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INTRODUCTION |
The biological advantages of iron in redox reactions and as an
oxygen carrier are offset by the interaction of ferrous ions with
reactive oxygen species to produce harmful radicals that damage
membranes, proteins, and nucleic acids and have been implicated in
neurodegenerative diseases and in apoptosis (reviewed in Ref. 1). With
the exception of yeast, most organisms rely on ferritin to buffer free
cytosolic iron. This highly conserved protein consists of large
multimeric shells that accommodate up to 4500 atoms of iron (reviewed
in Ref. 2). Iron is taken up in the ferrous form and deposited as
ferric hydroxides after oxidation by catalytic ferroxidase sites.
Release of iron is effected by reducing agents without shell breakdown
(3, 4). Mammalian ferritins consist of variable amounts of two subunit
types, H and L, in a 24-subunit shell (5). The H chain has the
ferroxidase activity that is responsible for the cytoprotective action
of ferritin and its central role in cellular processes (2).
Up-regulation of the H chain reduces free iron levels with a consequent
reduction in proliferation rate and increased resistance to oxidative
damage (6). Down-regulation increases free iron and is associated with
increased apoptosis and cell proliferation and is also critical for
cell transformation by c-MYC (7, 8). Inactivation of H ferritin
(HF)1 in knockout mice is
lethal at early stages of embryogenesis (9).
Mitochondrial iron toxicity should be a particular concern in erythroid
cells that have to process >80% of body iron flux, but it is not yet
known how this iron is maintained in a nontoxic form. Massive increases
in mitochondrial iron occur when heme synthesis is blocked in
sideroblastic anemia (10-12) and yet the mitochondria survive. Much of
this iron has the characteristic electron microscopic appearance of
ferritin (11), but this material does not stain with antibodies to
cytoplasmic H and L ferritins (12), and its form has remained an
enigma. Here we describe an unusual gene that encodes a mitochondrial
ferritin (MtF) that has ferroxidase activity. The levels of MtF
increase dramatically in sideroblastic anemia, suggesting that this new
ferritin is likely to be important in the trafficking of iron in
mitochondria as well as its detoxification.
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EXPERIMENTAL PROCEDURES |
PCR Analyses--
The following primers derived from
AA469940 were used to amplify fragments from human genomic DNA
or human cDNA libraries: T1, TATTTCCTTCACCAGTCCCGG (372); T2,
TGAAGATGGGGGCCCCGGATG (672); T1R, GCAGGAGACAGCTGACTTTGG
(824); and T3R, TTGGAGGAATAGTATAACAG (875). PCR
conditions were 94 °C for 2 min, followed by 30 cycles of 94 °C
for 30 s, 55 °C for 30 s, 72 °C for 45 s.
Genomic Southern--
Southern analysis of DNA from normal human
lymphocytes digested with EcoRI or XbaI was
performed as described (13). The blot was probed with the
-32P-labeled 3'-UTR from MtF generated from AA469940 by
PCR amplification using primers T2/T3R.
RNA Analyses--
A human poly(A)+ RNA Northern blot
from Origene (Rockville, MD) was hybridized with the 3'-UTR probe
(above). After overnight hybridization using the ultrahyb buffer
(Ambion) at 66 °C, the blot was washed twice with 0.5× SSC, 0.1%
SDS at 68 °C and exposed to Kodak Biomax Ms for 8 h.
Expression in HeLa Cells--
The DNA encoding the entire
precursor protein was cloned into pcDNA3 vector (Invitrogen). The
DNA fragment encoding the first 67 N-terminal amino acids was subcloned
and linked to the N terminus of the green fluorescent protein in the
pEGFP-N vector (CLONTECH). Cell culture,
transfection, and immunoprecipitation were all carried out as
described (14). GFP expression was visualized in living cells on
a fluorescence microscope (Axiovert S100TV, Zeiss) with a 507-nm
filter. To localize mitochondria, cells were preincubated with
MitoTracker Orange (Molecular Probes) following the manufacturer's instructions and visualized with a 576-nm filter. Cells transfected with pcDNA3MtF were incubated with MitoTracker Green FM (Molecular Probes) for 45 min and then fixed and permeabilized (12). The preparations were then overlaid with anti-r
9MtF antiserum at 1:200
dilution, followed by rhodamine-conjugated anti-rabbit IgG. Fluorescence was visualized on an Axiophot microscope (Zeiss) with a
554-nm filter for rhodamine and with a 516-nm filter for MitoTracker Green.
Expression in Erythroid Cells--
Erythroid cells from normal
marrow donors and patients with sideroblastic anemia were analyzed for
cytoplasmic HF using a monoclonal mouse antibody as described
(12) and for MtF with polyclonal rabbit antibody anti-r
9MtF
antiserum. Bound antibody was detected by an immunoalkaline phosphatase
method. For negative controls nonimmune mouse or rabbit serum was
substituted for the primary antibody.
Expression in Escherichia coli--
r
9MtF was produced in
E. coli by subcloning the DNA encoding amino acids 70-242
of the predicted precursor into pET expression vector (Novagen). Cell
transformation and protein expression were performed as described
(15). Antibodies to electrophoretically pure r
9MtF were
raised in rabbits.
Protein Labeling--
The pcDNA3MtF was transcribed and
translated in the TNT T7/T3-coupled reticulocyte lysate system L5010
obtained from Promega (Madison, WI) in the presence of
[35S]methionine, according to the manufacturer's
instructions. Ferritin was labeled in vivo by incubating
transfected HeLa cells with [35S]methionine for 18 h
and was isolated as described (14).
In Vitro Iron Uptake--
Apoferritins (1 µM
final concentration) were incubated aerobically with 1 mM
ferrous ammonium sulfate in 0.1 M HEPES buffer, pH 7.0, for
2 h at room temperature. The proteins were separated on 7% native
polyacrylamide gels and stained for protein or iron (14).
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RESULTS |
Gene--
BLAST searches of the GenBankTM human EST
data base with the cDNA for human HF, pHF16 (13), identified clones
AA469940, AI024273, and AI149710 from a testis library with about 80%
homology to human HF. The extended and corrected sequences indicated
that all represent the same mRNA and have the same predicted C
terminus as HF. Although none has a complete coding sequence, that in
AI49710 extended above the N terminus of HF. BLAST searches of the high
throughput gene sequence data base of GenBankTM with this
cDNA contig identified an identical 754-nt sequence in the BAC
clone (AC011181), which is now contained in Hs5_23264 mapping at
5q21.3.2 The initiating codon
in this genomic sequence was predicted to be 60 residues above that of
HF and was located about 30 nt downstream from the initiation site of
transcription of the H gene. Two mouse cDNAs (AK0105400 and
AK015346) from a testis library have recently been
reported3 that are similar to
the new human ferritin (see below). There is no corresponding gene yet
in public mouse data bases.
Amplification of human genomic DNA with primers T1/T1R from the
cDNA produced a single 452-base pair fragment with the same sequence as the published genomic sequence. Southern analyses of
EcoRI and XbaI digests of human genomic DNA with
a probe from the predicted 3'-UTR gave single hybridizing bands of
about 5.5 and 10.5 kb, respectively (Fig.
1A), consistent with the
restriction map of the BAC. These results confirm that this is a single
copy intronless gene.

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Fig. 1.
A, gene. Southern blot analyses of
EcoRI and XbaI digests of human genomic DNA
probed with the 3'-UTR probe from MtF. B, tissue
expression of MtF mRNA. Northern blot analyses of poly(A)-enriched
human RNA. The blot was probed with the 3'-UTR of MtF.
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The genomic sequence has a short poly(A) sequence in the 3'-UTR in the
same position as in the cDNA. This is immediately followed by a sequence, AAAGTTTTGCCCA, which has a possible counterpart, TCAGTTTCCCCA, 25 nt above the initiating ATG and close to the transcription initiation site of the H gene. Both features are characteristics of a processed pseudogene (18, 19). However, the mouse
cDNA has a similar sequence TCAGTTTCCCCT in the same position above
the ATG. This region therefore seems more likely to be part of the
human transcript than a flanking genomic repeat. There is no apparent
iron-responsive element (IRE) for translational control by iron (20) in
the 400-nt region above the coding region or in the mouse orthologs.
However, there is weak homology with the IRE of the H mRNA in the
Mt sequence immediately after the initiating AUG
(AUGCUGUCCUGCUUCAGGCUCCUCUCCAGGCACATC versus
GGGGUUUCCUGCUUCAACAGUGCUUGGACGGAACCC). 10 of the
first 14 bases of the 5' stem are conserved, but the canonical CAGUG
loop (underlined) and the 3' stem region are largely substituted. This
result suggests that much of the 5'-UTR of an H-like sequence,
including the IRE, mutated to form a leader sequence in MtF. The
lack of an apparent IRE indicates that the expression of MtF will not
be translationally controlled by iron.
Transcript--
Northern analysis of mRNA from different human
tissues using the 3'-UTR probe showed that the polyadenylated mRNA
corresponding to MtF was slightly above 1 kb (Fig. 1B). This
is similar in size to H- and L-mRNAs (21). However, in
contrast to the ubiquitous H- and L-mRNAs, MtF has a
different expression pattern. As expected from EST representation, the
transcript is expressed in testis, but levels are low in iron storage
organs such as the liver and spleen and in other tissues represented in
this blot.
Protein--
In overlapping coding regions, MtF is 79% identical
to HF and 63% to LF. The seven amino acids responsible for the
ferroxidase activity in HF are conserved in MtF (Fig.
2). In the coding region, the mouse
ortholog is 84% identical to human MtF and 78% identical to mouse HF
and human HF and also has a conserved ferroxidase center. The predicted
protein leader of 56 residues compares with that of 57 for human MtF.

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Fig. 2.
Alignment of human Mt, H, L, and mouse Mt
ferritins. The up arrows indicate residues of the
ferroxidase center conserved in H and Mt ferritins. The
arrow indicates the predicted cleavage site of the leader
peptide, and the open arrowhead indicates the N terminus of
r 9MtF. Residues present in all four ferritins are shaded
in black and those in two or three ferritins are in
gray. Mouse MtF is derived from clone AK0105400, which
differs from clone AK015346 in the substitutions G107E and K108R.
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Mitochondrial Localization--
The long N-terminal extension
suggested a leader sequence. A structural model in
helical
formation with the Insight II program (Molecular Simulations) predicted
four equally spaced arginines on one face, a characteristic of a
mitochondrial targeting sequence. The MitoProt II program (22) also
indicated mitochondrial targeting and predicted a cleavage site at
residue 57, three residues before the start of the H chain. The DNA
fragment encoding the first 67 amino acids was therefore fused to GFP
cDNA and the construct expressed in HeLa cells. Fluorescent imaging
of cells transfected with the parent GFP under the same promoter but
without the leader sequence showed a diffuse cytoplasmic stain (not
shown). By contrast the construct with the attached leader showed
accumulation of GFP in filamentous intracellular bodies characteristic
of mitochondria, and this pattern overlapped completely with that of a
specific mitochondrial stain (Fig.
3A). This localization was
confirmed by transfecting a construct containing the entire coding
region of MtF into HeLa cells and examining the distribution of the
expressed protein with anti-r
9MtF, prepared against, and specific
for, the new ferritin. This antiserum recognized r
9Mt MtF but not rHF or rLF (Fig. 4A) and
showed (Fig. 3B) that the distribution of the expressed MtF
coincided with that of the mitochondrial marker.

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Fig. 3.
Mitochondrial localization.
A, HeLa cells were transfected with a construct encoding the
first 67 residues of the MtF fused to GFP and its distribution
(i) compared with that of mitochondrial marker, MitoTracker
II orange (ii). B, distribution of MtF in HeLa
cells expressed from a construct, pcDNA3-MtF, encoding the complete
MtF precursor and analyzed 24 h after transfection. i,
stain with anti-r 9MtF antibody followed by rhodamine-labeled
secondary antibody. ii, the same field stained with
MitoTracker Green FM. The two stains overlap.
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Fig. 4.
Immunological properties, processing, and
ferroxidase activity of MtF. A, specificity
of anti-r 9MtF antibody. Purified rHF, rLF, and r 9-MtF were run in
duplicate sets on 7% nondenaturing PAGE. One portion of the gel was
stained with Coomassie Blue, and the other was immunoblotted, probed
with anti 9-Mt antibody (1/2000 dilution), and developed with ECL.
Samples: r 9-MtF (lanes 1 and 4), rHF
(lanes 2 and 5), and rLF (lanes 3 and
6). All wells were loaded with 1 µg of purified protein,
except for lane 1, which was loaded with 0.2 µg. Minor
bands in rHF and rLF represent higher polymers (23).
B, processing of MtF. SDS-PAGE analyses of
cytosolic and mitochondrial ferritins. HeLa cells were transfected with
pcDNA3-MtF and metabolically labeled with
[35S]methionine for 18 h. Extracts were first
cleared of cytosolic ferritins by precipitation with antibodies to
human L ferritin (lane 2) and then precipitated with
antibodies to MtF (lane 3). The ~22-kDa band in lane
3 results from in vivo processing of the ~30-kDa
precursor (P-Mt) obtained by in vitro translation
(lane 1) (Mt). C, iron incorporation into MtF.
Iron-free r 9MtF (lane 2) was incubated with Fe(II)
ammonium sulfate alone (lane 3) or with equimolar amounts of
rHF (lane 1) or rLF (lane 4). Samples were
separated on nondenaturing PAGE and stained for protein
(upper) or for ferric iron (lower).
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Processing--
To determine the size of mature MtF,
transfected HeLa cells were metabolically labeled with
[35S]methionine, immunoprecipitated with
anti-r
9MtF, and the precipitate separated on SDS-PAGE. This product
was compared with that obtained by in vitro translation of
mRNA from the same construct and in the absence of mitochondria.
These experiments showed that the 30-kDa precursor protein was
processed in cells to a 22-kDa peptide. The processed subunit was
slightly larger than the H subunit and was not recognized by antibodies
to human L ferritin that precipitated the cytoplasmic H and L ferritins
(Fig. 4B). We conclude that the precursor for the new
ferritin (MtF) is targeted to mitochondria and is appropriately processed.
To explore its structural properties, the fragment 70-242,
corresponding to residues 10-182 of the H chain, was expressed in
E. coli. The protein (r
9MtF) accumulated in the soluble
fraction as an assembled ferritin (see below) and could be purified
with procedures (14) used for recombinant H and L ferritins (rHF and
rLF). It was reversibly denatured in acidic 8 M guanidine HCl, and, when renatured together with H or L subunits, formed heteropolymers (not shown).
Iron Uptake--
The r
9MtF isolated from E. coli
stained with Prussian blue, indicating that it incorporated iron
in vivo, an index of ferroxidase activity (2). To compare
this activity with HF and LF, purified r
9MtF was made iron-free and
incubated at pH 7.0 with ferrous iron in the presence or absence of
equimolar amounts of iron-free rHF and rLF. Prussian blue staining of
the PAGE-separated shells showed that r
9MtF incorporated similar
amounts of iron to rHF and much more than rLF (Fig. 4C),
consistent with the conservation of residues Glu27,
Tyr34, Glu61, Glu62,
His65, Glu107, and Gln141
that act as iron ligands in the ferroxidase center of HF
(2).
Expression in Erythroid Cells--
MtF sequences were identified
in PCR products amplified with primers T1/T1R from a cDNA library
from the erythroleukemic cell line K562 cells. This finding, together
with the electron microscopic evidence from erythroid cells (11),
suggested that MtF might be expressed in erythroid cells. We therefore
examined levels of MtF in bone marrow smears from healthy donors and
from individuals with X-linked sideroblastic anemia (XLSA) arising from
mutations in the erythroid-specific 5-aminolevulinic acid
synthase. This mutation causes both anemia and mitochondrial
iron loading in marrow erythroblasts (12). As shown in Fig.
5A, normal erythroblasts showed very light staining. By contrast, erythroblasts from patients with XLSA showed a dense granular, perinuclear staining (Fig. 5B). This pattern is consistent with a mitochondrial
localization and is very similar to that given by ringed sideroblasts
by iron staining (11, 12). Staining with anti-HF antibodies showed a
diffuse cytoplasmic distribution (not shown) as reported previously (12). These results strongly suggest that much of the deposition of
iron in mitochondria is present in this new ferritin.

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Fig. 5.
Expression in erythroid cells.
Aspirated bone marrow cells were permeabilized and reacted with
antibodies to MtF, followed by an immunoalkaline phosphatase staining
(red color). A, normal; B,
sideroblastic anemia. In B about 90% of the erythroblasts
were ringed sideroblasts, and these were strongly stained by the MtF
antibody.
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DISCUSSION |
We describe a new human ferritin (MtF) that is expressed as
a precursor, targeted to mitochondria, and then processed into a
functional protein with a structure similar to the ferritins in
cytoplasm. Like the cytosolic HF, MtF incorporates iron in vivo and in vitro, consistent with its conserved
ferroxidase center. The major difference is in the quaternary
structure: the cytosolic ferritins are heteropolymers, while those in
mitochondria are homopolymers, since the subunits assemble only after
processing in the mitochondria. The discovery of MtF solves some old
puzzles. About 40 years ago, iron deposits in mitochondria were
identified as ferritin cores by electron microscopy (11), but this view was challenged because of negative immunological findings and because
it lacked a molecular basis for mitochondrial targeting. Our finding of
an immunologically distinct ferritin with a targeting leader sequence
resolves both paradoxes.
This is the first example of a mammalian ferritin that is specifically
targeted to an organelle, although it has parallels with
organelle-specific ferritins in other organisms such as the plastid
ferritin in plants, hemolymph ferritin in insects, and yolk ferritin in
snails (2). Compared with the ubiquitous
HF and LF, MtF appears to have a limited tissue distribution. Its relatively high expression in testis does not seem to reflect the
levels of mitochondria, since other mitochondria-rich tissues, such as
brain, have low MtF levels. Nor does its level correlate with cytosolic
iron content, since MtF is also low in the liver and spleen. Further
experiments in animal models would be helpful in elucidating expression
patterns in different physiological states.
Our findings may be relevant to iron trafficking in erythroid cells.
Ferrous iron is efficiently incorporated into heme by ferrochelatase.
If this reaction is temporarily blocked, the iron accumulated in the
mitochondrial matrix is available for heme synthesis when the block is
removed (24). This iron now seems likely to be in ferritin. Iron flux
through mitochondrial ferritin may therefore, in some situations, play
a role in regulating heme synthesis, a major determinant in the overall
control of hemoglobin synthesis (24). Since iron uptake into ferritin
requires oxygen and its release requires reductants, the trafficking of
iron to ferrochelatase or to ferritin may be affected by the redox
state of the mitochondria.
The finding of high levels of MtF in sideroblasts but not in normal
erythroblasts suggests that MtF expression increases with mitochondrial
iron loading. The response to iron is usually attributed to increased
rates of translation due to removal of a protein that binds the IRE in
the ferritin mRNA (20). However, there is no apparent IRE in the
gene for the mitochondrial ferritin, and some other mechanism, possibly
transcriptional, seems likely. The absence of an IRE in the transcript
for MtF but not for HF or LF has parallels with other differentially
expressed proteins of iron metabolism. Transferrin receptor and
5-aminolevulinic acid synthase are expressed as tissue-specific
transcripts with or without IREs, but in both cases it is the
nonerythroid form that lacks the IRE (25, 26).
Our findings may also be relevant to the mitochondrial iron
accumulation in Friedreich's ataxia linked to anomalies in frataxin. Aggregates of the yeast ortholog of frataxin, Yfh1p, can bind large amounts of iron in vitro, and it has been suggested
that frataxin substitutes for ferritin to detoxify mitochondrial iron in mammalian cells (16). It will be interesting to determine whether levels of MtF are also increased in this disorder and whether
the two proteins are metabolically related.
The origin of the MtF gene and its evolutionary relationship to H and L
genes remain a puzzle. The human genome contains multiple copies of H-
and L-like sequences, but only one member of each family is known to be
functional. Both have introns with similar arrangements (21). The other
members seen by Southern analyses with the H- and L-cDNAs are
intronless and appear to be processed pseudogenes (19). None has a
counterpart in cDNA data bases, and all are therefore presumed to
be nonfunctional. Although the MtF gene has some characteristics of a
processed pseudogene, it is clearly not an inactive relic whose
expression can be artificially forced by transfection, since we show
regulated expression of the protein in untransfected cells. It could
conceivably be derived from a putative bacterial precursor of
mitochondria, but its sequence is more similar to mammalian ferritins
(>75% identity) than to any known bacterial ferritin (<30%
identity). This suggests that it is more likely to have arisen from an
ancient H-like sequence that acquired a functional promoter. The
presence of MtF in rodents indicates that it is evolutionarily
relatively old and arose before the divergence of primates and rodents
about 112 million years ago (17). Preliminary analyses of the
putative promoter region show potential binding sites for
erythroid-specific factors such as GATA and NFE2, but the functionality
of these remains to be determined. In conclusion, the finding of
different ferritins in different cellular compartments, and under
different regulation, offers exciting prospects for selectively
modulating cellular iron homeostasis.