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• W2000381161 abstract "Mitochondrial ferritin (MtF) is a newly identified ferritin encoded by an intronless gene on chromosome 5q23.1. The mature recombinant MtF has a ferroxidase center and binds ironin vitro similarly to H-ferritin. To explore the structural and functional aspects of MtF, we expressed the following forms in HeLa cells: the MtF precursor (∼28 kDa), a mutant MtF precursor with a mutated ferroxidase center, a truncated MtF lacking the ∼6-kDa mitochondrial leader sequence, and a chimeric H-ferritin with this leader sequence. The experiments show that all constructs with the leader sequence were processed into ∼22-kDa subunits that assembled into multimeric shells electrophoretically distinct from the cytosolic ferritins. Mature MtF was found in the matrix of mitochondria, where it is a homopolymer. The wild type MtF and the mitochondrially targeted H-ferritin both incorporated the 55Fe label in vivo. The mutant MtF with an inactivated ferroxidase center did not take up iron, nor did the truncated MtF expressed transiently in cytoplasm. Increased levels of MtF both in transient and in stable transfectants resulted in a greater retention of iron as MtF in mitochondria, a decrease in the levels of cytosolic ferritins, and up-regulation of transferrin receptor. Neither effect occurred with the mutant MtF with the inactivated ferroxidase center. Our results indicate that exogenous iron is as available to mitochondrial ferritin as it is to cytosolic ferritins and that the level of MtF expression may have profound consequences for cellular iron homeostasis. Mitochondrial ferritin (MtF) is a newly identified ferritin encoded by an intronless gene on chromosome 5q23.1. The mature recombinant MtF has a ferroxidase center and binds ironin vitro similarly to H-ferritin. To explore the structural and functional aspects of MtF, we expressed the following forms in HeLa cells: the MtF precursor (∼28 kDa), a mutant MtF precursor with a mutated ferroxidase center, a truncated MtF lacking the ∼6-kDa mitochondrial leader sequence, and a chimeric H-ferritin with this leader sequence. The experiments show that all constructs with the leader sequence were processed into ∼22-kDa subunits that assembled into multimeric shells electrophoretically distinct from the cytosolic ferritins. Mature MtF was found in the matrix of mitochondria, where it is a homopolymer. The wild type MtF and the mitochondrially targeted H-ferritin both incorporated the 55Fe label in vivo. The mutant MtF with an inactivated ferroxidase center did not take up iron, nor did the truncated MtF expressed transiently in cytoplasm. Increased levels of MtF both in transient and in stable transfectants resulted in a greater retention of iron as MtF in mitochondria, a decrease in the levels of cytosolic ferritins, and up-regulation of transferrin receptor. Neither effect occurred with the mutant MtF with the inactivated ferroxidase center. Our results indicate that exogenous iron is as available to mitochondrial ferritin as it is to cytosolic ferritins and that the level of MtF expression may have profound consequences for cellular iron homeostasis. mitochondrial ferritin truncated MtF mitochondrial ferritin H-ferritin L-ferritin Dulbecco's modified Eagle's medium [55Fe]ferric ammonium citrate enzyme-linked immunosorbent assay desferrioxamine Ferritins are ubiquitous proteins made of 24 subunits that form a spherical shell that can accommodate up to 4,000 iron atoms (reviewed in Ref. 1Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar). In mammals, nearly all of the ferritin is found in cytoplasm, where its expression is controlled translationally by iron through an iron regulatory element in the mRNA (2Cairo G. Pietrangelo A. Biochem. J. 2000; 352: 241-250Crossref PubMed Scopus (276) Google Scholar, 3Hentze M.W. Kuhn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1137) Google Scholar). This ferritin is composed of two subunit types, H and L, with ∼50% sequence identity and very similar three-dimensional structures made of a bundle of four α-helices. H-ferritin shells have ferroxidase activity that results in the conversion of soluble ferrous ions into inert aggregates of ferric hydroxides (4Levi S. Santambrogio P. Cozzi A. Rovida E. Corsi B. Tamborini E. Spada S. Albertini A. Arosio P. J. Mol. Biol. 1994; 238: 649-654Crossref PubMed Scopus (163) Google Scholar, 5Santambrogio P. Levi S. Cozzi A. Rovida E. Albertini A. Arosio P. J. Biol. Chem. 1993; 268: 12744-12748Abstract Full Text PDF PubMed Google Scholar, 6Yang X. Chen-Barrett Y. Arosio P. Chasteen N.D. Biochemistry. 1998; 37: 9743-9750Crossref PubMed Scopus (130) Google Scholar). This ferroxidase activity is associated with di-iron binding sites coordinated by seven residues that are conserved in ferritins from animals, plants, and bacteria (1Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar,7Lawson D.M. Artymiuk P.J. Yewdall S.J. Smith J.M. Livingstone J.C. Treffry A. Luzzago A. Levi S. Arosio P. Cesareni G. Nature. 1991; 349: 541-544Crossref PubMed Scopus (669) Google Scholar). These sites catalyze Fe(II) oxidation, a rate-limiting step in iron incorporation, in a reaction that consumes one dioxygen molecule per two Fe(II) ions and produces hydrogen peroxide (1Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar, 6Yang X. Chen-Barrett Y. Arosio P. Chasteen N.D. Biochemistry. 1998; 37: 9743-9750Crossref PubMed Scopus (130) Google Scholar, 8Chasteen N.D Harrison P.M. J. Struct. Biol. 1999; 126: 182-194Crossref PubMed Scopus (682) Google Scholar). The L-subunit lacks the ferroxidase center, and L-homopolymers do not incorporate iron in vivo. However, the L-subunit provides efficient sites for iron nucleation and mineralization and somehow increases turnover at the H-ferroxidase centers (4Levi S. Santambrogio P. Cozzi A. Rovida E. Corsi B. Tamborini E. Spada S. Albertini A. Arosio P. J. Mol. Biol. 1994; 238: 649-654Crossref PubMed Scopus (163) Google Scholar, 5Santambrogio P. Levi S. Cozzi A. Rovida E. Albertini A. Arosio P. J. Biol. Chem. 1993; 268: 12744-12748Abstract Full Text PDF PubMed Google Scholar, 6Yang X. Chen-Barrett Y. Arosio P. Chasteen N.D. Biochemistry. 1998; 37: 9743-9750Crossref PubMed Scopus (130) Google Scholar). The ferroxidase activity of the H-chain is largely responsible for the biological activity of mammalian ferritins. Inactivation of H-chains in knockout mice is lethal at early stages of embryogenesis (9Ferreira C. Bucchini D. Martin M.E. Levi S. Arosio P. Grandchamp B. Beaumont C. J. Biol. Chem. 2000; 275: 3021-3024Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Overexpression of H-chains in stable transfectants of the mouse erythroleukemic (MEL) cell line (10Picard V. Renaudie F. Porcher C. Hentze M.W. Grandchamp B. Beaumont C. Blood. 1996; 87: 2057-2064Crossref PubMed Google Scholar, 11Epsztejn S. Glickstein H. Picard V. Slotki I.N. Breuer W. Beaumont C. Cabantchik Z.I. Blood. 1999; 94: 3593-3603Crossref PubMed Google Scholar, 12Picard V. Epsztejn S. Santambrogio P. Cabantchik Z.I. Beaumont C. J. Biol. Chem. 1998; 273: 15382-15386Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and HeLa cells results in an iron-deficient phenotype (13Cozzi A Corsi A. Levi S. Santambrogio P. Albertini A. Arosio P. J. Biol. Chem. 2000; 275: 25122-25126Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). This is accompanied by reductions in heme and hemoglobin synthesis and also in proliferation rate, a reduction multidrug resistance, and a reduction of oxidative damage from free iron (10Picard V. Renaudie F. Porcher C. Hentze M.W. Grandchamp B. Beaumont C. Blood. 1996; 87: 2057-2064Crossref PubMed Google Scholar, 11Epsztejn S. Glickstein H. Picard V. Slotki I.N. Breuer W. Beaumont C. Cabantchik Z.I. Blood. 1999; 94: 3593-3603Crossref PubMed Google Scholar, 12Picard V. Epsztejn S. Santambrogio P. Cabantchik Z.I. Beaumont C. J. Biol. Chem. 1998; 273: 15382-15386Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 13Cozzi A Corsi A. Levi S. Santambrogio P. Albertini A. Arosio P. J. Biol. Chem. 2000; 275: 25122-25126Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). These effects are abolished by iron supplementation or by the mutational inactivation of the ferroxidase center (13Cozzi A Corsi A. Levi S. Santambrogio P. Albertini A. Arosio P. J. Biol. Chem. 2000; 275: 25122-25126Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Other than facilitating iron deposition, little is yet known of the biological role of L-chains. Large increases in L-ferritin levels occur as a result of mutations in the iron regulatory element. These increases cause cataracts but no apparent abnormalities in body iron metabolism (14Beaumont C. Leneuve P. Devaux I. Scoazec J.Y. Berthier M. Loiseau M.N. Grandchamp B. Bonneau D. Nat. Genet. 1995; 11: 444-446Crossref PubMed Scopus (267) Google Scholar, 15Levi S. Girelli D. Perrone F. Pasti M. Beaumont C. Corrocher R. Albertini A. Arosio P. Blood. 1998; 91: 4180-4187Crossref PubMed Google Scholar). However, a mutation in the C-terminal sequence of the L-chain causes a neurological disorder with increased deposition of ferritin and iron in the basal ganglia of the brain (16Curtis A.R. Fey C. Morris C.M. Bindoff L.A. Ince P.G. Chinnery P.F. Coulthard A. Jackson M.J. Jackson A.P. McHale D.P. Hay D. Barker W.A. Markham A.F. Bates D. Curtis A. Burn J. Nat. Genet. 2001; 28: 350-354Crossref PubMed Scopus (445) Google Scholar). We have recently identified a new human ferritin, MtF,1 that is encoded by an intronless gene on chromosome 5q23.1 and a mouse ortholog (17Levi S. Corsi B. Bosisio M. Invernizzi R. Volz A. Sanford D. Arosio P. Drysdale J. J. Biol. Chem. 2001; 276: 24437-24440Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Human MtF is synthesized as a 242-amino acid precursor with a long N-terminal sequence for mitochondrial import (17Levi S. Corsi B. Bosisio M. Invernizzi R. Volz A. Sanford D. Arosio P. Drysdale J. J. Biol. Chem. 2001; 276: 24437-24440Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Experiments with transfectant cells showed that this precursor is efficiently targeted to mitochondria and processed into typical ferritin shells. The amino acid sequence of the predicted mature protein overlaps the H sequence with 77% identity and contains all the residues of the ferroxidase center. The mature protein produced in Escherichia coli incorporated iron in vitro, indicating that it has ferroxidase activity (17Levi S. Corsi B. Bosisio M. Invernizzi R. Volz A. Sanford D. Arosio P. Drysdale J. J. Biol. Chem. 2001; 276: 24437-24440Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). As judged from mRNA levels, MtF is expressed at low levels in most cells except testis. MtF is present at a low level in normal erythroblasts, but this level increases dramatically in iron-loaded erythroblasts from patients with sideroblastic anemia (17Levi S. Corsi B. Bosisio M. Invernizzi R. Volz A. Sanford D. Arosio P. Drysdale J. J. Biol. Chem. 2001; 276: 24437-24440Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). This increased expression does not appear to be due to the typical translational control since MtF mRNA lacks the classical stem-loop iron regulatory element. The function and regulation of this new ferritin have not been established. Mitochondria are exposed to a heavy traffic in iron for the synthesis of heme and Fe/S clusters. Mitochondria are also the major sites of reactive oxygen species production (18Lange H. Kispal G. Lill R. J. Biol. Chem. 1999; 279: 18989-18996Abstract Full Text Full Text PDF Scopus (135) Google Scholar, 19Ferreira G.C. Int J. Biochem. Cell Biol. 1999; 31: 995-1000Crossref PubMed Scopus (49) Google Scholar, 20Abbas A. Labbe-Bois R. J. Biol. Chem. 1993; 268: 8541-8546Abstract Full Text PDF PubMed Google Scholar) and presumably must have efficient mechanisms to segregate Fe(II) from reactive oxygen species (particularly H2O2) to prevent the production of highly toxic hydroxyl radicals in Fenton-type reactions. Iron homeostasis in mitochondria also differs from that in the cytoplasm. Iron deprivation affects mitochondrial iron enzymes less than cytosolic iron enzymes (21Thompson C.H. Kemp G.J. Taylor D.J. Radda G.K. Rajagopalan B. J Intern. Med. 1993; 234: 149-154Crossref PubMed Scopus (13) Google Scholar). By contrast, excess iron is not usually deposited in mitochondria but is deposited in the cytosol as ferritin. Although iron does not normally accumulate in mitochondria, defects in its transport or utilization in mitochondria can result in mitochondrial iron loading. Visible granular iron deposits are formed inside the mitochondria of erythroblasts with defective heme synthesis as in subjects with sideroblastic anemia (22Bottomley S.S. Lee G.R. Lee G.R. Foerster J. Lukens J. Gree J. Rodger G. Paraskevas F. Wintrobe's Clinical Hematology. Williams and Wilkins, Baltimore, MD1999: 1071-1108Google Scholar, 23Rademakers L.H. Koningsberger J.C. Sorber C.W. Baart de la Faille H. Van Hattum J. Marx J.J. Eur. J. Clin. Invest. 1993; 23: 130-138Crossref PubMed Scopus (59) Google Scholar). Much of this iron is probably present as MtF (17Levi S. Corsi B. Bosisio M. Invernizzi R. Volz A. Sanford D. Arosio P. Drysdale J. J. Biol. Chem. 2001; 276: 24437-24440Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Iron also accumulates in the mitochondria of patients with Friedreich's ataxia resulting from defects in the synthesis of frataxin (24Babcock M. de Silva D. Oaks R. Davis-Kaplan S. Jirlerspong S. Momtermini L. Pandolfo M. Kaplan J Science. 1997; 276: 1709-1712Crossref PubMed Scopus (831) Google Scholar) or in sideroblastic anemia with ataxia from defects in the Fe/S transporter ABC7 (25Allikmets R. Raskind W.H. Hutchinson A. Schueck N.D. Dean M. Koeller D.M. Hum. Mol. Genet. 1999; 8: 743-749Crossref PubMed Scopus (354) Google Scholar). The form of this iron is not known, but the iron overload is associated with a decrease in respiratory chain and aconitase activity, probably from iron-induced oxidative damage (26Lill R. Kispal G. Trends Biochem. Sci. 2000; 25: 352-356Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Very little is yet known about how iron is delivered to mitochondria and whether it is normally accessible to MtF. It is also not known whether MtF responds to changes in cellular iron or whether its level affects the partitioning of cellular iron. This report explores some of these issues through analyses of different forms of MtF and H-ferritins transfected into HeLa cells. We show that MtF readily incorporates iron inside mitochondria by a process similar to that of H-ferritins. Unlike cytoplasmic ferritins, the levels of MtF are not increased by exogenous iron. However, when increased by transfection, MtF retains a high proportion of available iron, and cells show signs of iron deficiency. We conclude that iron is potentially as accessible to MtF as it is to cytosolic ferritin and that the control of MtF levels may offer a powerful method for regulating cellular iron homeostasis. The vector pcDNA3.1 was purchased from Invitrogen. Monoclonal antibodies, rH02 and LF03, prepared against human ferritin H- and L-chains, respectively, have been described previously (27Luzzago A. Arosio P. Iacobello C. Ruggeri G. Capucci L. Brocchi E., De Simone F. Gamba D. Gabri E. Levi S. Biochim. Biophys. Acta. 1986; 872: 61-71Crossref PubMed Scopus (83) Google Scholar, 28Cozzi A. Levi S. Bazzigaluppi E. Ruggeri G. Arosio P. Clin. Chim. Acta. 1989; 184: 197-206Crossref PubMed Scopus (29) Google Scholar) as has the rabbit antiserum, anti-rΔ9MtF elicited by a truncated form of MtF corresponding to residues 10–182 of the H-chain (17Levi S. Corsi B. Bosisio M. Invernizzi R. Volz A. Sanford D. Arosio P. Drysdale J. J. Biol. Chem. 2001; 276: 24437-24440Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). A more potent antiserum, anti-MtF, was elicited in mice by injecting the full mature form of recombinant MtF. Monoclonal rH02 recognizes H- but not L-ferritins and also cross-reacts with MtF (17Levi S. Corsi B. Bosisio M. Invernizzi R. Volz A. Sanford D. Arosio P. Drysdale J. J. Biol. Chem. 2001; 276: 24437-24440Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), whereas LF03 is specific for L-ferritins. Both antisera recognized MtF, but neither recognized H- or L-ferritins. Anti-transferrin receptor antibody was purchased from Zymed Laboratories Inc.(San Francisco, CA). The pcDNA3MtF vector, encoding the entire precursor MtF protein, was described in Ref. 17Levi S. Corsi B. Bosisio M. Invernizzi R. Volz A. Sanford D. Arosio P. Drysdale J. J. Biol. Chem. 2001; 276: 24437-24440Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar. The MtF222 mutant (E62K, H65G, H-chain numbering) with an inactivated ferroxidase activity was produced by oligonucleotide-directed mutagenesis of pcDNA3MtF. The chimera Mt-HF was constructed by fusing the mitochondrial leader peptide of MtF (residues 1–60) to the full human H-ferritin chain sequence. The plasmid for the truncated MtF (T-MtF) was constructed by subcloning into pcDNA3 the sequence encoding residues −2 to 182 (H-chain numbering). To obtain stable transfectants, the full coding regions of MtF and of the MtF222 mutant were subcloned into pUDH10–3 vector (CLONTECH) (29Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89 ()): 5547-5551Crossref PubMed Scopus (4268) Google Scholar) under the control of the tTA promoter to obtain pUD-MtF and pUD-MtF222 plasmids. HeLa cells were transfected with calcium phosphate as in Ref. 30Corsi B. Perrone F. Bourgeois M. Beaumont C. Panzeri M.C. Cozzi A. Sangregorio R. Santambrogio P. Albertini A. Arosio P. Levi S. Biochem. J. 1998; 330: 315-320Crossref PubMed Scopus (42) Google Scholar and grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mm l-glutamine. Typically in transient experiments, 106 cells were transfected with 10 μg of pcDNA3 plasmid containing the ferritin cDNAs or with the pcDNA3 vector for a control. Transfection efficiency was monitored by immunofluorescence staining with anti-Δ9MtF antiserum and ranged between 20 and 30% of cells. A stable HeLa-tet Off cell line was generated and selected as described in Ref. 13Cozzi A Corsi A. Levi S. Santambrogio P. Albertini A. Arosio P. J. Biol. Chem. 2000; 275: 25122-25126Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar. The HeLa-tet Off cells (CLONTECH) were co-transfected with 3.8 μg of pUD-MtF or pUD-MtF222plasmids and with 1 μg of pTK-Hyg plasmid (5:1 molar ratio) (CLONTECH). Clones expressing MtF and MtF222 were selected and maintained in DMEM supplemented with 10% fetal bovine serum, 100 μg/ml G418 (Geneticin, Sigma), 150 μg/ml hygromycin D (CLONTECH), 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mm l-glutamine. In the presence of doxycycline (2 ng/ml, Sigma), the protein synthesis was repressed, whereas in its absence, the synthesis was induced. The levels of cytosolic ferritins were assayed in extracts of 106 cells with ELISA assays using the monoclonal antibody rH02 calibrated on the recombinant homopolymer (28Cozzi A. Levi S. Bazzigaluppi E. Ruggeri G. Arosio P. Clin. Chim. Acta. 1989; 184: 197-206Crossref PubMed Scopus (29) Google Scholar). Purified recombinant MtF was not recognized by L-ferritin ELISA, but it gave a signal in the H-ferritin ELISA that corresponded to 1% of the ferritin content and was also recognized by this antibody in Western blots. Protein concentration was evaluated by the BCA method (Pierce) calibrated on bovine serum albumin. In immunoblot experiments, 30 μg of soluble proteins were separated by PAGE in 7% non-denaturing gels. Nitrocellulose filters from the blotted gel were incubated with rabbit anti-Δ9MtF antiserum (dilution 1:2,000) or rHO2 monoclonal antibody (dilution 1:1,000) followed by peroxidase-labeled antibody (Sigma). The bound peroxidase was revealed by ECL (Amersham Biosciences). In experiments with transient transfectants, the cells (2 × 105) were transfected with 2 μg of DNA plasmid and grown for 30 h in complete medium. Stable transfectants were induced to express ferritin by omitting doxycycline for 7 days. The cells were then incubated for 18 h, or the indicated time, with 2 μCi/ml [55Fe]ferric ammonium citrate (FAC) (ratio 1:2), 200 μm ascorbic acid, or 1 μm55Fe-labeled transferrin in DMEM, 0.5% fetal calf serum, 0.5% bovine serum albumin. The cells were washed and lysed in 0.3 ml of lysis buffer. After centrifugation, 10 μl of the soluble fraction were mixed with 0.3 ml of Ultima Gold (Packard) and counted for 1 min in a scintillation counter (Packard). The soluble proteins were analyzed also by PAGE in 7% non-denaturing gels directly or after immunoprecipitation with anti-Δ9MtF or LF03 (13Cozzi A Corsi A. Levi S. Santambrogio P. Albertini A. Arosio P. J. Biol. Chem. 2000; 275: 25122-25126Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 17Levi S. Corsi B. Bosisio M. Invernizzi R. Volz A. Sanford D. Arosio P. Drysdale J. J. Biol. Chem. 2001; 276: 24437-24440Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Gels were dried and exposed to autoradiography. The intensity of ferritin subunit bands was quantified by densitometry in the linear range. Transfectant cells were grown for 18 h in the presence of 2 μCi/ml FAC (ratio 1:2), 200 μm ascorbic acid, and mitochondrial fraction enriched as described previously (31Fiskum G. Craig S.W. Decker G.L. Lehninger A.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3430-3434Crossref PubMed Scopus (215) Google Scholar). Briefly, the cells were washed twice in phosphate-buffered saline and lysed on the plate using 0.007% digitonin in 0.25 m sucrose, 10 mm Hepes, pH 7.4, 0.15% bovine serum albumin. Unbroken cells and nuclei were first cleared by centrifugation at 1,000 × g for 10 min, and the mitochondria were precipitated by a further centrifugation at 3,000 × g for 10 min at 4 °C. The cytosolic supernatants (post-mitochondrial fractions) and the mitochondrial pellet (mitochondrial fractions) were analyzed directly or heated at 75 °C for 10 min for ferritin enrichment. The heat-stable proteins were separated by PAGE in 7.5% non-denaturing gels and exposed to autoradiography. After transient transfection, the cells (5 × 105) were grown for 30 h, or the stable clones were grown for 7 days in the absence of doxycycline. Then, they were incubated for 1 h in DMEM, methionine, and cysteine-free (ICN) 0.5% fetal calf serum, 0.5% bovine serum albumin and were labeled for 18 h with 50 μCi/ml [35S]methionine, [35S]cysteine (ICN) in the same medium (13Cozzi A Corsi A. Levi S. Santambrogio P. Albertini A. Arosio P. J. Biol. Chem. 2000; 275: 25122-25126Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The cells were washed with phosphate-buffered saline and then lysed with 500 μl of lysis buffer (20 mm Tris-HCl, pH 8.0, 200 mm LiCl, 1 mm EDTA, 0.5% Nonidet P-40). Total radioactivity associated with the soluble proteins was determined by trichloroacetic acid precipitation. For immunoprecipitation studies, 4 × 106 cpm of cytosolic lysates were precleared by incubation with 30 μl of protein A-Sepharose 50% v/v (Sigma) for 1 h at 4 °C with gentle shaking and centrifuged for 1 min at 14,000 rpm. Then, anti-ferritin L-chain monoclonal antibody (LF03) or mouse anti-MtF antibody was added, incubated for 1 h followed by protein A-Sepharose (30 μl). The samples were then incubated for 1 h at 4 °C, and the precipitates were collected. The soluble fractions were further incubated for 1 h at 4 °C with 30 μg of anti-ferritin H-chain antibody (rH02) and protein A-Sepharose (30 μl) and precipitated (13Cozzi A Corsi A. Levi S. Santambrogio P. Albertini A. Arosio P. J. Biol. Chem. 2000; 275: 25122-25126Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The immunobeads were washed, resuspended in SDS buffer, boiled for 10 min, and loaded on 12% SDS-polyacrylamide gel. The gels were treated with autoradiography image enhancer (Amplify, Amersham Biosciences), dried, and exposed. Cells expressing MtF and its mutants were fixed and permeabilized (17Levi S. Corsi B. Bosisio M. Invernizzi R. Volz A. Sanford D. Arosio P. Drysdale J. J. Biol. Chem. 2001; 276: 24437-24440Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). The preparations were then overlaid with rH02 (0.5 μg/ml) antibody followed by rhodamine-conjugated anti-rabbit IgG and washed as described previously. Fluorescence was visualized on an Axiophot microscope (Zeiss) with a 554-nm filter for rhodamine. For immunoelectron microscopy, the cells were fixed for 15 min with 4% paraformaldehyde and 0.25% glutaraldehyde mixture, detached by scrubbing, and centrifuged. The pellets were infiltrated in 0.6m sucrose mixed with 7% polyvinylpyrrolidone and then brought to 1.86 m sucrose and 20% polyvinylpyrrolidone by successive increases of the infiltrating solution. Freezing was in a 3:1 mixture of propane and cyclopentane cooled with liquid nitrogen. Ultrathin cryosections (50–100 nm) were cut using an Ultracut ultramicrotome equipped with a Reichert FC4 cryosectioning apparatus and processed as described previously (32Villa A. Podini P. Nori A. Panzeri M.C. Martini A. Meldolesi J. Volpe P. Exp. Cell Res. 1993; 209: 140-148Crossref PubMed Scopus (37) Google Scholar). In brief, the cryosections were collected over nickel grids and covered with 2% gelatin. After treatment with 125 mm phosphate-buffered saline supplemented with 100 mm glycine, the sections were exposed for 2 h at 37 °C to anti-Δ9MtF in phosphate-glycine buffer, then washed with the buffer, and finally labeled with anti-IgG-coated gold particles (6 nm, dilution 1:60 in the same buffer). Cryosections were then examined by electron microscopy. The cDNA for the human MtF precursor was subcloned into pcDNA3 vector to transiently transfect HeLa cells. MtF protein expression was first analyzed using anti-Δ9MtF antibodies by Western blot after separating cell extracts on non-denaturing PAGE. No MtF was detected in the untransfected HeLa cells, but high levels were found in the transfectants (Fig1 A, lanes 1 and2). The single band in the transfectants had a similar, but slightly slower, mobility than that of the cytoplasmic ferritin shown in Fig. 1 A (lane 3), indicating that MtF has a similar multimeric structure. To explore iron uptake into MtF, cells were incubated with the 55Fe label, supplied as FAC, for 18 h. Cells and organelles were lysed with 0.5% Nonidet P-40, and the proteins in supernatant fractions were separated on non-denaturing gels and then exposed to autoradiography. The untransfected parent cells gave a single radioactive band corresponding to the cytosolic ferritin and none in the position of MtF (Fig 1A, lane 3). The MtF transfectants showed uptake into cytosolic ferritin but also into a slower band in the position of MtF (Fig. 1 A, lane 6). To confirm the identity of the bands, cytosolic ferritin heteropolymers were first precipitated from the cell extracts with an excess of anti-L-chain LF03 antibody (13Cozzi A Corsi A. Levi S. Santambrogio P. Albertini A. Arosio P. J. Biol. Chem. 2000; 275: 25122-25126Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). This treatment eliminated the band corresponding to the cytosolic ferritin but left the MtF band (Fig. 1 A, lanes 5and 8). In contrast, anti-Δ9MtF antibody essentially eliminated the upper band specific to the transfected cells but had no effect on the lower band (Fig. 1 A, lanes 4 and7). These results identify MtF and show that it is a homopolymer, as predicted from its compartmentalization, and that it actively incorporates iron in vivo. To confirm the cellular compartmentalization of the ferritins, the plasma membranes of the cells were lysed with digitonin, and the mitochondrial fractions of 55Fe-labeled cells were separated from the cytosolic fraction by differential centrifugation (31Fiskum G. Craig S.W. Decker G.L. Lehninger A.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3430-3434Crossref PubMed Scopus (215) Google Scholar). Ferritin was partially purified from both fractions by heat extraction and identified by its electrophoretic mobility and the bound radioactive iron. Autoradiography showed that the MtF band was highly enriched in the mitochondrial fraction (Fig. 1 B,MF) of the transfected cells, whereas cytosolic ferritins were almost exclusively associated with the post-mitochondrial fractions (Fig. 1 B, PMF) of the control and transfected cells (Fig. 1 B). We conclude that MtF is restricted to mitochondria, where it assembles into ferritin-like structures that incorporate iron. To explore structural and functional elements for iron uptake into MtF, different constructs were expressed in HeLa cells. MtF222 has Glu-62 → Lys and His-65 → Gly (H-chain numbering), which inactivate the ferroxidase activity of human H-ferritin (13Cozzi A Corsi A. Levi S. Santambrogio P. Albertini A. Arosio P. J. Biol. Chem. 2000; 275: 25122-25126Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). T-MtF represents the predicted mature protein lacking the mitochondrial targeting sequence and starting at position −2 (H-chain numbering). Finally, Mt-HF has the N-terminal MtF sequence (residues 1–60) fused to the H-chain and predicted to be cleaved at residue 58. Transfectant ferritins were identified wit" @default.
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