the RNA-binding activity of IRP1 does not increase in animals on a low-iron diet that is sufficient to activate IRP2. In animal tissues, most of the bifunctional IRP1 is in the form of cytosolic aconitase rather than an RNA-binding protein. Our findings indicate that the small RNA-binding fraction of IRP1, which is insensitive to cellular iron status, contributes to basal mammalian iron homeostasis, whereas IRP2 is sensitive to iron status and can compensate for the loss of IRP1 by increasing its binding activity. Thus, IRP2 dominates post-transcriptional regulation of iron metabolism in mammals. Introduction Iron regulatory protein 1 (IRP1) is a ubiquitously expressed bifunctional protein that functions either as a cytosolic aconitase or as an RNA-binding protein involved in post-transcriptional regulation of iron metabolism (Hentze and Kuhn, 1996; Rouault and Klausner, 1997; Schneider and Leibold, 2000). Similar to the related citric acid cycle enzyme (mitochondrial aconitase) and other members of the aconitase family (Gruer et al, 1997), the enzymatic form of IRP1 contains a (4Fe–4S)2+ cluster prosthetic group at its catalytic center, and interconverts citrate and isocitrate (Kennedy et al, 1992). Although IRP1 is only 22% identical to mitochondrial aconitase in peptide sequence (Rouault et al, 1991), its enzymatic specific activities with all three substrates are very similar to those of mitochondrial aconitase (Kennedy et al, 1992). However, unlike mitochondrial aconitase, the role of cytosolic aconitase in mammalian cellular metabolism remains poorly understood. IRP1 homologs are found in numerous organisms, including Caenorhabditis elegans (Gourley et al, 2003) and Drosophila melanogaster (Muckenthaler et al, 1998), but there are no IRP1 homologues in yeast. In mammals, IRP1 is one of two iron regulatory proteins (IRP1 and IRP2) that bind to RNA stem-loop elements known as iron-responsive elements (IREs) found within transcripts that encode several iron metabolism proteins. IRP binding to IREs within 5′UTRs of transcripts such as ferritin results in translational repression, whereas binding to IREs in the 3′UTR of the transferrin receptor (TfR) results in stabilization of the transcript. Thus, IRPs coordinate the cellular response to iron depletion by decreasing iron storage and increasing iron uptake (Hentze and Kuhn, 1996; Rouault and Klausner, 1997; Schneider and Leibold, 2000). IRP1 and IRP2 share high sequence homology and exhibit very similar biochemical activities with respect to binding affinity and in vitro regulation of IRE-containing transcripts (Kim et al, 1995; Allerson et al, 1999). Overexpression of a form of IRP1 that constitutively binds IREs results in repression of ferritin synthesis and increased TfR expression (DeRusso et al, 1995; Wang and Pantopoulos, 2002), indicating that IRP1 can regulate its proposed target transcripts in cells. Furthermore, although both IRPs are ubiquitously expressed, IRP1 was the focus of much early interest because it appeared to be much more abundant than IRP2 in most cells and tissues (Rouault et al, 1990; Hirling et al, 1992; Mullner et al, 1992; Patino and Walden, 1992; Yu et al, 1992; Henderson et al, 1993). Thus, based on binding affinities, regulatory activities and abundance, IRP1 would be expected to be the predominant mammalian regulator of post-transcriptional iron metabolism. We have previously reported that IRP2 plays an important physiological role in systemic iron homeostasis, as indicated by the fact that IRP2−/− mice develop a progressive neurodegenerative disease associated with misregulation of iron metabolism in specific areas of the brain and the small intestine (LaVaute et al, 2001). These results indicate that IRP1 alone is unable to regulate appropriately iron metabolism in mammalian tissues, and they are consistent with a report that IRP1 is not required for normal regulation of IRP targets in a lymphocyte cell line (Schalinske et al, 1997). To evaluate the role of IRP1 in mammalian iron physiology, we have genetically ablated IRP1 in mice. IRP1−/− mice show no overt pathology and show compromise of normal iron metabolism only in tissues in which the level of IRP1 greatly exceeds that of IRP2. We demonstrate that IRP1 predominantly exists in the cytosolic aconitase form, and that iron starvation sufficient to activate IRP2 does not increase the IRE-binding activity of IRP1. Thus, although IRP1 represents a large pool of potential IRE-binding activity, IRP2 is the primary post-transcriptional regulator of mammalian iron metabolism in response to iron status. Results Targeted deletion of IRP1 Mice with a targeted disruption of IRP1 were generated by homologous recombination with a construct that inserted the neomycin resistance gene into exon 13. Genotyping by Southern analysis using the probe indicated in Figure 1A showed a 10.1 kb wild-type (wt) band, a 4.1 kb mutant band or both bands in heterozygous mice (Figure 1B). The absence of IRP1 can be confirmed by two functional assays. Electrophoretic separation on cellulose acetate membranes, followed by a coupled enzymatic assay for aconitase activity, revealed two distinct bands, which represent cytosolic and mitochondrial aconitase. Targeted deletion of IRP1 abolished cytosolic aconitase activity without affecting mitochondrial aconitase activity (Figure 1C). Likewise, by gel retardation assay, we distinguished IRP1 from IRP2 with a radiolabeled IRE probe and demonstrated the absence of IRP1-dependent IRE-binding activity in embryonic fibroblasts of IRP1−/− mice (Figure 1D). In addition, IRP1 was not detectable on Western blots of IRP1−/− animals (not shown). Figure 1.Targeted deletion of IRP1 in mouse. (A) Schematic diagram of the IRP1 deletion construct (top), IRP1 genomic clone (middle) and IRP1 deletion allele (bottom). (B) Southern analysis of BamHI digest of genomic DNA isolated from mouse tailcuts. In lane 1, wt genomic DNA shows a band of 10.1 kb. In lane 2, genomic DNA from a heterozygote mouse shows a wt band of 10.1 kb and a mutant band of 4.1 kb. In lane 3, DNA from a homozygous mutant shows only a 4.1 kb band. In all three lanes, a wt band for IRP2 is visible at 15.1 kb. (The probe used for IRP1 detection is indicated in (A).) (C) Cytosolic and mitochondrial aconitase activities in mouse liver and kidney lysates were assayed after cellulose acetate electrophoresis. Note that lysates from wt mice show both cytosolic and mitochondrial aconitase activity, while in lysates from IRP1−/− mice cytosolic aconitase activity is absent. (D) Gel retardation assays of IRPs. Lysates of wt and IRP1−/− mouse embryonic fibroblasts (15 μg protein/lane) were incubated with 32P-IRE and resolved on a 10% nondenaturing gel. Both IRPs respond to decreased iron concentration with increased IRE-binding activity. In lysates from IRP1−/− cells, IRP1 IRE-binding activity is absent (C: control; F: 100 μg/ml ferric ammonium citrate; D: 50 μM deferoxamine). Download figure Download PowerPoint Viability and health of IRP1−/− animals Offspring from IRP1+/− matings had a distribution of 27% wt, 27% IRP1−/− and 46% IRP1+/− (n=190), which is consistent with the genotype distribution predicted for this type of mating (25% wt, 25% IRP1−/− and 50% IRP1+/−). Litter sizes of wt matings were comparable to litter sizes of IRP1−/− matings (6.3±2.7 and 8.05±2.6, respectively). Furthermore, all major tissues and glands were histologically normal when stained with standard hematoxylin and eosin (H&E) stains or iron stains. Complete blood cell counts and serum chemistries showed no difference between wt and IRP1−/− mice. Thus, IRP1−/− mice develop and reproduce normally. Expression patterns of IRP1 and IRP2 We have analyzed ferritin and TfR expression in primary cultures of embryonic fibroblasts of the IRP1−/− and IRP2−/− genotype, and found that each cell line is able to iron-dependently regulate ferritin and TfR expression despite the striking phenotypical difference between IRP1−/− and IRP2−/− mice (LaVaute et al, 2001; EG Meyron-Holtz, unpublished). As these results suggested that each IRP was capable of mediating iron-dependent regulation, we asked whether the difference in phenotype of the two IRP−/− mouse models was attributable to differences in tissue-specific expression levels of the two IRPs. In situ hybridization assays revealed that both IRPs were ubiquitously expressed, but the relative expression levels of the two mRNAs differed in a cell- and tissue-specific manner. In newborn pups (P0), IRP1 mRNA was highly expressed in the brown fat, liver and small intestine, whereas IRP2 mRNA expression was highest in the forebrain, cerebellum, dorsal root ganglia, thymus (insert) and retina (Figure 2A). This expression pattern was consistent with the expression pattern detected in adult tissues. IRP2 mRNA was highly expressed in many areas of the adult brain such as the substantia nigra, the granule cell layer of the cerebellum and the hippocampus (Figure 2B). In contrast, IRP1 was highly expressed in several peripheral tissues, including the liver and kidney, whereas IRP1 expression was low in the spleen and heart (Figure 2C). Notably, the expression pattern varies among cell types within specific tissues, a fact that may complicate interpretation of tissue lysate results. Figure 2.Expression patterns of IRP1 and IRP2 determined by in situ hybridization and Western blotting. In situ hybridization was performed using DIG-labeled cRNA probes and AP detection. Purple/brown staining develops in RNA-positive areas. (A) Fresh frozen sections of newborn pups at postpartum day 0 (P0); inset: thymus. Note that IRP1 is highly expressed in the brown fat and liver, whereas IRP2 expression is high in the brain, thymus and retina. (B) Adult brain coronal sections reveal that IRP2 is highly expressed in several brain regions. IRP1 is also expressed in these regions. (C) In adult peripheral tissues, IRP1 is highly expressed in the kidney, liver and epididymis, whereas IRP2 is most highly expressed in the testis. (D) Protein analysis by Western blotting. Lysates from each of the tissues indicated (40 μg protein/lane) were separated by SDS–PAGE, transferred to membranes, and IRP levels were compared between tissues in one gel that is representative of three separate experiments. Note that IRP1 levels are highest in the kidney, liver and brown fat, whereas IRP2 levels are highest in the cerebellum and forebrain and are hardly detectable in the brown fat. Download figure Download PowerPoint To verify that levels of mRNA expression of IRP1 and IRP2 in different tissues correlated with levels of protein expression, we measured IRP1 and IRP2 protein levels from wt tissue lysates by Western blot. The protein levels were consistent with the results of the in situ hybridizations, showing the highest IRP1 expression predominantly in the kidney, liver and brown fat, whereas the highest IRP2 expression was in the forebrain and cerebellum (Figure 2D). Tissue-specific misregulation of ferritin in IRP1−/− mice To examine whether tissue-specific misregulation of ferritin in IRP−/− mice was attributable to differences in tissue-specific expression levels of the two IRPs, levels of H and L subunits of ferritin and TfR levels in various tissues of IRP1−/− or IRP2−/− mice were compared to wt mice. All tissues examined from IRP2−/− mice showed an elevation of steady-state ferritin levels compared to wt tissues, consistent with derepression of ferritin synthesis in all of these tissues (Figure 3A and B). Likewise, TfR decreased in the cerebellum and forebrain from IRP2−/− mice, consistent with lack of protection of the TfR transcript by IRP binding. These results indicate that in the absence of IRP2, IRP1 alone is unable to maintain normal TfR and ferritin levels in any of the examined tissues, and that IRP2 plays an essential role in the regulation of iron metabolism in living animals. In contrast, IRP1−/− mice showed no apparent misregulation of ferritin expression in the cerebellum, forebrain and liver. Elevated levels of ferritin were found only in the brown fat and kidney. IRP1, therefore, is needed for full regulation of iron metabolism in a few specific tissues, whereas in other tissues, IRP2 alone is sufficient for the regulation of ferritin levels (Figure 3B). In summary, we find that the high expression of IRP1 relative to IRP2 in the kidney and brown fat correlates with misregulation of ferritin in these tissues in the IRP1−/− mouse. However, loss of IRP2 disrupts iron metabolism in virtually all tissues, even including those in which its expression is very low, such as brown fat. Figure 3.Ferritin levels are elevated in multiple tissues from IRP2−/− mice, but are elevated only in the kidney and brown fat of IRP1−/− mice. (A) Western blot analysis of ferritin in the liver, kidney and brown fat was performed on tissue lysates of wt, IRP1−/− and IRP2−/− animals using antibodies against mouse liver ferritin. (B) Western blot analysis of ferritin or TfR from the forebrain or cerebellum was performed on tissue lysates of wt, IRP1−/− and IRP2−/− animals using antibodies to mouse ferritin H-chain (forebrain), mouse liver ferritin (cerebellum) or TfR antibodies. The slower running band in forebrain ferritin is L chain that crossreacts with anti-H-chain antibody. Results are presented in duplicate and are representative of at least three experiments. Download figure Download PowerPoint The fraction of IRP1 with cytosolic aconitase activity is much greater than the fraction with IRE-binding activity Since Western analysis of IRP1 detects total IRP1 protein and cannot distinguish between its two functional states, we performed cytosolic aconitase and gel retardation assays to investigate the amount of IRP1 that was either a functional enzyme or an IRE-binding protein. Detection of significant amounts of cytosolic aconitase in the kidney and liver and a smaller amount in brown fat correlated well with the IRP1 expression levels determined in the Western blot (Figure 4A). In comparison, cytosolic aconitase activity was much lower in the cerebellum, forebrain, skeletal muscle and heart. Also, the gel retardation assay reflected this tissue-specific distribution. In vitro addition of 2-mercaptoethanol (2-ME) can reduce and convert IRP1 from its non-IRE-binding to its IRE-binding form, thus providing a means to measure total activatable IRP1. Comparison of IRE-binding activity of IRP1 to the total 2-ME-activatable IRE-binding activity of IRP1 indicated that between 4 and 18% of IRP1 was in an IRE-binding state in various tissues (Figure 4B). Figure 4.IRP1 activity levels in tissues determined by aconitase and gel retardation assays. (A) Cytosolic and mitochondrial aconitase activities in mouse tissue lysates were assayed after cellulose acetate electrophoresis. Note that relative amounts of the two aconitases vary greatly between tissues and that cytosolic aconitase is mainly detected in the kidney, liver and brown fat. (B) Gel retardation assays of IRPs. Lysates from wt tissues (9 μg protein/lane) were incubated with 32P-IRE and resolved on a 10% nondenaturing gel. Assay without 2-ME reflects IRE-binding activity in vivo (top panel). Assay with 2% 2-ME activates IRP1 that was functionally in a non-IRE-binding state to bind IREs in vitro. A representative example of at least three experiments is shown. In the bottom panel, the small percentage of IRP1 that is in the IRE-binding form is shown as cross-hatched blocks in each column. Notably, 4–18% of IRP1 is in the IRE-binding state prior to 2-ME activation in the seven tissues analyzed. Results shown are representative of three independent experiments. Download figure Download PowerPoint Since more than 80% of endogenous IRP1 was not in the IRE-binding form, we were interested in estimating the fraction of IRP1 that functions as an active aconitase in animal tissues. To assess how much IRP1 contributes to total cellular aconitase activity, we separated mitochondrial from cytosolic aconitase on cellulose acetate membranes and calculated a mitochondrial to cytosolic aconitase ratio of 3.8 (n=3) (Figures 1C and 4A). The total aconitase activity in the kidney was measured with a coupled aconitase assay and, using the ratio of 3.8, cytosolic aconitase in the kidney lysate was calculated to be 1.1±0.3 ng/μg protein (n=5). Using IRP1 standard curves in Western blots, we calculated that kidney lysates contained 1.8±0.5 ng IRP1/μg total protein (n=12). Comparing the amount of cytosolic aconitase to the amount of total IRP1 protein detected by Western blots, we calculated that approximately 60% of all IRP1 protein in the kidney was in the cytosolic aconitase form. In the liver, a similar calculation showed that virtually all IRP1 was in the cytosolic aconitase form. These results imply that, at steady-state conditions, most IRP1 functions as cytosolic aconitase, rather than as an IRE-binding protein, or as an intermediate that does not possess either function (Brown et al, 2002). IRE-binding activity of IRP1 is not activated by dietary-induced iron deficiency in animals The substantial fraction of IRP1 that is in the cytosolic aconitase form represents an enormous store of potential regulatory power. To test if this pool of latent IRE-binding activity can be activated by iron starvation, mice were weaned to low- or high-iron diets, and IRP activity and ferritin levels were assessed after 4 months. Surprisingly, the IRE-binding activity of IRP1 was not activated in any tissue examined from mice maintained on a low-iron diet in comparison to the IRE-binding activity in tissues of mice fed a high-iron diet (Figure 5, top and center panels). In contrast, IRP2 in these mice was activated in a diet-dependent manner as expected (Figure 5, top and bottom panels). The diet-induced activation of IRP2 was especially evident in the gel shift assays of IRP1−/− tissues. Figure 5.IRE-binding activity of IRP1 is not recruited by a low-iron diet. Mice were maintained on a low (no iron added)- or high (0.3 g ferric ammonium citrate/kg chow)-iron diet after weaning for a period of 4 months prior to killing. Tissues were prepared, and gel retardation assays and Western blot analysis for ferritin were carried out as described previously. Duplicates are shown of lysates from age-, sex- and diet-matched mice. Note that although IRP1 IRE-binding activity is not recruited by the low-iron diet, IRP2 binding activity increases significantly under the same conditions. Results are presented in duplicate and are representative of at least three experiments. Download figure Download PowerPoint To assess whether the ability to regulate IRP targets was intact, we assessed whether iron-deficient IRP1−/− and IRP2−/− mice were able to repress ferritin synthesis. In peripheral tissues of wt mice, ferritin levels were significantly decreased in animals on the low-iron diet (Figure 5). In agreement with the results in Figure 3, IRP2−/− animals on a low-iron diet did not appropriately repress ferritin synthesis in the liver, kidney and brown fat. Ferritin levels were markedly elevated in the cerebellum of IRP2−/− mice compared to wt on both the low- and high-iron diets. These results imply that IRP1 in IRP2−/− animals is not only unable to maintain the steady-state level of ferritin found in wt animals (Figure 3), but it is also unable to register and respond to iron deficiency by inhibiting ferritin synthesis. In contrast to IRP2−/− animals, elevated ferritin levels in IRP1−/− mice were found only in the kidney and brown fat of animals that were on the low-iron diet. In the liver of IRP1−/− mice, ferritin regulation was comparable to wt regulation, and in the cerebellum, ferritin levels were similar to those of wt. Thus, in tissues such as brain and liver, lack of IRP1 does not result in misregulation of ferritin synthesis, whereas in brown fat and kidney, lack of IRP1 compromises but does not completely abolish appropriate ferritin repression under iron starvation conditions. It is interesting to note that the impact of the low-iron diet on ferritin in the cerebellum was minimal, perhaps because the blood–brain barrier protects the brain from extreme iron conditions. IRP2 levels increase in the cerebellum and spleen of IRP1−/− animals, suggesting that IRP2 compensates for loss of IRP1 On multiple occasions, levels of IRP2 detected in gel retardation assays appeared to be elevated in tissues of IRP1−/− animals relative to wt animals. To better assess whether IRP2 levels increase in cells that lack IRP1, we quantitated total IRP2 levels by Western blot in tissues of wt, IRP1−/− and IRP2−/− animals. We consistently observed an approximately two-fold increase in IRP2 levels in the cerebellum and spleen of IRP1−/− animals (Figure 6A). Results of four separate experiments for the cerebellum and three separate experiments for the spleen were quantitated, and a statistically significant increase (P=0.0001 and 0.035) of IRP2 in IRP1−/− cerebellum and spleen, respectively, is shown in Figure 6B. As IRP2 constitutes over one-third of the total IRE-binding activity in the cerebellum (Figure 4B, lane 4), the measured increase of IRP2 in the cerebellum of IRP1−/− mice substantially compensates for the loss of IRE-binding activity normally provided by IRP1 in the cerebellum. Figure 6.Steady-state levels of IRP2 increase in the cerebellum and spleen of IRP1−/− mice. (A) Western blots of equal amounts of cerebellar or spleen lysate from two wt (lanes 1,2), IRP2−/− (lanes 3,4) and IRP1−/− (lanes 5,6) mice show a two-fold increase in IRP1−/− animals compared to control. The blot shows duplicates that are representative of four separate experiments from the cerebellum and three separate experiments from the spleen, and results are represented in a bar graph in (B) after quantitation and statistical comparison (P=0.0001 for cerebellum and P=0.035 for spleen). Download figure Download PowerPoint Discussion Analyses of IRP1−/− and IRP2−/− mice allow us to make a distinction between the major in vivo functions of each IRP. Unlike the IRP2−/− mice, the IRP1−/− mice do not develop neurodegeneration or profound aberrations of iron homeostasis. Our results indicate that IRP1 in animal tissues is mostly in the form of a cytosolic aconitase that is not recruited to regulate iron metabolism in iron-deficient animals. The viability and health of IRP1−/− animals indicate that IRP1 is not critical for either of its activities under normal physiological conditions. It is likely that the viability of the IRP1−/− animals can be attributed to redundancy for each of the two functions of IRP1. In the absence of cytosolic aconitase, mitochondrial aconitase can interconvert citrate and isocitrate, and precursors and products of this reaction can cross the mitochondrial membrane to the cytosol if they are needed (Chen et al, 1998). Similarly, our results reveal that IRP2 appears to be able to regulate fully post-transcriptional iron metabolism in most tissues. A surprising conclusion of our studies is that IRP1 is not recruited to the IRE-binding form in iron-deficient animals. Although the low-iron diet led to markedly increased IRP2 activity in the wt and IRP1−/− mice, IRP1 was not activated by iron starvation, even in mice that expressed no IRP2. Moreover, expression of both subunits of ferritin was misregulated in IRP2−/− animals in all tissues examined. These results strongly imply that IRP2 is poised to sense iron status in animal tissues, whereas IRP1 is not. Interestingly, most IRP1 remains in the non-IRE-binding form, even when iron starvation is sufficient to stabilize and thereby activate IRP2. The inability of IRP1 to convert to the IRE-binding form in iron-deficient animal tissues differs from results that have been repeatedly observed in tissue culture, where IRP1 is usually the major IRE-binding protein activated by treatment of cells with the iron chelator desferal (Pantopoulos and Hentze, 1995, and Figure 1D of this paper). One major difference between animal tissues and tissue culture may be the degree to which the iron–sulfur cluster of IRP1 is destabilized by exposure to oxygen (Rouault and Klausner, 1996; Beinert et al, 1997). Once formed, iron–sulfur clusters may be more stable in cells exposed to low tissue oxygen concentrations than in cell cultures, which are exposed to atmospheric oxygen concentrations (Beckman and Ames, 1998). In addition, the iron–sulfur cluster assembly machinery may continue to function even when cellular iron deficiency is sufficient to activate IRP2. The iron concentration threshold necessary to trigger changes in regulation of proteins involved with iron uptake and sequestration through IRP2 activation may be set differently than the threshold for certain vital functions such as iron–sulfur c