Báo cáo khoa học: Evidence for the presence of ferritin in plant mitochondria pdf

The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochon-dria and cross-sections of pea stem cells.. While animal ferritins ar

Evidence for the presence of ferritin in plant mitochondria

Marco Zancani1, Carlo Peresson1, Antonino Biroccio2, Giorgio Federici2, Andrea Urbani3, Irene Murgia4, Carlo Soave4, Fulvio Micali5, Angelo Vianello1and 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` – IRCCS, Roma, Vatican State; 3 Centro Studi sull’Invecchiamento (Ce.S.I), Facolta` di Medicina e Chirurgia, Dipartimento di Scienze Biomediche, Universita` ‘G D’Annunzio’, Chieti, Italy;

4 Dipartimento di Biologia, Sezione di Fisiologia e Biochimica delle Piante, Universita` di Milano, Italy; 5 Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Universita` di Trieste, Italy

In this work, evidence for the presence of ferritins in plant

mitochondria is supplied Mitochondria were isolated from

etiolated pea stems and Arabidopsis thaliana cell cultures

The proteins were separated by SDS/PAGE A protein, with

an apparent molecular mass of approximately 25–26 kDa

(corresponding to that of ferritin), was cross-reacted with an

antibody raised against pea seed ferritin The mitochondrial

ferritin from pea stems was also purified by

immunopre-cipitation The purified protein was analyzed by

MALDI-TOF mass spectrometry and the results of both mass finger

print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P < 0.05) The mitochondrial localization of ferritin was also confirmed by 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 amplify the damaging

effect of reactive oxygen species (ROS) on 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, plant mitochondria seem to have only systems to scavenge

H2O2or to prevent its generation [7,9], but not to sequester iron Only recently a mitochondrial ferritin, encoded by an intronless gene, has been described in erythroblasts of subjects with impaired heme synthesis [10] The gene, expressed in HeLa cells, has permitted to reveal that exogenous iron is available to mitochondrial ferritin as it is

to cytosolic ferritin [11]

Ferritins are highly conserved proteins consisting of large multimeric shells that can store up to 4500 atoms of iron [12,13] The latter is taken up in the ferrous form and immobilized after oxidation catalyzed by ferroxidase sites as ferric hydroxides or as amorphous hydrous ferric oxyphos-phate [12] Iron can also be released from the core of ferritins This process is affected by reducing agents, but does not imply shell breakdown [13] Nevertheless, an in vitro degradation of plant ferritin, induced by iron exchange, was described [14] Therefore, ferritins can play a critical role in the cellular regulation of iron storage and homeostasis Soluble (ferritins) and insoluble (phytosiderin) iron-storage proteins have been described in dry pea seeds [14] While animal ferritins are mainly cytosolic proteins, the plant ones appear to be localized in chloroplasts of plant cells or, more in general, in plastids [8,15] In this work it is shown that ferritins are also present in plant mitochondria

Materials and methods

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 Percoll 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: biolveg@uniud.it

Abbreviations: CMt, crude mitochondria; IDP, inosine

5¢-diphos-phate; MP, mitochiondrial matrix proteins; PAAF, polyclonal

anti-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)

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 mM HEPES/Tris (pH 7.6), 40 mM sucrose Mitoplasts

were collected from the pellet after centrifugation at

13 000 g for 10 min and resuspended in 20 mM HEPES/

Tris (pH 7.5), 0.25Msucrose

Crude and purified mitochondria of Arabidopsis thaliana

were isolated from liquid cell cultures resuspended in 10 mL

of 10 mM HEPES/Tris (pH 7.6), 0.5M mannitol, 10 mM

EDTA, 2 mMcystein, homogenized with a Turrax at 4C,

diluted with 20 mL of the above buffer, further

homo-genized by a Potter homogenizer and centrifuged at 1000 g

for 10 min The supernatant was centrifuged at 13 000 g for

10 min and the final pellet was resuspended in 30 mL of

10 mM HEPES/Tris (pH 7.6), 0.5M mannitol and

centri-fuged at 2000 g for 5 min The supernatant was collected

and centrifuged at 13 000 g for 10 min The final pellet

(CMt) was resuspended in 20 mM HEPES/Tris, 0.25 M

sucrose To obtain A thaliana purified mitochondria, the

final pellet was resuspended in 20 mM

3-[N-morpholino]pro-panesulfonic acid/KOH (pH 7.2), 0.3M mannitol, 1 mM

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 A1

-sensi-tive, marker enzyme for tonoplast; oligomycin-sensi-sensi-tive,

marker enzyme for mitochondria) were assayed as

previ-ously described [18] Latent IDPase (marker enzyme for

Golgi), antimycin 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 antibody raised against pea seed ferritin (PAAF, described

in [22]) was added After incubation for 1 h at 4C,

50 lL of protein-A sepharose (50% v/v slurry, washed twice in 50 mM Tris/HCl, pH 8.0), was added and incubated for 1 h at 4C The immune complex was precipitated by centrifugation at 12 000 g for 20 s and the pellet washed thrice with 50 mM Tris/HCl (pH 8.0) The pellet was resuspended in 50 mM Tris/HCl (pH 7.5),

100 mM dithioerythritol and 1% (w/v) SDS, and then boiled at 95C for 3 min The sample for electrophoresis analysis was obtained by collecting the supernatant (
30 lL) after centrifugation at 12 000 g for 20 s and addition of 10 lL of 75% (w/v) glycerol plus 1 lL of 0.1% (w/v) bromophenol blue

Analytical electrophoresis Gel electrophoresis was carried out in 12% (w/v) polyacryl-amide gels containing 0.1% (w/v) SDS [23] After SDS/ PAGE, the gels were either stained with Coomassie Brilliant Blue R-250, or layered onto a nitrocellulose membrane to transfer the proteins by electroblotting The nitrocellulose membranes were incubated with either PAAF or antibodies raised against the a/b-subunit of mitochondrial ATPase (1 : 5000 dilution) [24] and the reaction was developed by the activity of the 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 described in [40], was 570 nmolÆ(mg proteinÆmin))1; the activity of the glucose-6-phosphate dehydrogenase (marker for plastids) detected in pea stem etioplast, prepared as described in [41], was 235 nmol NADPH reduced (mg proteinÆmin))1 n.d., Not determined.

Marker enzyme

CMt nmolÆ(mg proteinÆmin))1

Percentage

of control

PMt nmolÆ(mg proteinÆmin))1

Percentage

of control ATPase

Latent IDPase

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 40 mMammonium bicarbonate at 37C for

6–8 h The reaction was stopped by freezing the samples at

)80 C Tryptic peptides were extracted by ZipTip C18

(Millipore) reverse phase material, directly eluted 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 with 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/z¼ 842.50 and m/z ¼

2211.10

Database search with the peptide masses was performed

against the NCBInr, taxon Viridiplantae, database using the

peptide search algorithm MASCOT (Matrix Science)

Fragments generated by post source decay (PSD)

experi-ments were fitted using the database search algorithm

MASCOT (Matrix Science) and analyzed by the de novo

sequencing routine of Biotools (Bruker-Daltonik)

Immunochemical electron microscopy

Cross-sections of etiolated pea stem and isolated

mitochon-dria were fixed with 4% (v/v) paraformaldehyde and 0.5%

(v/v) glutaraldehyde in 0.17M phosphate, 0.17M sucrose

buffer (pH 7.0) for 3 h at 4C The cross-sections or the

mitochondria were washed several times in 0.17M

phos-phate, then dehydrated in ethanol and embedded in LR

White M acrylic resin (Sigma) Immunolabelling of

ultra-thin sections (120 nm on 300 mesh nickel grids) was carried

out by grids flotation technique at room temperature for 1 h

on drops of blocking buffer: 1% (w/v) bovine serum

albumin, 20% (v/v) normal goat serum in 0.1M

buffered saline (pH 7.4), and then incubated for 2 h in

Tris-buffered saline (pH 7.4) containing PAAF (diluted 1 : 5),

1% (w/v) bovine serum albumin, 4% (v/v) fetal bovine

serum, and 0.1% (v/v) Tween-20 After several washes in

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 counterstained with uranyl acetate (2% w/v) for 3 min and lead citrate solution (0.25% w/v) for 2 min and observed with Philips EM 208 electron microscope at 80

kV accelerating voltages Anti-ferritin Ig was omitted in the controls

Fig 1 Identification of ferritin in pea stem mitochondria (A) SDS/ PAGE (12%) analysis of proteins (25 lg) from crude mitochondria (CMt), purified mitochondria (PMt), matrix from pea stem purified mitochondria (MP), and total pea seed proteins (CP, control proteins); molecular mass of protein standards is indicated in kDa (Std) (B) Immunoblotting of the same proteins with polyclonal antibody against ferritin (PAAF) (C) Immunoblotting of recombinant human mito-chondrial ferritin (rHuMt, 10 ng), protein extract from HeLa cells overexpressing human mitochondrial ferritin (MtF-HeLa, 30 lg) and matrix proteins from pea stem purified mitochondria (MP, 35 lg) with antihuman mitochondrial ferritin polyclonal 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 the 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.

Protein assay

Protein concentration was determined by the method of

Bradford [26], using bovine serum albumin as a standard

Results

Purified pea stem mitochondria, particularly when

com-pared with CMt, were almost devoid of contamination from

different types of cellular components (Table 1) ATPase

activity of this fraction was, indeed, uninhibited or only very

slightly inhibited by vanadate (plasmalemma ATPase

inhibitor), bafilomycin A1 (tonoplast ATPase inhibitor)

and molybdate (soluble phosphatase inhibitor), but strongly

inhibited by oligomycin (mitochondrial ATPase inhibitor)

In addition, PMt showed a low level of latent IDPase (Golgi

membrane marker enzyme) Cytochrome c reductase

activ-ity was assayed in the presence and absence of antimycin A

to assess the contamination from endoplasmic reticulum In

purified mitochondria (in the presence of antimycin A), the

activity was 4.35 times and almost six-fold lower than that

recovered in control mitochondria and microsomes,

respect-ively On the other hand, the activity of cytochrome c

reductase, still detected in the presence of antimycin A,

could depend on the presence of a similar enzyme on the

outer membrane of plant mitochondria [27] Finally, this

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 localization 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 the same electron micrograph showing labeled mitochondria Arrows indicate electron-dense particles after immunolabeling with PAAF followed by gold-conjugated secondary antibody Bars correspond to 300 lm Fig 2 Localization of ferritin in pea stem purified mitochondria (A)

Immunoblotting with PAAF 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)1trypsin for 30 min at 25 C and stopped by the

addition of 1 m M PMSF (B) Immunoblotting of PMt (25 lg) and

Mpl (25 lg) with monoclonal antibody raised against cytochrome

c (Cyt c), polyclonal antibody raised against the a/b-subunit of

mito-chondrial ATPase (a/b-subunit) or PAAF (ferritin).

(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

25–26 kDa, a value similar to that of ferritins This

reactivity was achieved in all types of preparations In

particular, in some cases two very close bands were evident

As already suggested, they represent ferritin and a product

of degradation of the same protein [14] The same

cross-reactivity was also detected for a protein of twofold purified

pea stem mitochondria (result not shown) In addition, the

pea stem mitochondrial protein, after nondenaturing

elct-rophoresis and blotting, cross-reacted with the polyclonal

antibody anti-human mitochondrial ferritin [10] (Fig 1C)

The matrix proteins from purified pea mitochondria were

also subjected to purification by immunoprecipitation SDS/

PAGE analysis of the immunoprecipitated revealed, after

Coomassie staining, a protein band at 25–26 kDa (Fig 1D)

The tryptic peptides of this band were analyzed with a

MALDI-TOF mass spectrometer and the monoisotopic

masses of each singly charge species were annotated with

their intensities These data were fitted on the NCBI

non-redundant Viridiplantae database returning, with a confidence

score greater than 95% (P < 0.05) accuracy, the pea ferritin

1 chloroplast precursor (NCBI accession gi/417006;

Swiss-Prot accession P19975) This assignment was confirmed by fragmentation analysis employing a MALDI-TOF post source decay experiment, selecting the ion species at 1078.52 amu (MH+) with a time gate ion selector The resulting fragmentation pattern was characteristic the y and b ion series of the sequence ISEYVAQLR (223–231) The PSD fragments were fitted on the NCBI nonredundant Viridi-plantae database 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 digestion 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 stem mitochondria, subjected to immunogold decoration in the presence (A and B) or absence (C) of PAAF and at lower magnification (D) Arrows indicate electron-dense particles after immunolabeling with PAAF followed by gold-conjugated secondary antibody Bars correspond to 300 lm.

against cytochrome c, polyclonal antibodies raised against

the a/b-subunit of mitochondrial ATPase and PAAF,

respectively (Fig 2B) These results show that Mpl partially

lost the cytochrome c; the densitometric analysis of the

immunodecoration show a decrease 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

indicates that both the a/b-subunit and the ferritin are still

retained in Mpl, thus confirming the localization of ferritin

in the mitochondrial matrix

The ultrastructural localization of the pea stem

mito-chondrial ferritin was further confirmed in ultra-thin

sections of pea stem cross-sections (Fig 3) and fixed

mitochondria (Fig 4), both immunolabeled with PAAF,

and followed by gold-conjugated secondary antibody

Figure 3 shows that mitochondria, selected from a

trans-mission electron microscopic micrograph of pea stem cells (Fig 3A), have some electron-dense particles (Fig 3B,C; arrows) In agreement, such particles were also detected in isolated mitochondria (Fig 4A,B) The electron-dense par-ticles were not detected when PAAF was omitted in both cross-sections (result not shown) and isolated mitochondria (Fig 4C) According to the low level of plastidial enzymatic marker, detected in PMt (Table 1), the electron micro-graphs show that the purified mitochondrial fraction was almost free from etioplast contamination (Fig 4D) Figure 5A shows the protein patterns of control proteins and of CMt and PMt from A thaliana When these separated proteins were subjected to cross-reaction with PAAF (Fig 5B), again a band with an apparent molecular mass of 25–26 kDa was revealed, thus suggesting the presence of this iron-storage protein also in mitochondria from this type of plant cells Furthermore, preliminary results indicate that ferritin was also present in Percoll-purified mitochondria isolated from soybean hypocotyls (results not shown)

The genome of A thaliana contains a family of nuclear 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 the 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),

purified mitochondria (PMt), and total pea seed proteins (CP, control

proteins) (B) Immunoblotting of the same proteins with PAAF.

Molecular mass of protein standards is indicated in kDa (Std).

Table 3 Calculated values for prediction of mitochondrial targeting for some plant ferritins Scores were obtained from different programs available

on the net; values higher than 0.6 are highlighted in bold; the output for IPSORT is given as mitochondrial target peptide (mTP) or chloroplast transit peptide (cTP).

Plant species

Prediction programs SwissProt accession PREDOTAR MITOPROT II PSORT IPSORT

Arabidopsis thaliana Q39101 (AtFer1) 0.020 0.5482 0.360 cTP

protein [10] This 30 kDa protein is targeted to

mitochon-dria and processed to a 22 kDa subunit This ferritin,

expressed in HeLa cells, is available to exogenous iron,

similarly to the cytosolic ferritin, suggesting that this

mitochondrial protein may have profound consequences

on cellular iron homeostasis [11] As shown in this paper, a

ferritin has also been identified in higher plant

mitochon-dria This evidence arises from the following findings First,

a mitochondrial matrix protein of 25–26 kDa, cross-reacted

with a polyclonal antibody of pea seed ferritin in both pea

stem (Fig 1) and A thaliana (Fig 5) mitochondria Such

organelles were highly purified by discontinuous Percoll

gradient, providing a very low interference in the

immuno-decoration from other cellular components, 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 just etioplast ferritin, these organelles have 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 PMt from other organelles Second,

ferritin was immunocytochemically identified in etiolated

pea stem cross-sections (Fig 3) and in isolated pea

mito-chondria (Fig 4) The pea stem mitomito-chondrial 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 of 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–

4 possess at the N-terminus the typical presequences of the

chloroplastic protein transit peptide [28], similarly to what

reported for pea ferritin [29,30] On the other hand, the

analysis of the presequence of AtFer4 reveals a high score

for its mitochondrial localization, especially when compared

with AtFer1 (Table 3) The same analysis for pea ferritin

shows that the programs PSORT and IPSORT give a high

probability for this protein to be targeted to mitochondria

(Table 3)

The data presented in this paper strongly indicate a

mitochondrial localization for ferritins in P sativum and

A thalianaand could be rationalized as follows: the protein

may be targeted to both plastids and mitochondria,

similarly to what shown for several plant proteins [31] This

feature can be accomplished by alternative transcription,

alternative translation starts, alternative exon splicing (or

a combination of the above), or the presence in the

N-terminus of an ambiguous presequence [31]; prediction

programs could just be unable to detect such dual targeting

On the other hand, a similar situation has been described

for ferrochelatase-I, an enzyme involved in heme

biosyn-thesis and, probably, in protection against oxidative stress in

A thaliana[32] This enzyme has been recently reported to

be present also in pea mitochondria [33] The presence of ferrochelatase-I and -III in A thaliana mitochondria has been recently questioned, while their presence in pea 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 may 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 with 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 this damage

Acknowledgements

We thank Dr J.F Briat, Centre National de la Recherche Scientifique, Montpellier, France, for a generous gift of pea seed ferritin antibody.

We also thank very much Dr Sonia Levi, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), H San Raffaele, Milan, Italy, for the cross-reactivity analysis with human mitochondrial ferritin antibody Thanks are also due to Dr Sonia Patui for her help during Percoll-purified mitochondria preparation and to Mr Claudio Gamboz for his help with electron microscopy analysis This research was supported by

‘Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica’ (Cofin 2000–01) in the frame of the program entitled: Nitric Oxide and Plant Resistance to Pathogens.

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