Cytosolic Aconitase and Ferritin Are Regulated by Iron inCaenorhabditis elegans
2003; Elsevier BV; Volume: 278; Issue: 5 Linguagem: Inglês
10.1074/jbc.m210333200
ISSN1083-351X
AutoresBrett L. Gourley, Samuel B. Parker, Barbara J. Jones, Kimberly B. Zumbrennen, Elizabeth A. Leibold,
Tópico(s)Genetics, Aging, and Longevity in Model Organisms
ResumoIron regulatory protein-1 (IRP-1) is a cytosolic RNA-binding protein that is a regulator of iron homeostasis in mammalian cells. IRP-1 binds to RNA structures, known as iron-responsive elements, located in the untranslated regions of specific mRNAs, and it regulates the translation or stability of these mRNAs. Iron regulates IRP-1 activity by converting it from an RNA-binding apoprotein into a [4Fe-4S] cluster protein exhibiting aconitase activity. IRP-1 is widely found in prokaryotes and eukaryotes. Here, we report the biochemical characterization and regulation of an IRP-1 homolog in Caenorhabditis elegans(GEI-22/ACO-1). GEI-22/ACO-1 is expressed in the cytosol of cells of the hypodermis and the intestine. Like mammalian IRP-1/aconitases, GEI-22/ACO-1 exhibits aconitase activity and is post-translationally regulated by iron. Although GEI-22/ACO-1 shares striking resemblance to mammalian IRP-1, it fails to bind RNA. This is consistent with the lack of iron-responsive elements in the C. elegans ferritin genes, ftn-1 and ftn-2. While mammalian ferritin H and L mRNAs are translationally regulated by iron, the amounts of C. elegans ftn-1 and ftn-2 mRNAs are increased by iron and decreased by iron chelation. Excess iron did not significantly alter worm development but did shorten their life span. These studies indicated that iron homeostasis in C. elegans shares some similarities with those of vertebrates. Iron regulatory protein-1 (IRP-1) is a cytosolic RNA-binding protein that is a regulator of iron homeostasis in mammalian cells. IRP-1 binds to RNA structures, known as iron-responsive elements, located in the untranslated regions of specific mRNAs, and it regulates the translation or stability of these mRNAs. Iron regulates IRP-1 activity by converting it from an RNA-binding apoprotein into a [4Fe-4S] cluster protein exhibiting aconitase activity. IRP-1 is widely found in prokaryotes and eukaryotes. Here, we report the biochemical characterization and regulation of an IRP-1 homolog in Caenorhabditis elegans(GEI-22/ACO-1). GEI-22/ACO-1 is expressed in the cytosol of cells of the hypodermis and the intestine. Like mammalian IRP-1/aconitases, GEI-22/ACO-1 exhibits aconitase activity and is post-translationally regulated by iron. Although GEI-22/ACO-1 shares striking resemblance to mammalian IRP-1, it fails to bind RNA. This is consistent with the lack of iron-responsive elements in the C. elegans ferritin genes, ftn-1 and ftn-2. While mammalian ferritin H and L mRNAs are translationally regulated by iron, the amounts of C. elegans ftn-1 and ftn-2 mRNAs are increased by iron and decreased by iron chelation. Excess iron did not significantly alter worm development but did shorten their life span. These studies indicated that iron homeostasis in C. elegans shares some similarities with those of vertebrates. iron regulatory protein iron-responsive element untranslated region nematode growth medium ferric ammonium citrate deferoxamine green fluorescent protein Iron is an essential element required for growth and survival of most organisms. The importance of iron is implicit in the role it plays in oxygen transport and heme synthesis as well as its ability to serve as a cofactor for enzymes involved in a variety of biological processes including DNA synthesis, energy production, and neurotransmitter synthesis. Abnormally high concentration of cellular iron is toxic due to its ability to catalyze the generation of free radicals that damage DNA, lipids, and proteins. In humans, the accumulation of excess cellular iron can result in cirrhosis, arthritis, cardiomyopathy, diabetes mellitus, and increased risk of cancer and heart disease. To provide adequate iron for cellular needs yet prevent the accumulation of excess iron, the concentration of iron within cells is tightly controlled. In vertebrates, the iron regulatory proteins 1 and 2 (IRP-1 and IRP-2)1 regulate iron homeostasis. IRPs are cytosolic RNA-binding proteins that regulate the translation or the stability of mRNAs encoding proteins involved in iron and energy homeostasis (1Hentze M.W. Kühn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Google Scholar, 2Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Google Scholar, 3Schneider B.S. Leibold E.A. Curr. Opin. Clin. Nutr. Metab. Care. 2000; 3: 267-273Google Scholar, 4Theil E.C. Eisenstein R.S. J. Biol. Chem. 2000; 275: 40659-40662Google Scholar). IRPs bind to RNA stem-loop structures, known as iron-responsive elements (IREs), that are located in either the 5′- or 3′-untranslated regions (UTRs) of specific mRNAs. These mRNAs encode proteins involved in iron storage (ferritin), iron utilization (erythroid aminolevilunate synthase and mitochondrial aconitase), and iron transport (transferrin receptor and divalent metal transporter-1). When iron is scarce, IRP binding to the 5′ IRE in ferritin mRNA represses translation, whereas IRP binding to the 3′ IREs in the transferrin receptor mRNA stabilizes this mRNA. When iron is abundant, IRPs lose affinity for the IREs, leading to enhanced ferritin synthesis and to the rapid degradation of transferrin receptor mRNA. By regulating the amount of iron taken up by transferrin receptor and the amount of iron sequestered by ferritin, cellular iron concentration is maintained, and iron toxicity is avoided. Iron regulates the RNA binding activity of IRP-1 and IRP-2, but the mechanism of regulation differs. In the presence of iron, a [4Fe-4S] cluster assembles in IRP-1, converting it from an RNA-binding protein into a cytosolic aconitase. Aconitases are [4Fe-4S] cluster proteins that are found in the cytosol, mitochondria, and glyoxysomes and catalyze the reversible isomerization of citrate and isocitrate viacis-aconitate (5Gruer M.J. Artymiuk P.J. Guest J.R. Trends Biochem. Sci. 1997; 22: 3-6Google Scholar). Despite information regarding the role of aconitase in mitochondria and in the glyoxylate cycle in microorganisms and plants, the function of cytosolic aconitase in higher eukaryotes is not clear. Unlike IRP-1, IRP-2 lacks a [4Fe-4S] cluster and consequently lacks aconitase activity (6Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Google Scholar). Rather, iron regulation of IRP-2 involves iron-induced IRP2 degradation by the proteasome (7Guo B. Phillips J.D., Yu, Y. Leibold E.A. J. Biol. Chem. 1995; 270: 21645-21651Google Scholar, 8Iwai K. Klausner R.D. Rouault T.A. EMBO J. 1995; 14: 5350-5357Google Scholar, 9Iwai K. Drake S.K. Wehr N.B. Weissman A. LaVaute T. Minato N. Klausner R.D. Levine R.L. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4924-4928Google Scholar). IRP-1s have been identified in a wide variety of organisms, including bacteria (10Prodromou C. Artymiuk P.J. Guest J.R. Eur. J. Biochem. 1992; 204: 599-609Google Scholar, 11Alen C. Sonenshein A.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10412-10417Google Scholar, 12Mengaud J.M. Horwitz M.A. J. Bacteriol. 1993; 175: 5666-5676Google Scholar), plants (13Peyret P. Perez P. Alric M. J. Biol. Chem. 1995; 270: 8131-8137Google Scholar, 14Hayashi M. DeBellis L. Alpi A. Nishimura M. Plant Cell Physiol. 1995; 36: 669-680Google Scholar, 15Navarre D.A. Wendehenne D. Durner J. Noad R. Klessig D.F. Plant Physiol. 2000; 122: 573-582Google Scholar), and animals (16Muckenthaler M. Gunkel N. Frishman D. Cyrklaff A. Tomancak P. Hentze M.W. Eur. J. Biochem. 1998; 254: 230-237Google Scholar, 17Saas J. Ziegelbauer K. von Haeseler A. Fast B. J. Biol. Chem. 2000; 275: 2745-2755Google Scholar). IRP-1s share a high degree of amino acid identity among different species. For example, mammalian IRP-1s are >90% identical and are highly homologous to IRP-1s from other organisms, includingCaenorhabditis elegans (64% identity) (16Muckenthaler M. Gunkel N. Frishman D. Cyrklaff A. Tomancak P. Hentze M.W. Eur. J. Biochem. 1998; 254: 230-237Google Scholar),Arabidopsis thaliana (59% identity) plants (13Peyret P. Perez P. Alric M. J. Biol. Chem. 1995; 270: 8131-8137Google Scholar),Trypanosome brucei (64% identity) (17Saas J. Ziegelbauer K. von Haeseler A. Fast B. J. Biol. Chem. 2000; 275: 2745-2755Google Scholar), andEscherichia coli (52% identity) (10Prodromou C. Artymiuk P.J. Guest J.R. Eur. J. Biochem. 1992; 204: 599-609Google Scholar). In contrast, IRP-1s share only ∼20% amino acid identity to mitochondrial aconitases. Although these IRP-1s show striking similarity to mammalian IRP-1 and in most cases exhibit aconitase activity, only vertebrate and insect IRP-1s (18Rothenberger S. Mullner E.W. Kühn L.C. Nucleic Acids Res. 1990; 18: 1175-1179Google Scholar, 19Kohler S.A. Henderson B.R. Kuhn L.C. J. Biol. Chem. 1995; 270: 30781-30786Google Scholar, 20Gray N.K. Pantopoulous K. Dandekar T. Ackrell B.A. Hentze M.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4925-4930Google Scholar, 21Zhang D. Albert D.W. Kohlhepp P., D- Pham D.Q. Winzerling J.J. Insect Mol. Biol. 2001; 10: 531-539Google Scholar) bind RNA. Exceptions are Bacillus subtilisand Plasmodium falciparum IRP-1s, where studies show that these proteins are capable of binding to a mammalian consensus IRE (11Alen C. Sonenshein A.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10412-10417Google Scholar,22Loyevsky M. LaVaute T. Allerson C.R. Stearman R. Kassim O.O. Cooperman S. Gordeuk V.R. Rouault T.A. Blood. 2001; 15: 2555-2562Google Scholar). Here, we report on the biochemical properties and regulation ofC. elegans cytosolic aconitase (GEI-22/ACO-1). C. elegans is a multicellular organism that shares many basic cellular mechanisms with vertebrates, and consequently, it has been used to study development, neurobiology, stress, and aging. Many of the same genes that are involved in iron and energy homeostasis in vertebrates are conserved in C. elegans, including aconitases, ferritin, divalent metal transporter-1, frataxin, and iron sulfur cluster assembly proteins, suggesting that iron and energy homeostasis are also conserved. These features prompted us to characterize the biochemical properties and the regulation of GEI-22/ACO-1 and mechanisms regulating iron homeostasis in C. elegans. Wild-type C. elegans (variety Bristol, strain N2) were cultivated on nematode growth medium (NGM) agar plates or in large scale liquid cultures seeded with E. coli strain OP50 at 22 °C (23Sulston J.E. Brenner S. Genetics. 1974; 77: 95-104Google Scholar). For large scale cultures, mixed stage worms were placed in complete S-basal medium (500 ml) containing OP50 bacteria (15 g) and grown until the bacteria were gone. S-basal medium was supplemented with 0.003–6.6 mg/ml ferric ammonium citrate (FAC) or 100 μm deferoxamine (DF), an iron chelator. Because FAC lowered the pH of S-basal medium, it was adjusted to pH 7.0. Worms were grown for 4 days with gentle shaking at 180 rpm and were collected and washed free of bacteria by sucrose flotation. The toxic concentration of FAC and DF was determined by iron toxicity assays (see below) and by growth of worms in different DF concentrations followed by assaying worm survival. RNA for Northern blots was made from L4 worms prepared by alkaline hypochlorite of gravid adults (24Sulston J. Hodgkins J. Wood W.B. The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 603-605Google Scholar). Embryos were grown on NGM agar plates (12 × 60-mm plates) supplemented with FAC (0.003–6.6 mg/ml) and adjusted to pH 7.0 or DF (100 μm) for 4 days at 22 °C. DF-supplemented plates were prepared by seeding NGM plates with 0.1 ml of OP50 bacteria (380 mg/ml) to which 100 μmDF was added. For iron toxicity life span analysis, 10 L4 larval stage worms were placed on NGM agar plates supplemented with FAC (0.03–6.6 mg/ml) at 22 °C. Each day, the worms were moved to a new plate and were scored as dead if they failed to move when provoked or lacked pharyngeal pumping. Three distinct experiments were carried out, with 10 worms in each experiment. Statistical comparisons were made using a Cox regression model testing for trend in survival explained by the dose of iron. C. elegans gei-22/aco-1 cDNA was synthesized from RNA isolated from mixed stage worms using TRIzol reagent (Invitrogen) and Superscript II reverse transcriptase (Invitrogen). PCR was performed usingPfu polymerase and an upstream primer containing aKpnI site followed by the first 18 nt of thegei-22/aco-1 coding sequence (5′-GCGCGGTACCGCCATGCGTTTCAACAACCTT-3′) and a downstream primer containing the last 18 nt of gei-22/aco-1 coding region in frame with a FLAG epitope, a stop codon, and anXbaI site (5′-GCGCTCTAGATTACTTGTCATCGTCGTCCTTGTAGTCTTGGATCAACTTTCTGATCAT-3′). These primers were chosen based on the alignment of the GEI-22/ACO-1 amino acid sequence predicted from the ZK455.1 with those of other IRP-1s using the ClustalW multiple sequence alignment program (Fig. 1). The 2.9-kb fragment was cloned into the KpnI-XbaI sites of pcDNA3 (Invitrogen), yielding the mammalian expression construct pACO-1FLAG. A translational green fluorescent protein (GFP) construct GEI-22/ACO-1::GFP was constructed by digesting ZK455.1 withEco47III-NruI and inserting the 3,895-bp fragment into the SmaI site of the promoterless GFP reporter vector pPD95.77 (Dr. Andrew Fire). This construct contained 939 nt of 5′ regulatory sequences and 2.9 kb of gei-22/aco-1coding and intronic sequences (7 introns) fused in frame to GFP. The construct lacks sequences encoding the last 110 amino acids of GEI-1/ACO-1 and the 3′-UTR, resulting in a nonfunctional protein. pGFP-ACO-1 (30 ng/μl) was co-injected with pRF4 DNA (30 ng/μl) containing a dominant mutant gene for rol-6 allele, and transformed worms were selected by their rolling behavior. Four transgenic worms were generated, and all showed the same GFP expression pattern. A His8 tag was cloned onto the N terminus of pACO-1FLAG, and the insert was subcloned into theKpnI-XbaI sites of pYes2 (Invitrogen), yielding the yeast expression construct pYhis-ACO-1FLAG. pYhis-ACO-1FLAG was transformed into yeast strain JEL1 (25Lindsley J.E. Wang J.C. J. Biol. Chem. 1993; 268: 8096-8104Google Scholar). Yeast were grown overnight in synthetic complete medium minus uracil (SC-Ura−) containing 2% dextrose. Yeast were then diluted 1:100 into SC-Ura− containing 3% glycerol, 2.0% lactic acid and grown to an A 600 of 0.8. 2% galactose was added to the culture for 10–18 h to induce GEI-22/ACO-1 expression. Yeasts were harvested, and the pellet was resuspended in 3 ml/g, wet weight, lysis extraction buffer (0.3 m NaCl, 0.05m NaH2PO4, pH 8.0, 10 mm β-mercaptoethanol, 0.025% Nonidet P-40, 0.1 mm EDTA) containing a protease inhibitor tablet (Roche Molecular Biochemicals). Cells were disrupted by vortexing with glass beads, and the lysate was centrifuged at 27,000 × gfor 30 min. The lysate was mixed with 2.0 ml of equilibrated Ni2+-nitrilotriacetic acid resin (Qiagen) for 1 h at 4 °C. The slurry was poured into a column and was washed with column buffer (0.3 m NaCl, 0.05 mNaH2PO4, pH 8.0) containing 0.07 mimidazole. GEI-22/ACO-1 was eluted with column buffer containing 0.2m imidazole, and fractions containing GEI-22/ACO-1 were dialyzed against assay buffer (0.05 m Tris, pH 7.3, 0.1m NaCl, 1 mm dithiothreitol). For antibody preparation, GEI-22/ACO-1 was further purified by preparative 8% SDS-PAGE. The protein was visualized using a cold solution of 2.5m KCl, and the band containing GEI-22/ACO-1 was excised and used to inject rabbits. The polyclonal antibodies detected a band on SDS-PAGE with a molecular mass of ∼100 kDa, which corresponds to the size of the predicted gene product ofgei-22/aco-1 mRNA. Preimmune serum did not detect this band. A 10-cm plate of HEK 293 cells was cotransfected with 8 μg of pACO-1FLAG and 2 μg of pEGFP (Clontech) DNAs. The cells were equally split after 5 h to six 35-mm plates, and duplicate plates received either FAC (50 μg/ml) or DF (50 μm) or no addition. The cells were incubated for 16 h and then harvested in 0.125 ml of lysis buffer (0.02 mHEPES, pH 7.6, 0.025 m KCl, 1 mmdithiothreitol, 0.25% Nonidet P-40) containing a protease inhibitor tablet (Roche Molecular Biochemicals). The lysate was centrifuged at 15,000 × g for 20 min at 4 °C, and the supernatant was assayed for protein using the Coomassie Blue Plus Protein Reagent (Pierce). Worms were harvested from 500-ml cultures containing 0.33 mg/ml FAC or 100 μm DF. The worms were washed free of bacteria by sucrose flotation and suspended in a 2× volume of homogenization buffer (0.05m Tris-HCl, pH 7.9, 25% glycerol, 0.1 mm EDTA, 0.32 m NH4SO4) containing a protease inhibitor tablet (Roche Molecular Biochemicals). The lysates were homogenized on ice using a Brinkmann Instruments Polytron homogenizer at full power for 15 s, repeated seven times. The homogenates were centrifuged at 160,000 × g for 1 h, and the supernatants were assayed for protein using the Coomassie Blue Plus protein reagent (Pierce). RNA-binding assays were performed as described (26Leibold E.A. Munro H.N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2171-2175Google Scholar), using protein (12 μg) from yeast, HEK 293 cells, or C. elegans lysates and a32P-labeled rat ferritin L-IRE (R. norvegicus fer-l) (25Lindsley J.E. Wang J.C. J. Biol. Chem. 1993; 268: 8096-8104Google Scholar) or a C. elegans ferritin-2 (ftn-2) RNA. The ftn-2 RNA was synthesized from DNA that corresponded to 530 nt 5′ to the start codon offtn-2 gene (C. elegans cosmid D1037.3). The forward primer contained a T7 promoter sequence (19 nt) followed by 20 nt of ftn-2 sequence (5′-TAATACGACTCACTATAGGGGTTGAAGCATAATACTATTACG-3′), and the reverse primer contained the first 20 nt 5′ to the start site inftn-2 (5′-GGTAGTTTGTGGCTGGTAAG-3′). After incubation of the RNA-protein complexes for 20 min, RNase T1 (1 unit) was added to the reaction for 5 min followed by the addition of 1.5 μg of heparin (50 mg/ml) for 5 min. A 5% native polyacrylamide gel was used to resolve the RNA-protein complexes. Aconitase assays were performed using lysates obtained from worms and from HEK 293 cells transfected with pcACO-1FLAG. Aconitase activity was assayed by the addition of cis-aconitate (0.2 mmfinal concentration) to 50 μg of protein in 0.5 ml of aconitase buffer (0.05 m Tris-HCl, pH 7.5, 0.1 m NaCl) (27Kennedy M.C. Emptage M.H. Dreyer J.L. Beinert H. J. Biol. Chem. 1983; 258: 11098-11105Google Scholar). The disappearance of cis-aconitate was measured at 240 nm over time. For aconitase assays in worms, four separate experiments were carried out. The differences between FAC- and DF-treated worms were determined by paired Student's t test, andp < 0.05 was considered significant. Protein from HEK 293 cells (50 μg), yeast (20 μg), and worms (50 μg) was separated by 8% SDS-PAGE, and the protein was transferred onto nitrocellulose membranes. Membranes were incubated with the following primary antibodies: rabbit anti-FLAG (Sigma) (1:5,000), rabbit anti-GEI-22/ACO-1 (1:5,000), mouse anti-GFP (1:2,000), chicken anti-Rattus norvegicus IRP-1 (1:6,000) (6Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Google Scholar), and rabbit anti-R. norvegicus IRP-2 (1:8,000) (6Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Google Scholar). The appropriate goat horseradish peroxidase-conjugated secondary antibodies (Pierce) were used at 1:10,000. Antibodies were visualized using the Renaissance detection system (PerkinElmer Life Sciences). Total RNA was isolated from age-synchronized L4 worms grown on FAC (0.003–6.6 mg/ml)-supplemented or DF (100 μm)-supplemented NGM plates for 4 days. Worms (∼100 mg) were homogenized in TRIzol (1 ml) using a Dounce homogenizer. Total RNA (25 μg) was resolved using a 1.2% formaldehyde agarose gel and transferred to a nylon membrane. The membrane was hybridized with 32P-labeled C. elegans ftn-1, ftn-2, gei-22/aco-1, andact-1 DNA probes prepared by the amplification of worm genomic DNA. DNA templates for amplification were prepared by washing worms in PCR lysis buffer (50 mm KCl, 10 mmTris-HCl, pH 8.2, 2.5 mm MgCl2, 0.45% Nonidet P-40, 0.45% Tween 20, 0.01% gelatin). The worms were placed in 25 μl of lysis buffer containing proteinase K (1 mg/ml) for 1 h at 65 °C followed by a 15-min incubation at 95 °C. The lysate (1 μl) was added to a PCR (50 μl) containing 20 μm of the appropriate forward and reverse primers. Forward and reverse primers, respectively, for PCR were obtained from sequences of the following C. elegans cosmid clones: ftn-1(C54F6.14), 5′-ACGTAGAACTCTACGCCTCC-3′ and 5′-CTCCGAGTCCTGGGCCGG-3′; ftn-2 (D1037.3), 5′-TCCGAGGTTGAAGCTGCC-3′ and 5′-CGGAAAAGTGTTCCTTATCG-3′; andact-1 (TO4C12.6), 5′-GACAATCCATCCGGAATGTGCAAGGCC-3′ and 5′-GAAGCACTTGCGGTGAATGGAT-3′. A probe forgei-22/aco-1 was obtained by excising the DNA insert from pACO-1FLAG. PCRs were resolved on 1% agarose gels, and the DNA bands were purified and 32P-labeled using the RadPrime DNA Labeling System (Invitrogen). 32P-Labeled DNA was hybridized with membranes for 18 h at 42 °C and washed with 2× SSC, 0.5% SDS at 50 °C. Band intensity was normalized toact-1 and quantified by PhosphorImager analysis. The C. elegans genome possesses two aconitase genes, which are designatedgei-22/aco-1 and aco-2. gei-22/aco-1 encodes a protein that shares ∼63% identity with mammalian IRP-1 (16Muckenthaler M. Gunkel N. Frishman D. Cyrklaff A. Tomancak P. Hentze M.W. Eur. J. Biochem. 1998; 254: 230-237Google Scholar), D. melanogaster IRP-1A and 1B (16Muckenthaler M. Gunkel N. Frishman D. Cyrklaff A. Tomancak P. Hentze M.W. Eur. J. Biochem. 1998; 254: 230-237Google Scholar), and A. thaliana (13Peyret P. Perez P. Alric M. J. Biol. Chem. 1995; 270: 8131-8137Google Scholar) and T. brucei(17Saas J. Ziegelbauer K. von Haeseler A. Fast B. J. Biol. Chem. 2000; 275: 2745-2755Google Scholar) IRP-1s while sharing only ∼24% identity to porcine (28Zheng L. Andrews P.C. Hermodson M.A. Dixon J.E. Zalkin H. J. Biol. Chem. 1990; 265: 2814-2821Google Scholar) andC. elegans mitochondrial aconitases (Fig. 1). aco-2 encodes a protein that shares ∼74% identity to human and porcine mitochondrial aconitases. To study the iron homeostasis in C. elegans, agei-22/aco-1 cDNA was generated by reverse transcription-PCR using sequences obtained from the C. elegans cosmid clone ZK455.1. GEI-22/ACO-1 contains all 24 active-site residues required for aconitase activity, including the 3 cysteines that serve as ligands for the [4Fe-4S] cluster (29Robbins A.H. Stout C.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3639-3643Google Scholar, 30Robbins A.H. Stout C.D. Proteins. 1989; 1989: 289-312Google Scholar, 31Lauble H. Kennedy M.C. Beinert H. Stout C.D. Biochemistry. 1992; 31: 2735-2748Google Scholar). GEI-22/ACO-1 also contains residues that are similar to those identified in mammalian IRP-1 implicated in RNA binding, including amino acids 121–130, 685–689, and 732–737 (32Basilion J.P. Rouault T.A. Massinople C.M. Klausner R.D. Burgess W.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 574-578Google Scholar, 33Kaldy P. Menotti E. Moret R. Kuhn L.C. EMBO J. 1999; 18: 6073-6083Google Scholar). The expression pattern of GEI-22/ACO-1 in living worms was determined using GFP-reporter fusions constructs. Agei-22/aco-1::GFPtranslational fusion gene was made that contained ∼1 kb of 5′ regulatory sequences and 2.9 kb of coding and intronic sequences ofgei-22/aco-1 fused in-frame to gfp(Fig. 2 A). This construct lacks 3′-UTR sequences and those encoding the last 110 amino acids GEI-22/ACO-1. Transgenic embryos, L2 larval stage, and adult animals carrying the reporter construct showed high levels of cytosolic GFP expression in the hypodermal seam cells and in the intestine (Fig. 2,B–D). No significant GFP expression was observed in muscle cells or in neurons. Mammalian IRP-1 exhibits the mutually exclusive activities of RNA binding and aconitase. Iron causes IRP-1 to switch from an RNA-binding apoprotein form to a non-RNA binding aconitase form containing a [4Fe-4S] cluster. The switch between these forms occurs without changes in IRP-1 protein levels. Since GEI-22/ACO-1 shows significant amino acid identity with mammalian IRP-1, we questioned whether GEI-22/ACO-1 binds RNA. The 5′ sequences flanking the C. elegans genes encoding ferritin-1 (C. elegans FTN-1) and ferritin-2 (C. elegans FTN-2), succinate dehydrogenase, and ACO-2 were inspected for IREs, since 5′ IREs are found in homologous genes in other organisms (19Kohler S.A. Henderson B.R. Kuhn L.C. J. Biol. Chem. 1995; 270: 30781-30786Google Scholar, 34Leibold E.A. Munro H.N. J. Biol. Chem. 1987; 262: 7335-7341Google Scholar, 35Hentze M.W. Caughman S.W. Rouault T.A. Barriocanal J.G. Dancis A. Harford J.B. Klausner R.D. Science. 1987; 238: 1570-1573Google Scholar, 36Zheng L. Kennedy M.C. Blondin G.A. Beinert H. Zalkin H. Arch. Biochem. Biophys. 1992; 299: 356-360Google Scholar). No consensus IREs was identified in these genes. It was possible, however, that nonconsensus IREs might be present in the 5′-UTRs of these genes. To test for this, IRE binding activity was measured in extracts of worms grown in FAC or the iron chelator DF using 32P-labeled RNAs corresponding to sequences located ∼500 nt upstream of the start site of the ftn-1, ftn-2, aco-2, and succinate dehydrogenase genes. Since C. elegans exons and introns are generally shorter than in vertebrates (37Blumenthal T. Steward K. Riddle D.L. Blumenthal T. Meyer B.J. Priess J.R. C. Elegans II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 117-145Google Scholar), we reasoned that if these genes harbor 5′-IREs, then they should be present within these sequences. Worms were grown in FAC- or DF-supplemental medium, because previous studies showed that FAC and DF can decrease and increase, respectively, IRP-1 RNA binding activity (1Hentze M.W. Kühn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Google Scholar, 2Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Google Scholar, 4Theil E.C. Eisenstein R.S. J. Biol. Chem. 2000; 275: 40659-40662Google Scholar). The concentration of FAC and DF was chosen based on testing the growth of worms in different concentrations of FAC and DF (see “Experimental Procedures”). Fig. 3 B shows that no specific RNA binding activity was detected in worm extracts by RNA-band shift gels with the 32P-labeled ftn-2RNA or with other 32P-labeled C. elegans RNAs (data not shown), although GEI-22/ACO-1 was detected in these extracts by immunoblotting using an anti-GEI-22/ACO-1 antibody (Fig. 3 C). As controls, HEK 293 cells were treated with FAC or DF, and IRP RNA binding activity was measured using the32P-labeled R. norvegicus fer-l IRE (Fig. 3 A). DF increased IRP RNA binding activity as expected, whereas FAC had little effect on IRP RNA binding activity due to the high iron concentration in these cells. No RNA-protein complexes were formed with a 32P-labeled R. norvegicus fer-l IRE in worm extracts (Fig. 3 A). C. elegans extracts spiked with R. norvegicus IRP-1 showed that 32P-labeled R. norvegicus fer-l IRE bound to R. norvegicus IRP-1, indicating that an inhibitor of RNA binding activity was not present in these extracts (data not shown). To confirm that GEI-22/ACO-1 lacks RNA binding activity, we expressed His/FLAG-tagged GEI-22/ACO-1 in yeast using a galactose-inducible promoter. The advantage of yeast is that they do not express endogenous IRPs, which might interfere with the detection of small amounts of RNA binding activity. As controls, yeast strains expressing His-taggedR. norvegicus IRP-1 and IRP-2 were also assayed for RNA binding activity. Galactose induced the expression of GEI-22/ACO-1,R. norvegicus IRP-1, and R. norvegicus IRP-2, but only R. norvegicus IRP-1 and R. norvegicus IRP-2 bound to the R. norvegicus fer-l IRE (Fig. 4, A and B). Taken together, these data indicate that GEI-22/ACO-1 does not bind to a mammalian consensus IRE or to C. elegans RNAs that might be expected to harbor functional IREs. The data indicate that GEI-22/ACO-1 lacks RNA binding activity. The 24 active sites in mitochondrial aconitases (29Robbins A.H. Stout C.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3639-3643Google Scholar, 30Robbins A.H. Stout C.D. Proteins. 1989; 1989: 289-312Google Scholar, 31Lauble H. Kennedy M.C. Beinert H. Stout C.D. Biochemistry. 1992; 31: 2735-2748Google Scholar) are present in GEI-22/ACO-1, suggesting that GEI-22/ACO-1 is an aconitase. To determine whether GEI-22/ACO-1 exhibits aconitase activity and whether it is regulated by iron, total aconitase activity was measured in HEK 293 cells transfected with FLAG-tagged GEI-22/ACO-1 or pcDNA3 control. Some cells were treated with either FAC or DF for 16 h before assaying aconitase activity. Cells transfected with FLAG-tagged GEI-22/ACO-1 showed a ∼2-fold increase in total aconitase activity compared with pcDNA3-transfected cells (Fig. 5 A). When cells were treated with FAC, total aconitase activity increased ∼4-fold in GEI-22/ACO-1-transfected cells compared with pcDNA3- transfected cells. Endogenous aconitase activity did not significantly increase in pcDNA3-transfected cells treated with FAC, indicating that these cells are iron-sufficient, and that the majority of IRP-1 is in the aconitase form. In contrast, DF dramatically reduced aconitase activity in GEI-22/ACO-1- and pcDNA3-transfected cells. The decreased aconitase activity observed in pcDNA3-transfected cells is due to decreased endogenous cytosolic and mitochondrial aconitase activities. GEI-22/ACO-1 was expressed at similar levels in all extracts, suggesting that the change in aconitase activity was due to the post-translational conversion of the apoprotein form to the [4Fe-4S] form (Fig. 5 B). The fact that iron caused a ∼3-fold increase in aconitase activity in GEI-22/ACO-1-transfected cells compared with GEI-22/ACO-1-untreated cells suggested that iron is limiting under conditions of overexpression. Taken together, these data showed that GEI-22/ACO-1 is an aconitase a
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