The Aconitase Function of Iron Regulatory Protein 1
2000; Elsevier BV; Volume: 275; Issue: 21 Linguagem: Inglês
10.1074/jbc.m910450199
ISSN1083-351X
AutoresJanaki Narahari, Rong Ma, Man Wang, William E. Walden,
Tópico(s)Biofuel production and bioconversion
ResumoIron 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. 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. iron regulatory proteins iron-responsive element IRE-binding protein cytoplasmic aconitase mitochondrial aconitase erythroid aminolevulinate synthase transferrin receptor bathophenanthroline sulfonate NADP+-dependent isocitrate dehydrogenase 5-fluoro-orotic acid base pair(s) kilobase pair(s) protein kinase C nitric oxide polymerase chain reaction enhanced cytosolic aconitase-dependent growth The iron regulatory proteins (IRP)1 are a small family of sequence-specific RNA-binding proteins that mediate gene regulation by binding to iron-responsive elements (IRE) located in either the 5′- or 3′-untranslated region of a variety of animal cell mRNAs. IRE-containing mRNAs encode proteins of iron storage and transport as well as proteins involved in iron utilization or intermediary metabolism (for review see Refs. 1.Eisenstein R.S. Kennedy M.C. Beinert H. Silver S. Walden W. Metal Ions in Gene Regulation. International Thomson Publishing, New York1997: 157-216Google Scholar and 2.Hentze M.W. Kühn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1126) Google Scholar). IRPs exert their effect through translational regulation (for ferritin, m-aconitase, and erythroid aminolevulinate synthase (eALAS) mRNAs) or by controlling mRNA stability (for transferrin receptor (TfR) mRNA), depending on the location of the IRE (1.Eisenstein R.S. Kennedy M.C. Beinert H. Silver S. Walden W. Metal Ions in Gene Regulation. International Thomson Publishing, New York1997: 157-216Google Scholar, 2.Hentze M.W. Kühn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1126) Google Scholar, 3.Theil E. Sigel A. Sigel H. Metal Ions in Biological Systems. Marcel Dekker, Inc., New York1998: 403-434Google Scholar). Two IRPs have been identified to date, called IRP1 and IRP2 (4.Samaniego F. Chin J. Iwai K. Rouault T.A. Klausner R.D. J. Biol. Chem. 1994; 269: 30904-30910Abstract Full Text PDF PubMed Google Scholar, 5.Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Abstract Full Text PDF PubMed Google Scholar, 6.Guo B. Brown F.M. Phillips J.D., Yu, Y. Leibold E.A. J. Biol. Chem. 1995; 270: 16529-16535Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 7.Henderson B.R. Seiser C. Kühn L.C. J. Biol. Chem. 1993; 268: 27327-27334Abstract Full Text PDF PubMed Google Scholar). Both bind similar IRE sequences and appear to be capable of mediating iron-responsive gene regulation, although each has a distinct IRE preference (8.Henderson B.R. Menotti E. Kühn L.C. J. Biol. Chem. 1996; 271: 4900-4908Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9.Butt J. Kim H.-Y. Basilion J.P. Cohen S. Iwai K. Philpott C.C. Altschul S. Klausner R.D. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4345-4349Crossref PubMed Scopus (129) Google Scholar, 10.Ke Y. Wu J. Leibold E.A. Walden W.E. Theil E.C. J. Biol. Chem. 1998; 273: 23637-23640Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). IRP1 is a bifunctional protein, having activity as an IRE-binding protein (IRE-BP) or as the cytosolic isoform of aconitase (c-aconitase) (1.Eisenstein R.S. Kennedy M.C. Beinert H. Silver S. Walden W. Metal Ions in Gene Regulation. International Thomson Publishing, New York1997: 157-216Google Scholar,11.Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (296) Google Scholar). The two activities of IRP1 are mutually exclusive (1.Eisenstein R.S. Kennedy M.C. Beinert H. Silver S. Walden W. Metal Ions in Gene Regulation. International Thomson Publishing, New York1997: 157-216Google Scholar, 11.Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (296) Google Scholar, 12.Kaptain S. Downey W.E. Tang C. Philpott C. Haile D. Orloff D.G. Harford J.B. Rouault T.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10109-10113Crossref PubMed Scopus (157) Google Scholar, 13.Haile D.J. Rouault T.A. Tang C.K. Chin J. Harford J.B. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7536-7540Crossref PubMed Scopus (214) Google Scholar). Interconversion of IRP1 between an IRE-BP and c-aconitase is itself regulated by iron, through the assembly/disassembly of a [4Fe-4S] cluster (1.Eisenstein R.S. Kennedy M.C. Beinert H. Silver S. Walden W. Metal Ions in Gene Regulation. International Thomson Publishing, New York1997: 157-216Google Scholar, 11.Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (296) Google Scholar, 12.Kaptain S. Downey W.E. Tang C. Philpott C. Haile D. Orloff D.G. Harford J.B. Rouault T.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10109-10113Crossref PubMed Scopus (157) Google Scholar, 13.Haile D.J. Rouault T.A. Tang C.K. Chin J. Harford J.B. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7536-7540Crossref PubMed Scopus (214) Google Scholar). Fe-S cluster assembly occurs under conditions of excess iron, converting IRP1 to c-aconitase and stimulating synthesis of ferritin, m-aconitase, and eALAS, while TfR synthesis is depressed. Iron depletion promotes cluster disassembly, conversion of c-aconitase to IRE-BP, and repression of ferritin, m-aconitase, and eALAS synthesis, while stimulating TfR expression. In contrast to IRP1, IRP2 has only the IRE binding activity and is regulated by iron through protein degradation via the proteasome pathway (1.Eisenstein R.S. Kennedy M.C. Beinert H. Silver S. Walden W. Metal Ions in Gene Regulation. International Thomson Publishing, New York1997: 157-216Google Scholar, 14.Iwai K. Drake S.K. Wehr N.B. Weissman A.M. LaVaute T. Minato N. Klausner R.D. Levine R.L. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4924-4928Crossref PubMed Scopus (264) Google Scholar, 15.Iwai K. Klausner R.D. Rouault T.A. EMBO J. 1995; 14: 5350-5357Crossref PubMed Scopus (192) Google Scholar, 16.Guo B. Phillips J.D., Yu, Y. Leibold E.A. J. Biol. Chem. 1995; 270: 21645-21651Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar).The control of IRP activity is also subject to regulation by other factors. Both IRP1 and IRP2 are phosphorylated in vivo, apparently through the action of protein kinase C (PKC) (17.Eisenstein R.S. Tuazon P.T. Schalinske K.L. Anderson S.A. Traugh J.A. J. Biol. Chem. 1993; 268: 27363-27370Abstract Full Text PDF PubMed Google Scholar, 18.Schalinske K.L. Eisenstein R.S. J. Biol. Chem. 1996; 271: 7168-7176Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Treatment of cells with PKC stimulators results in an increase in IRE binding activity and TfR mRNA abundance concomitant with an increase in IRP phosphorylation (17.Eisenstein R.S. Tuazon P.T. Schalinske K.L. Anderson S.A. Traugh J.A. J. Biol. Chem. 1993; 268: 27363-27370Abstract Full Text PDF PubMed Google Scholar, 18.Schalinske K.L. Eisenstein R.S. J. Biol. Chem. 1996; 271: 7168-7176Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The Fe-S cluster may be the target of phosphoregulation of IRP1. Mutation of serine 138 of IRP1, a site of PKC phosphorylation, to amino acids that mimic phosphoserine results in oxygen-dependent Fe-S cluster instability (19.Brown N.M. Anderson S.A. Steffen D.W. Carpenter T.B. Kennedy M.C. Walden W.E. Eisenstein R.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15235-15240Crossref PubMed Scopus (73) Google Scholar). This observation suggests that the Fe-S cluster of phosphorylated c-aconitase is more susceptible to cluster disassembly in response to oxidants, which likely is part of the normal mechanism of cluster turnover in IRP1 (19.Brown N.M. Anderson S.A. Steffen D.W. Carpenter T.B. Kennedy M.C. Walden W.E. Eisenstein R.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15235-15240Crossref PubMed Scopus (73) Google Scholar). Exposure of cells to nitric oxide (NO) or hydrogen peroxide enhances IRE binding activity and inhibits c-aconitase activity in animal cells (reviewed in Ref. 2.Hentze M.W. Kühn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1126) Google Scholar). Both NO and H2O2 cause Fe-S cluster disruption (20.Kennedy M.C. Antholine W.E. Beinert H. J. Biol. Chem. 1997; 272: 20340-20347Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 21.Brazzolotto X. Gaillard J. Pantopoulos K. Hentze M.W. Moulis J.-M. J. Biol. Chem. 1999; 274: 21625-21630Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The consequence of these regulatory processes would be to modulate the expression of genes regulated by IRP1 and c-aconitase activity, similar to the effect of iron on IRP1.The dichotomy between the role of IRP1 in the post-transcriptional regulation of genes involved in iron metabolism and its direct function as an enzyme of intermediary metabolism is intriguing. To gain insight into the significance of c-aconitase activity in vivo, we expressed IRP1 in aconitase-deficient (aco1)Saccharomyces cerevisiae and investigated conditions that affected its ability to provide c-aconitase activity for cell growth. We found that hyperexpression of cytosolic NADP+-dependent isocitrate dehydrogenase (Idp2p) enhanced the ability of IRP1 to provide aconitase function toaco1 yeast and altered the extent of interconversion of IRP1 between IRE-BP and c-aconitase. These results provide insight into the possible role of c-aconitase in animal cells.DISCUSSIONThe role of IRP1 as c-aconitase in animal cells has been overshadowed by the focus on its function as a regulator of gene expression. In fact, IRP1 exists predominantly as c-aconitase in some tissues, particularly liver (11.Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (296) Google Scholar, 39.Chen O.S. Schalinske K.L. Eisenstein R.S. J. Nutr. 1997; 127: 238-248Crossref PubMed Scopus (101) Google Scholar). Reconstituted in yeast, the ability of IRP1 to function physiologically to provide c-aconitase activity is evident. Here we show that a mutation that leads to hyperexpression of cytosolic isocitrate dehydrogenase enhances the ability of yeast to utilize IRP1 as c-aconitase. Because animal cells also have a cytosolic isocitrate dehydrogenase, c-aconitase in animal cells may well contribute normally to glutamate biosynthesis and other metabolic processes such as fatty acid metabolism (40.Minard K.I. Jennings G.T. Loftus T.M. Xuan D. McAlister-Henn L. J. Biol. Chem. 1998; 273: 31486-31493Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 41.Jennings G.T. Sadleir J.W. Stevenson P.M. Biochim. Biophys. Acta. 1990; 1034: 219-227Crossref PubMed Scopus (23) Google Scholar, 42.Farrell H.M. Wickham E.D. Reeves H.C. Arch. Biochem. Biophys. 1995; 321: 199-208Crossref PubMed Scopus (12) Google Scholar, 43.van Roermund C.W.T. Hettema E.H. Kal A.J. van den Berg M. Tabak H.F. Wanders R.J.A. EMBO J. 1998; 17: 677-687Crossref PubMed Scopus (121) Google Scholar).Depletion of available iron in yeast growth media inhibited c-aconitase activity and growth of IRP1-transformed aco1 yeast on glutamate-free media. That iron may regulate pathways involving c-aconitase in animal cells raises questions regarding the role and significance of iron in regulating metabolic pathways involving c-aconitase. In addition to converting isocitrate to α-ketoglutarate, which is a precursor in glutamate biosynthesis (38.Zhao W.-N. McAlister-Henn L. Biochemistry. 1996; 35: 7873-7878Crossref PubMed Scopus (40) Google Scholar), the reaction catalyzed by Idp2p also produces NADPH. Idp2p is an important source of this cofactor in the cytosol (40.Minard K.I. Jennings G.T. Loftus T.M. Xuan D. McAlister-Henn L. J. Biol. Chem. 1998; 273: 31486-31493Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 42.Farrell H.M. Wickham E.D. Reeves H.C. Arch. Biochem. Biophys. 1995; 321: 199-208Crossref PubMed Scopus (12) Google Scholar, 43.van Roermund C.W.T. Hettema E.H. Kal A.J. van den Berg M. Tabak H.F. Wanders R.J.A. EMBO J. 1998; 17: 677-687Crossref PubMed Scopus (121) Google Scholar, 44.Minard K.I. McAlister-Henn L. J. Biol. Chem. 1999; 274: 3402-3406Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). NADPH is a key cofactor in cellular defenses against oxidative stress, particularly through its involvement in the thioredoxin and glutathione redox cycles (44.Minard K.I. McAlister-Henn L. J. Biol. Chem. 1999; 274: 3402-3406Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 45.Bodaness R.S. Biochem. Biophys. Res. Commun. 1982; 108: 1709-1715Crossref PubMed Scopus (20) Google Scholar, 46.Winkler B.S. DeSantis N. Solomon F. Exp. Eye Res. 1986; 43: 829-847Crossref PubMed Scopus (83) Google Scholar). We propose a model whereby regulation of IRP1/c-aconitase by iron coordinates NADPH levels with iron uptake, utilization, and storage. This would provide the cell with the reducing power to deal with the increased oxidative stress brought on by higher intracellular iron and an additional source of NADPH as a cofactor for ferric reductase (47.Shatwell K.P. Dancis A. Cross A.R. Klausner R.D. Segal A.W. J. Biol. Chem. 1996; 271: 14240-14244Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar,48.Finegold A.A. Shatwell K.P. Segal A.W. Klausner R.D. Dancis A. J. Biol. Chem. 1996; 271: 31021-31024Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Ferric reductase has been shown to be an important component of iron transport systems in eukaryotes (49.Dancis A. Klausner R.D. Hinnebusch A.G. Barriocanal J.G. Mol. Cell. Biol. 1990; 10: 2294-2301Crossref PubMed Scopus (256) Google Scholar, 50.Jordan I. Kaplan J. Biochem. J. 1994; 302: 875-879Crossref PubMed Scopus (82) Google Scholar, 51.Akompong T. Inman R.S. Wessling-Resnick M. J. Biol. Chem. 1995; 270: 20937-20941Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 52.Inman R.S. Coughlan M.M. Wessling-Resnick M. Biochemistry. 1994; 33: 11850-11857Crossref PubMed Scopus (71) Google Scholar, 53.Parkes J.G. Olivieri N.F. Templeton D.M. Toxicology. 1997; 117: 141-151Crossref PubMed Scopus (43) Google Scholar, 54.Han O. Failla M.L. Hill A.D. Morris E.R. Smith J.C. J. Nutr. 1995; 125: 1291-1299PubMed Google Scholar). The increase in NADPH predicted to accompany the rise in c-aconitase activity in iron replete cells would provide additional reducing equivalents, allowing animal cells to maintain the redox balance in the cytoplasm during intensive iron transport. Moreover, the effect of iron on c-aconitase activity and downstream steps catalyzed by Idp2p would provide animal cells with a means to modulate NADPH production specifically. Increased c-aconitase-driven NADPH levels also would favor the Fe(II) state and thereby promote ferritin-mediated iron biomineralization and iron incorporation into heme (55.Waldo G.S. Theil E.C. Suslick K.S. Comprehensive Supramolecular Chemistry. Pergamon Press, Oxford1996: 65-89Google Scholar, 56.Ferreira G.C. Int. J. Biochem. Cell Biol. 1999; 31: 995-1000Crossref PubMed Scopus (48) Google Scholar). The evolution of c-aconitase as an iron-responsive regulator of ferritin synthesis may have been prompted by this redox-dependent regulation of iron storage and utilization.It is not surprising that a mutation in the gene encoding the cytosolic isoform of isocitrate dehydrogenase alters glutamate synthesis in cells utilizing IRP1 as c-aconitase. Idp2p most likely drives the reactions toward glutamate by mass action in Idp2p-hyperexpressing cells. On the other hand, we would have predicted that hyperexpression of Idp2p would not effect the interconversion of IRP1 between the IRE-BP and c-aconitase. However, a higher percentage of IRP1 was converted to c-aconitase in strains that hyperexpressed Idp2p. This suggests that the increase in Idp2p activity either enhanced conversion of IRP1 to c-aconitase or stabilized c-aconitase once it was formed. At present, we cannot distinguish between these possibilities. The proportion of IRP1 that exists as c-aconitase is affected by oxidants produced during normal, aerobic metabolism in yeast (19.Brown N.M. Anderson S.A. Steffen D.W. Carpenter T.B. Kennedy M.C. Walden W.E. Eisenstein R.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15235-15240Crossref PubMed Scopus (73) Google Scholar). Therefore, it is possible that the anti-oxidant effects of elevated NADPH may have protected c-aconitase in these yeast. Alternatively, conversion of apo-IRP1 to c-aconitase could have been enhanced in these strains. Elevated NADPH would effect levels of reduced thioredoxin, which has been shown to reduce oxidized apo-IRP1 generated upon Fe-S cluster removal (57.Oliveira L. Bouton C. Drapier J.-C. J. Biol. Chem. 1999; 274: 516-521Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Protein thiol reduction appears to be a necessary step in the assembly of Fe-S clusters in aconitases (36.Kennedy M.C. Emptage M.H. Dreyer J.-L. Beinert H. J. Biol. Chem. 1983; 258: 11098-11105Abstract Full Text PDF PubMed Google Scholar). Increased NADPH may also enhance Fe-S cluster assembly by increasing availability of Fe(II).In animal cells, interconversion of IRP1 and c-aconitase and iron-responsive gene regulation respond to chelatable iron levels (58.Rogers J. Munro H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2277-2281Crossref PubMed Scopus (113) Google Scholar). In yeast, the transcription factor Aft1p responds to chelatable iron levels by tightly regulating iron uptake by controlling the expression of genes encoding the components of the high affinity iron transport system (59.Hassett R.F. Romeo A.M. Kosman D.J. J. Biol. Chem. 1998; 273: 7628-7636Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 60.Yamaguchi-Iwai Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Crossref PubMed Scopus (288) Google Scholar, 61.Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (313) Google Scholar). Therefore, we might expect that overexpression of IRP1, which could deplete the chelatable iron pool, would cause a net increase in iron accumulation in yeast. We did not observe a consistent increase in iron accumulation in strains overexpressing IRP1, suggesting that iron was not the limiting factor for cluster assembly in IRP1 in yeast (not shown). On the other hand, assembly of an Fe-S cluster in IRP1 in the cytosol of yeast could be limiting. We cannot rule out this possibility at the present time, but other cytosolic Fe-S proteins do exist in yeast, and so it is expected that the machinery for assembling Fe-S clusters in cytosolic proteins is present (62.Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (583) Google Scholar).Growth of aco1 strains that hyperexpress Idp2p on glutamate-free media was very sensitive to the level of c-aconitase activity. This was most evident when iron availability was reduced in the growth media, a condition that significantly inhibited c-aconitase activity (Table I). These yeast strains were hypersensitive to iron depletion, in fact, suggesting that growth of these strains became limited by c-aconitase activity in low iron media. We have also observed effects on growth of an Idp2p hyperexpressing strain when expressing IRP1 mutants that have defects in c-aconitase function (19.Brown N.M. Anderson S.A. Steffen D.W. Carpenter T.B. Kennedy M.C. Walden W.E. Eisenstein R.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15235-15240Crossref PubMed Scopus (73) Google Scholar). Mutations in IRP1 that decreased c-aconitase activity in vivo strongly reduced growth of these yeast strains on glutamate-free media. Moreover, reduction in c-aconitase activityin vivo by lowering IRP1 expression in these strains also led to a much slower growth rate. 2J. Narahari, unpublished observations. The responsiveness of the strains that hyperexpress Idp2p to the level of c-aconitase activity makes them very useful for the study of factors and conditions that affect IRP1 function in vivo. The iron regulatory proteins (IRP)1 are a small family of sequence-specific RNA-binding proteins that mediate gene regulation by binding to iron-responsive elements (IRE) located in either the 5′- or 3′-untranslated region of a variety of animal cell mRNAs. IRE-containing mRNAs encode proteins of iron storage and transport as well as proteins involved in iron utilization or intermediary metabolism (for review see Refs. 1.Eisenstein R.S. Kennedy M.C. Beinert H. Silver S. Walden W. Metal Ions in Gene Regulation. International Thomson Publishing, New York1997: 157-216Google Scholar and 2.Hentze M.W. Kühn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1126) Google Scholar). IRPs exert their effect through translational regulation (for ferritin, m-aconitase, and erythroid aminolevulinate synthase (eALAS) mRNAs) or by controlling mRNA stability (for transferrin receptor (TfR) mRNA), depending on the location of the IRE (1.Eisenstein R.S. Kennedy M.C. Beinert H. Silver S. Walden W. Metal Ions in Gene Regulation. International Thomson Publishing, New York1997: 157-216Google Scholar, 2.Hentze M.W. Kühn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1126) Google Scholar, 3.Theil E. Sigel A. Sigel H. Metal Ions in Biological Systems. Marcel Dekker, Inc., New York1998: 403-434Google Scholar). Two IRPs have been identified to date, called IRP1 and IRP2 (4.Samaniego F. Chin J. Iwai K. Rouault T.A. Klausner R.D. J. Biol. Chem. 1994; 269: 30904-30910Abstract Full Text PDF PubMed Google Scholar, 5.Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Abstract Full Text PDF PubMed Google Scholar, 6.Guo B. Brown F.M. Phillips J.D., Yu, Y. Leibold E.A. J. Biol. Chem. 1995; 270: 16529-16535Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 7.Henderson B.R. Seiser C. Kühn L.C. J. Biol. Chem. 1993; 268: 27327-27334Abstract Full Text PDF PubMed Google Scholar). Both bind similar IRE sequences and appear to be capable of mediating iron-responsive gene regulation, although each has a distinct IRE preference (8.Henderson B.R. Menotti E. Kühn L.C. J. Biol. Chem. 1996; 271: 4900-4908Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9.Butt J. Kim H.-Y. Basilion J.P. Cohen S. Iwai K. Philpott C.C. Altschul S. Klausner R.D. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4345-4349Crossref PubMed Scopus (129) Google Scholar, 10.Ke Y. Wu J. Leibold E.A. Walden W.E. Theil E.C. J. Biol. Chem. 1998; 273: 23637-23640Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). IRP1 is a bifunctional protein, having activity as an IRE-binding protein (IRE-BP) or as the cytosolic isoform of aconitase (c-aconitase) (1.Eisenstein R.S. Kennedy M.C. Beinert H. Silver S. Walden W. Metal Ions in Gene Regulation. International Thomson Publishing, New York1997: 157-216Google Scholar,11.Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (296) Google Scholar). The two activities of IRP1 are mutually exclusive (1.Eisenstein R.S. Kennedy M.C. Beinert H. Silver S. Walden W. Metal Ions in Gene Regulation. International Thomson Publishing, New York1997: 157-216Google Scholar, 11.Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (296) Google Scholar, 12.Kaptain S. Downey W.E. Tang C. Philpott C. Haile D. Orloff D.G. Harford J.B. Rouault T.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10109-10113Crossref PubMed Scopus (157) Google Scholar, 13.Haile D.J. Rouault T.A. Tang C.K. Chin J. Harford J.B. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7536-7540Crossref PubMed Scopus (214) Google Scholar). 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