Evidence for the presence of ferritin in plant mitochondria
Marco Zancani
1
, Carlo Peresson
1
, Antonino Biroccio
2
, Giorgio Federici
2
, Andrea Urbani
3
, Irene Murgia
4
,
Carlo Soave
4
, Fulvio Micali
5
, Angelo Vianello
1
and Francesco Macrı
`
1
1
Dipartimento di Biologia ed Economia Agro-Industriale, Sezione di Biologia Vegetale, Universita
`
di Udine, Italy;
2
Laboratorio di
Biochimica Clinica, Ospedale Pediatrico del Bambino Gesu
`

immunocytochemistry experiments on isolated mitochon-
dria and cross-sections of pea stem cells. The possible role of
ferritin in oxidative stress of plant mitochondria is discussed.
Keywords: ferritin; iron; mitochondria; Arabidopsis thaliana;
Pisum sativum.
Iron is an essential element for all living organisms [1]. In
green plants, its importance mainly derives from the
presence at the active sites of metalloproteins involved in
the electron transport chains linked to both oxygen
evolution (photosynthesis) and consumption (respiration).
However, iron(II) ions may also a mplify t he damaging
effect of reactive oxygen species (ROS) o n membranes,
proteins and nucleic acids [2]. This happens particularly
during the response of plants to diseases and other
environmental stresses accompanied by an excess of ROS
production (oxidative stress) [ 3,4]. The intracellular concen-
tration of free iron has therefore to be tightly controlled at
both the uptake and storage levels [5].
In the plant cell, chloroplasts and mitochondria are two
of the major sites of ROS generation [6,7]. In both cases, the
direct transfer of one electron from the electron transport
chain to oxygen (univalent reaction) generates superoxide
anion, which then dismutates, spontaneously or enzymat-
ically, to hydrogen peroxide. The latter can react with
iron(II) ion (Fenton reaction) generating the highly reactive
hydroxyl radical. To prevent this risk, plant cells have
evolved two strategies, namely scavenging of hydrogen
peroxide or sequestration of iron [2]. Chloroplasts possess
both systems, the scavenging (e.g. ascorbate peroxidase) [6]
and the iron-buffering proteins (ferritins) [8]. Conversely,

Isolation of Percoll-purified plant mitochondria
Crude mitochondria (CMt) were isolated from etiolated pea
(Pisum sativum L., cv. Alaska) stems as previously described
[16], and purified by a P ercoll discontinuous gradient (PMt)
Correspondence to F. Macrı
`
, Dipartimento di Biologia ed Economia
Agro-Industriale, Sezione di Biologia Vegetale, Universita
`
di Udine,
via Cotonificio 108, I-33100 Udine, Italy. Fax: +39 0432558784,
Tel.: +39 0432558781/82, E-mail: [email protected]
Abbreviations: CMt, crude mitochondria; IDP, inosine 5¢-diphos-
phate; MP, mitochiondrial matrix proteins; PAAF, polyclonal a nti-
body against pea seed ferritin; PMt, Percoll-purified mitochondria;
PSD, post source decay; ROS, reactive oxygen species;
TOF, time-of-flight.
(Received 24 June 2004, accepted 23 July 2004)
Eur. J. Biochem. 271, 3657–3664 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04300.x
as described in [17]. Where indicated, to obtain extremely
pure pea mitochondria, PMt were subjected to a second
discontinuous Percoll gradient. Matrix proteins (MP) were
obtained from PMt as described in [16]. Mitoplasts (Mpl)
were obtained from PMt after osmotic shock for 10 min in
10 m
M
HEPES/Tris (pH 7.6), 40 m
M
sucrose. Mitoplasts
were collected from the pellet after centrifugation at

HEPES/Tris, 0 .25
M
sucrose. To obtain A. thaliana purified mitochondria, the
final pellet was resuspended in 20 m
M
3-[N-morpholino]pro-
panesulfonic acid/KOH (pH 7.2), 0.3
M
mannitol, 1 m
M
EDTA and handled as described for pea stem PMt [17].
Enzyme assay
ATPase activities (vanadate-sensitive, marker enzyme for
plasma membrane; molybdate-sensitive, marker enzyme
for cytosolic soluble phosphatases; bafilomycin A
1
-sensi-
tive, marker enzyme for tonoplast; oligomycin-sensitive,
marker enzyme for mitochondria) were assayed as previ-
ously described [18]. Latent IDPase (marker enzyme for
Golgi), antimyc in A-insensitive cytochrome c reductase
(marker enzyme for endoplasmic reticulum) and glucose-
6-phosphate dehydrogenase (marker enzyme for plastids)
activities were detected as described in [19–21],
respectively.
Immunoprecipitation
The immunoprecipitate was obtained from purified mito-
chondria that had been frozen and thawed three times
and then centrifuged at 12 000 g for 15 min. The super-
natant ( 50 lL) was taken and 2 lL of rabbit polyclonal

(1 : 5000 dilution) [24] and the reaction was developed by
the activity of t he alkaline phosphatase conjugated to anti-
(rabbit IgG) Ig. For the immunodecoration, in the presence
of the monoclonal antibodies against cytochorme c
(PharMingen International, 1 : 10 000 dilution), the reac-
tion was developed by the activity of alkaline phosphatase
conjugated to anti-(mouse IgG) Ig.
The cross-reactivity with the antihuman mitochondrial
ferritin (HuMtF) was performed as described in [11].
Table 1. Marker enzyme activity in crude (CMt) and purified pea mitochondria (PMt). The activity of antimycin A-insensitive cytochrome c
reductase ( marker for endoplasmic reticulum) detected in pea microsomes, prepared as describe d in [40], was 570 nmolÆ(mg proteinÆmin)
)1
;the
activity of the glucose-6-phosphate dehydrogenase (marker for plastids) detected in pea s tem etioplast, prepared as described in [ 41], was 235 nmol
NADPH reduced (mg pro teinÆmin)
)1
. n.d., Not determined.
Marker enzyme
CMt
nmolÆ(mg proteinÆmin)
)1
Percentage
of control
PMt
nmolÆ(mg proteinÆmin)
)1
Percentage
of control
ATPase
1m

Cytochrome c reductase n.d. 457
+2l
M
Antimycin A n.d. 105
Glucose-6-phosphate dehydrogenase 35 6
3658 M. Zancani et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Mass spectrometry analysis
Identification of polypeptides from polyacrylamide gel
plugs was pursued by the trypsin mass fingerprint technique
on a MALDI-TOF mass spectrometer. In short, the protein
band was excised from a Coomassie-stained SDS/PAGE,
cysteines were reduced and alkylated with iodoacetamide
[25]. The samples were then digested with porcine trypsin
(Promega) in 4 0 m
M
ammonium bicarbonate at 37 °Cfor
6–8 h. The reaction was sto pped by freezing the samples at
)80 °C. Tryptic peptides were extracted by ZipTip C18
(Millipore) reverse phase material, d irectly e luted and
crystallized in a 50% (v/v) acetonitrile/water saturated
solution of a-cyano-4-hydroxycinnamic acid.
MALDI mass spectra were recorded in the positive ion
mode with delayed extraction on a Reflex IV time-of-flight
instrument equipped w ith a multiprobe inlet and a 337 nm
nitrogen laser. Mass spectra were obtained by averaging
50–200 individual laser shots. Calibration of the spectra was
internally performed by a two-point linear fit using the
autolysis products of trypsin at m/z ¼ 842.50 and m/z ¼
2211.10.
Database search with the peptide masses was performed

Tris-buffered saline to remove the antibody excess, the
sections were incubated for 2 h in the same incubation
medium, but at pH 8.4, containing secondary antibody
gold-conjugated 10 nm goat anti-(rabbit IgG) Ig (British
BioCell, Cardiff, UK) diluted 1 : 100. Finally, the sections
were counterst ained with uranyl a cetate ( 2% w/v) for 3 min
and l ea d citrate solution (0.25% w/ v) for 2 min and
observed with Philips EM 208 electron microscope at 80
kV accelerating voltages. Anti-fe rritin Ig was omitted in t he
controls.
Fig. 1. Identification of ferritin in pea stem mitochond ria. (A) SDS/
PAGE(12%)analysisofproteins(25lg) from crude mitochondria
(CMt), purified mitochondria (PMt), matrix from pea stem purified
mitochondria (MP), and to tal pea seed proteins (CP , control p roteins);
molecular mass of protein standards is indicated in kDa ( Std). (B)
Immunoblotting of the same proteins with polyclonal antibod y against
ferritin (PAAF). (C) Immunoblotting of recomb inant h uman mito-
chondrial ferritin (rHuMt, 10 ng), protein extract from HeLa cells
overexpressing human mitoc hondrial ferritin ( MtF-HeLa, 30 lg) and
matrix proteins from pea stem purified mito chondria (MP, 35 lg) with
antihuman mitochondrial ferritin polyclon al antibody after native 6%
PAGE. (D) SDS/PAGE (12%) of the 25–26 kDa protein purified by
immunoprecipitation.
Table 2. Sequence coverage by trypsin digestion peptide mass fingerprint of the pea ferritin, purified from mitochondria, with t he translated sequence
precursor of a pea ferritin (SwissProt accession number P19975). In bold are reported the protein regions covered in the mass fingerprint. Peptide
sequences confirmed by fragmentation analysis by post source decay (PSD) are underlined. The putative peptide leader sequence located at the
N-terminus is highlighted in black.
1
MALSSSKFSS FSGFSLSPVS GNGVQKPCFC DLRVGEKWGS RKFRVSATTA
51 PLTGVIFEPF EEVKKDYLAV PSVPLVSLAR QNFADECESV INEQINV EYN

preparation exhibited a negligible glucose-6-phosphate
dehydrogenase activity (plastid marker enzyme), partic-
ularly when compared to that of a sample of etioplasts
isolated from the same plant material.
The proteins of CMt, PMt, and the relative matrix
components were subjected to SDS/PAGE, in compari-
son with a pea seed protein extract containing ferritin
Fig. 3. Immunocytological l ocalization of ferritin in etiolated pea stem.
(A) Cross-section of etiolated pea stem; cw, cell wall, v, vacuole, m,
mitochondria. (B) and (C) Higher magnification of t he same electron
micrograph showing labeled mitochondria. Arrows in dicate electron-
dense particles after immunolabeling with PAAF followed by gold-
conjugated se condary a ntibo dy. Bars co rres pond to 30 0 lm.
Fig. 2. Localization of ferritin in pea stem purified mitochondria. (A)
Immunoblotting with P AAF of PMt (25 lg) incubated (+) or not ( –)
with 0.5% (w/v) Triton X-100 for 10 min, then subjected to proteolysis
with 125 lgÆmL
)1
trypsinfor30minat25°C and stopped by the
addition of 1 m
M
PMSF. (B) Imm unoblotting of PMt (25 lg) and
Mpl (25 lg) with monoclonal antibody raised against cytochrome
c (Cyt c), polyclonal antibody raised a gainst the a/b-subunit of mito-
chondrial ATPase (a/b-subunit) or PAAF (ferritin).
3660 M. Zancani et al. (Eur. J. Biochem. 271) Ó FEBS 2004
(Fig. 1A). Proteins, thus separated, were then subjected to
an immunoblot assay by using PAAF (Fig. 1B). The results
show that this antibody cross-reacted with a protein
exhibiting an apparent molecular mass of approximately

plantae d atabase returning again the ferritin sequence
ISEYVAQLR (223–231). The overall mass fingerprint data
cover about the 30% of the assigned sequence and details are
reported in Table 2. The theoric molecular mass, 23.6 kDa,
calculated from the database sequence after removal of the
N-terminus signal peptide, is in agreement with the value of
25–26 kDa estimated from the SDS/PAGE.
The localization of ferritin in pea stem purified mito-
chondria was investigated (Fig. 2). Figure 2A shows an
immunoblot of ferritin in PMt, treated (lane +) or
untreated (lane –) with Triton X -100, which were then
subjected to trypsin d igestion. The intensity of the immuno-
labeled band was lower in the presence of the detergent,
demonstrating that ferritin is localized inside the mito-
chondrial membranes. Furthermore, Mpl were obtained
by osmotic shock of PMt to remove the outer mito-
chondrial membrane. Mitoplast and PMt proteins were
then cross-reacted with monoclonal antibodies raised
Fig. 4. Ultrastructural localization of ferritin in pea stem mitochondria. Electron micrograph from fixed Percoll-purified pea st em mitochondria,
subjected to immunogold decoration in th e presence (A and B) or absence (C) of PAAF and at lower magnification (D). Arrows indicate ele ctron-
dense particles after immunolabeling with PAAF followe d by gold-conj ugated secondary ant ibody. Bars correspond to 300 lm.
Ó FEBS 2004 Plant mitochondrial ferritin (Eur. J. Biochem. 271) 3661
against cytochrome c, polyclonal antibodies raised against
the a/b-subunit of mitochondrial A TPase and PAAF,
respectively (Fig. 2B). These results show that Mpl partially
lost the cytochrome c; the densitometric a nalysis of the
immunodecoration show a d ecrease of approximately 50%
in the Mpl proteins. On the other hand, the immunodec-
oration of PMt and Mpl proteins with antibodies against
the a/b-subunit and PAAF was comparable (Fig. 2B). This

genes for ferritins (AtFer1–4) [28]. These genes encode the
ferritin subunit precursors, each containing a transit peptide.
The structural analysis of the presequences of the corres-
ponding polypeptides suggests that all are targeted to plastids
[28]. Table 3 shows the scores for t he mitochondrial/plasti-
dial localization of some plant ferritins from P. sativum
(SwissProt accession P19975), cowpea (Vigna unguiculata,
SwissProt accession T08124), soybean (Glycine max,
SwissProt accession BAB64536) and AtFer1 and AtFer4
from A. thaliana. While it is clear that AtFer1 is a poor
candidate for a mitochondrial localization, for the other
proteins significant scores were found. In particular,
PSORT
and
IPSORT
programs predicted high probability for the
presence of a mitochondrial target peptide in pea ferritin.
Remarkably, the ferritins from cowpea, soybean and AtFer4
exhibit values corresponding to a high probability for a
mitochondrial targeting from at least three programs.
Discussion
Animal and plant ferritins are encoded by nuclear gene
families, which diverge in their exon/intron organization
[13]. This suggests that they derive from a common ancestor,
albeit animal ferritins display a cytoplasmic localization,
whereas the plant ones are plastidic [8,15]. However, as seen,
an unusual intronless gene on human chromosome 5q23.1
encodes a 242 amino acid precursor of a ferritin H-like
Fig. 5. Identification of ferritin in A. thaliana mitochondria. (A) SDS/
PAGE (12%) of proteins (25 lg) from crude mitochondria (CMt),

stem (Fig. 1) and A. thaliana (Fig. 5) mitochondria. Such
organelles were highly purified by discontinuous Percoll
gradient, providing a v ery low interference in the immuno-
decoration from other cellular c omponents, especially from
etioplast contamination. On the basis of densitometric
analysis of immunoblots obtained with etioplasts isolated
from pea stem (results not shown), we calculated that if
PAAF detects j ust etioplast ferritin, these organelles h ave to
be present, in purified mitochondrial fractions, as a heavy
contamination (estimated to be approximately 25% of the
total protein). The results shown here demonstrate that this
is not the case, because the low enzymatic activity of
glucose-6-phosphate dehydrogenase in PMt (Table 1) con-
firms that the purified mitochondrial fractions possess a
maximum of 2 .5% of plastid proteins and, in addition , the
electron micrographs (Fig. 4) clearly show a very limited
contamination of P Mt from other o rganelles. Second,
ferritin was immunocytochemically identified in etiolated
pea stem cross-sections (Fig. 3) and in isolated pea mito-
chondria (Fig. 4). The pea stem mitochondrial ferritin is
present in the mitochondrial matrix as demonstrated by its
colocalization in Mpl with the a/b-subunit of mitochondrial
ATPase (Fig. 2). Finally, the 25–26 kDa soluble protein
was purified by immunoprecipitation (Fig. 1D); the primary
structure o f the polypeptide chain, inferred by Mass Finger
Print experiments on MALDI-TOF mass spectrometry, fits
to a high degree with the sequence of the ferritin from
P. sativum (SwissProt accession P19975, Table 2).
In A. thaliana, four ferritin genes (AtFer1–4) have been
reported and it has been suggested that the proteins AtFer1–

mitochondria has been related to the fact that the latter
organelles import a variety of (but not all) chloroplastic
proteins [34].
Plant mitochondria possess an electron transport chain
where superoxide anion may be generated by univalent
reactions at the level of complex I or III [35]. For this
reason, mitochondria have evolved systems to scavenge
ROS, or to prevent their formation [7,9], but sequestration
of potential harmful ferrous ions has not yet been
described.
Metal tolerance and homeostasis in plant cells is accom-
plished by different mechanisms [36]. In this context, the
main role of ferritins could concern iron sequestration.
Overexpression of this protein, in either the cytoplasm or
plastids of transgenic tobacco, leads to an increase of iron
sequestration that induces an activation of the iron trans-
port systems [37]. Therefore, they are crucial in controlling
iron storage and homeostasis in the plant cells. Other
functions of plant ferritins are, on the other hand, still
obscure. It has been suggested that sequestering of intracel-
lular iron m ay protect from oxidative damage induced by a
wide range of stresses [38]. Indeed, an increase of ferritin
mRNA has been observed in A. thaliana leaves photo-
inhibited w ith high light or fumigated with ozone [39].
Therefore, the sequestration of iron by ferritins in chloro-
plasts and mitochondria, two of the major sites of ROS
generation in plant cells [6,7], can constitute an additional
strategy to prevent th is damage.
Acknowledgements
We thank Dr J.F. Briat, Centre National d e l a R echerche S cientifique,

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