Dual mitochondrial localization and different roles of the
reversible reaction of mammalian ferrochelatase
Masayoshi Sakaino
1
, Mutsumi Ishigaki
1
, Yoshiko Ohgari
1
, Sakihito Kitajima
1
, Ryuichi Masaki
2
,
Akitsugu Yamamoto
3
and Shigeru Taketani
1,4
1 Department of Biotechnology, Kyoto Institute of Technology, Japan
2 The First Department of Physiology, Kansai Medical University, Moriguchi, Osaka, Japan
3 Faculty of Bioscience, Nagahama Institute of Bioscience and Technology, Nagahama, Shiga, Japan
4 Insect Biomedical Center, Kyoto Institute of Technology, Japan
Keywords
ferrochelatase; inner membrane; iron
removal; mitochondrial outer membrane;
phosphorylation
Correspondence
S. Taketani, Department of Biotechnology,
Kyoto Institute of Technology, Sakyo-ku,
Kyoto 606-8585, Japan
Fax: +81 75 724 7789
Tel: +81 75 724 7789

MINT-7233234: Ferrochelatase (uniprotkb:P22315), Abcb7 (uniprotkb:Q61102) and b5 reduc-
tase (uniprotkb:
Q9DCN2) colocalize (MI:0403)bycosedimentation through density gradients
(
MI:0029)
l
MINT-7233207: b5 reductase (uniprotkb:Q9DCN2 ), COXIV (uniprotkb:P19783), Abcb7 (uni-
protkb:
Q61102) and Ferrochelatase (uniprotkb:P22315) colocalize (MI:0403)bycosedimenta-
tion through density gradients (
MI:0029)
l
MINT-7233195: ATP synthase (uniprotkb:Q50DL5) and Ferrochelatase (uniprotkb:P22315)
colocalize (
MI:0403)byfluorescence microscopy (MI:0416)
Abbreviations
AIF, apoptosis inducible factor; b
5
-reductase, NADH-cytochrome b
5
reductase; COX IV, cytochrome c oxidase subunit IV; MDH, malate
dehydrogenase; MEL, mouse erythroleukemia; PKC, protein kinase C; TPA, 12-O-tetradecanoyl-phorbol 13-acetate.
FEBS Journal 276 (2009) 5559–5570 ª 2009 The Authors Journal compilation ª 2009 FEBS 5559
Introduction
In the last step in the heme biosynthetic pathway,
ferrochelatase catalyzes the insertion of ferrous ions
into protoporphyrin IX to form protoheme. The mam-
malian enzyme is nuclear encoded, synthesized as a
precursor form (48 kDa), and translocated into the
mitochondrion, where it is proteolytically processed to

presequence corresponding to the mitochondrial recog-
nition signal is present at the N-terminus of the trans-
lation product, resulting in the targeting of the enzyme
to mitochondria. Dual localizations of some mitochon-
drial proteins have been reported previously [9,10],
although the mechanisms involved in the differential
localization of the same translational product have not
been demonstrated.
The phosphorylation of various mitochondrial pro-
teins has been established [11]. The presence of protein
kinases in the inner membrane of mitochondria may
play a role in the modulation of mitochondrial func-
tions in various tissues. For example, some subunits of
cytochrome oxidase are phosphorylated both in vivo
and in vitro [12]. NADH dehydrogenase and pyruvate
dehydrogenase are phosphorylated and their activities
are changed for physiological purposes [13,14].
Although ferrochelatase activity is modulated by lipids
and heavy metal ions [1,15], the post-translational
modification of ferrochelatase to address these differ-
ent functions has not been reported. The present study
reports the localization of ferrochelatase in the outer
and inner membranes of mitochondria and the possible
regulation of its reversible enzyme activity by phos-
phorylation. Phosphorylation of the enzyme may relate
to the activities and differential localization of ferr-
ochelatase. A new recycling pathway of heme that
includes the iron-removal reaction of heme at the sur-
face of mitochondria is proposed.
Results

tase was found in the supernatants, indicating that ferr-
ochelatase is a peripheral membrane protein, as revealed
by the deduced amino acid sequence of mammalian
Mitochondrial location of ferrochelatase M. Sakaino et al.
5560 FEBS Journal 276 (2009) 5559–5570 ª 2009 The Authors Journal compilation ª 2009 FEBS
ferrochelatase [1,16]. Next, mitochondria were purified
from the crude mitochondrial fraction, and intact
mitochondria were treated with trypsin or Na
2
CO
3
.As
shown in Fig. 2A, an immunoblot analysis revealed
that the amount of NADH-cytochrome b
5
reductase
(b
5
-reductase), a protein located in the outer mem-
brane, was markedly decreased by trypsin treatment,
whereas inner membrane proteins cytochrome c oxi-
dase subunit IV (COX IV) and ABCB7 remained
unchanged, indicating that the surface of outer mem-
brane was digested by trypsin. The amount of ferroch-
elatase in the mitochondria was decreased by trypsin
treatment, suggesting that a part of the ferrochelatase
protein is located at the surface of mitochondria. Alka-
line (0.1 m Na
2
CO

(Fig. 3B). When immunostaining by anti-ferrochelatase
ATP synthase Ferrochelatase
Merged
0
0.2
0.4
0.6
0.8
1
1.2
Mitochondria Cytosol
MDH
Cytochrome oxidase
Zinc insertion
Iron removal
Relative specific activity (ratio to control)
1
Supernatants
Pellets
43 kDa-
43 kDa-
2 3
A B
C
Fig. 1. (A) Mitochondrial localization. Cos-7 cells were transfected with pcDNA-HA-FECH, and incubated for 23 h. They were then fixed, per-
meabilized and reacted simultaneously with anti-ATP synthase and anti-HA sera to demonstrate localization of ATP synthase and ferrochela-
tase. The merged exposure confirms that the dots co-localize. Scale bar = 10 lm. (B) Subcellular distribution of the ferrochelatase activity of
mouse liver. After mouse liver was homogenized, the cell debris and nuclear fraction were removed. Mitochondria were separated by centri-
fugation and washed. Cytosol was obtained from the post-mitochondrial supernatant by centrifugation at 105 000 g for 60 min. Ferrochela-
tase activities, including zinc-insertion and iron-removal reactions, were measured. The activities of MDH and cytochrome c oxidase were

viously, ferrochelatase was separately purified by the
conventional iron-insertion activity using Blue-Sepha-
rose [4,15] and iron-removal activity using Red-Aga-
rose [7], and 2D gel analysis of the purified enzyme
showed the ferrochelatase bound to blue dye was more
basic than that bound to red-dye (Fig. 4E). When
comparing the peptides from these two ferrochelatase
enzymes by MALDI-TOF MS, three tryptic peptides
containing serine residues at positions 130, 303 and
330 were found to be different. These serine residues
were conserved among yeast, bacteria and mammalian
enzymes. It is possible that these serine residues can be
phosphorylated. Therefore, three mutated ferrochelata-
ses were constructed, expressed and purified from Esc-
herichia coli. When ferrochelatase was phosphorylated
in E. coli, (Fig. 4F, lower), the intensity of phospho-
ferrochelatase of S130A and S303A was decreased,
and the band was not detected in the double mutant
S130A and S303A, indicating that ferrochelatase was
phosphorylated at positions 130 and 303. The reaction
of ferrochelatase with anti-phosphoserine sera was
unchanged by the S330A mutation, indicating that ser-
ine at position 330 is not phosphorylated. When the
conventional zinc-chelating activity in these mutants
Ferrochelatase
COXIV
ABCB7
B5-reductase
1.6
0.2

ABCB7
B5-reductase
None Trypsin Na
2
CO
3
Mitochondria
Whole Inner
membrane
Outer
membrane
Ferrochelatase
COXIV
ABCB7
B5-reductase
Mitochondria Inner
membrane
Outer
membrane
Protoporphyrin formed (pmol·mg
–1
protein·h
–1
)
50
0
100
Zn-mesoporphyrin formed
(nmol·mg
–1

Mitochondrial location of ferrochelatase M. Sakaino et al.
5562 FEBS Journal 276 (2009) 5559–5570 ª 2009 The Authors Journal compilation ª 2009 FEBS
was examined, S130A and S330A decreased to 20%
and 68% of wild-type, respectively, and S303A did not
show any activity (Fig. 4F, upper). The iron-removal
activity of S130A was similar to that of control, but
that of S330A was 55% of the control. No activity
was observed in S303A. These results suggest that ser-
ine at position 303 is essential for the catalytic activity
and that phosphorylation of serine at position 130
may be involved in the regulation of the forward reac-
tion of ferrochelatase.
An increase in the acidic form of ferrochelatase
in 12-O-tetradecanoyl-phorbol 13-acetate
(TPA)- or hemin-treated mouse erythroleukemia
(MEL) cells
Finally, we attempted to clarify the possible regulation
of phosphorylation of ferrochelatase. When MEL cells
are treated with hemin, the cells can utilize exoge-
nously added heme and initiate erythroid differentia-
tion [17,18]. Accordingly, cell extracts from 50 lm
hemin-treated MEL cells were analyzed by 2D gel elec-
trophoresis. The phosphorylation of ferrochelatase was
examined by treatment of the cells with TPA, a typical
activator of protein kinase C (PKC), for 6 h, as a posi-
tive control. As shown in Fig. 5A, most ferrochelatase
in TPA-treated cells appeared as a single spot at an
acidic site, whereas major two spots were observed in
untreated cells. MEL cells were then treated with
50 lm hemin and ferrochelatase was analyzed by 2D

HEK293T cells were transfected with
pcDNA-HA-FECH and cryo-ultrathin sections
were double stained by immunogold meth-
ods. Anti-HA (10 nm gold particles) and anti-
TOM 20 (arrows, 5 nm gold particles) were
used. Scale bars = 0.1 lm. (B) Cryo-ultrathin
sections of HEK293T cells, as above, were
labeled with anti-HA (10 nm gold particles)
and anti-AIF (arrows, 5 nm gold particles).
(C) Cryo-ultrathin sections of mouse liver
were labeled with anti-ferrochelatase serum
and 10 nm immunogold particles.
M. Sakaino et al. Mitochondrial location of ferrochelatase
FEBS Journal 276 (2009) 5559–5570 ª 2009 The Authors Journal compilation ª 2009 FEBS 5563
Lysates Control IgG Anti-HA
HA
Immunoprecipitation
Immunoblots
P- Serine
P- Serine
P- Serine
P- Serine
Ferrochelatase
Mitochondria
Outer
Membrane
Whole Inner
Membrane
Outer membrane
Inner membrane

Protoporphyrin formed
(pmol·mg
–1
protein·h
–1
)
0
0.5
1
1.5
2
2.5
3
AB
EF
C
D
Fig. 4. 2D gel analysis of ferrochelatase. (A) Mitochondria were fractionated into the inner and outer membranes. The mitochondrial proteins
from both membrane fractions were analyzed by 2D gel electrophoresis. Immunoblotting with anti-ferrochelatase serum was performed. (B)
Mitochondrial proteins were analyzed by 2D gel electrophoresis and immunoblotting was performed with anti-ferrochelatase and anti-phos-
phoserine sera. (C) HEK293T cells were transfected with pcDNA-HA-FECH and solubilized using 1% Triton X-100. After centrifugation at
15 000 g for 20 min, immunoprecipitation with anti-HA serum was carried out, followed by immunoblotting with ant-HA and anti-phospho-
serine sera. (D) Mitochondrial proteins from the inner and outer membranes were analyzed by SDS-PAGE and labeled with anti-ferrochela-
tase and anti-phosphoserine. (E) Ferrochelatases purified from Blue-Sepharose and Red-Agarose were analyzed by 2D gel electrophoresis.
Immunoblotting was performed with anti-ferrochelatase serum. (F) Wild-type and mutated (S130A, S303A and S330A) ferrochelatases were
expressed in E. coli. Cellular proteins were analyzed and immunoblotting was performed with anti-phosphoserine and anti-ferrochelatase
sera (lower panel). The zinc-insertion and iron-removal activities of ferrochelatase were measured (upper panel). Data are the mean ± SD of
three independent experiments.
Mitochondrial location of ferrochelatase M. Sakaino et al.
5564 FEBS Journal 276 (2009) 5559–5570 ª 2009 The Authors Journal compilation ª 2009 FEBS

rochelatase was purified from various tissues using
blue dye, but did not bind to red dye. Conversely, the
enzyme catalyzing removal of iron from heme was
purified using Red-Agarose and identified as ferroch-
elatase. Analysis of the purified ferrochelatases from
red and blue dyes by 2D gel analysis revealed that they
exhibited different isoelectric points (Fig. 4E), indicat-
ing the occurrence of post-translational modification of
ferrochelatase. Various mitochondrial enzymes, such as
cytochrome c oxidase and aconitase, are phosphory-
lated, and reversible phosphorylation may play an
important role in mitochondrial function [11]. The
present data clearly showed that one of the phophory-
lated proteins is ferrochelatase. Considering that fer-
rochelatase located in the outer membrane exhibited
an acidic isoelectric point by 2D gel analysis (Fig. 4A),
the enzyme in the outer membrane is mainly phos-
phorylated. The newly-synthesized ferrochelatase con-
tains a pre-sequence at the N -terminus, which is
cleaved during the processing into the inner membrane
of mitochondria [1]. Because ferrochelatase in the
outer membrane has a molecular mass similar to that
of the enzyme in the inner membrane, the movement
of the enzyme to the outer membrane may occur after
the cleavage of the pre-sequence, and may relate to the
phosphorylation.
Mutation studies with ferrochelatase showed that
serine residues at positions 130 and 303 were phos-
phorylated (Fig. 4F). The zinc-insertion activity of
S130A mutant was low compared to that of the wild-

(pmol·mg
–1
protein·h
–1
)
30
20
10
0
Zn-mesoporphyrin formed
(nmol·mg
–1
protein·h
–1
)
TPA
A
B
Fig. 5. Phosphorylation of ferrochelatase in hemin- and TPA-treated
MEL cells. (A) MEL cells were treated with 50 l
M hemin and
10 n
M TPA for 6 h. The cellular proteins were analyzed by 2D gel
electrophoresis and immunoblotting was performed using anti-fer-
rochelatase serum. (B) The zinc-insertion and iron-removal activities
of ferrochelatase with extracts from cells untreated or treated with
hemin and TPA were measured. Data are the mean ± SD of three
independent experiments.
M. Sakaino et al. Mitochondrial location of ferrochelatase
FEBS Journal 276 (2009) 5559–5570 ª 2009 The Authors Journal compilation ª 2009 FEBS 5565

treated with hemin for 2–3 days also increased [1,32].
By contrast to data demonstrating that ferrochelatase
levels increased in hemin-treated MEL cells [18,30], the
data obtained in the present study showed that treat-
ment of cells with hemin for 6 h resulted in a decrease
in activity. Because a short period of treatment of the
cells with hemin caused an increase in the phosphory-
lation of ferrochelatase, with a concomitant decrease
in the zinc-insertion reaction, but not the iron-removal
reaction, phosphorylated ferrochelatase prefers to
remove iron from heme of exogenously added hemin,
suggesting that the iron-removal activity plays a role
in decreasing the level of uncommitted heme in cells.
The discrepancy between short- and long-period treat-
ments with hemin has not been explained, although it
is possible that additional regulation may exist in the
expression of ferrochelatase, which plays a role in the
iron-removal reaction of exogenous heme and the
change in position of the heme-moiety of hemopro-
teins. The protoporphyrin ring of the heme-moiety in
hemoproteins is re-used and utilized for the new syn-
thesis of hemoproteins after the re-insertion of ferrous
ions. This recycling system of protoporphyrin-heme is
markedly induced, accompanied by the induction of
de novo biosynthesis of heme [7] during erythroid
differentiation, indicating that this may be necessary
for the supply of heme to apo-proteins located in com-
partments different from those of the original proteins.
Experimental procedures
Materials

cells were transfected using Lipofectamine (Invitrogen Co.,
San Jose, CA, USA) or calcium phosphate with pcDNA-
HA-FECH and were then incubated in the presence of fetal
bovine serum at 37 ° C for the specified period [34].
Isolation and subfractionation of mouse liver
mitochondria
Mouse liver mitochondria were isolated by differential
centrifugation [7,15] and purified further by a self-forming
Percoll gradient centrifugation according to the method of
Hoppel et al. [35]. To separate the outer membrane from
Mitochondrial location of ferrochelatase M. Sakaino et al.
5566 FEBS Journal 276 (2009) 5559–5570 ª 2009 The Authors Journal compilation ª 2009 FEBS
the inner membrane, the mitochondria pellet was resus-
pended in 20 mm potassium phosphate ⁄ 0.2% defatted
BSA ⁄ 1mm NaVO
4
(pH 7.2) (0.2 mg proteinÆmL
)1
) and
incubated on ice with gentle stirring to induce swelling and
rupture of the mitochondrial outer membrane. After
20 min, ATP and MgCl
2
were added at final concentrations
of 1 mm each, and the suspension was stirred for a further
5 min on ice. The swelling ⁄ shrunk mitochondria were cen-
trifuged for 20 min at 4 °C at 22 550 g and the pellet was
gently resuspended in 50 mL of 20 mm potassium phos-
phate ⁄ 0.2% defatted BSA ⁄ 1mm NaVO
4

for 30 min on ice and then trypsin inhibitor
(300 lgÆmL
)1
) was added. The trypsin-treated mitochondria
were collected by centrifugation at 9000 g for 10 min. To
collect membrane proteins from mitochondria, mitochon-
dria were treated with 0.1 m Na
2
CO
3
for 30 min on ice,
and the membrane fraction was collected by centrifugation
at 9000 g at 4 °C for 10 min [37].
2D gel analysis
Proteins were first analyzed on the basis of charge by IEF
and then by size, using SDS-PAGE. Briefly, mitochondrial
proteins were separated by IEF using an ATTO 2D agar
gel (pH 3.5–10) (ATTO Corp., Tokyo, Japan). IEF ran at
300 V for 150 min. After the first-dimension IEF, the tube
was removed from the glass tube and loaded onto a slab
SDS-polyacrylamide gel (10%) for electrophoresis in the
second dimension at 100 V for 2 h.
Immunoblotting
Cellular and mitochondrial proteins were separated by
SDS-PAGE and transferred to a poly(vinylidene difluoride)
membrane (Bio-Rad Laboratories, Hercules, CA, USA).
Conditions for immunoblotting for ferrochelatase and other
antigens, and the detection of cross-reacted antigens, were
performed as described previously [7,34]. The relative level
of proteins was quantitated by scanning the band using

viously [38] with slight modifications. Briefly, HEK293T cells
were transfected with pcDNA-HA-FECH and the pellet of
HEK293T cells was fixed in 4% paraformaldehyde in 0.1 m
sodium phosphate buffer (pH 7.4) for 30 min. Mouse liver
was perfusion-fixed through the heart with 4% paraformal-
dehyde in 0.1 m phosphate buffer (pH 7.4) for 10 min. Fixed
HEK293T cells and liver tissue were processed for ultrathin
cryosectioning. Frozen sections of HEK293T cells were incu-
bated with mixture of monoclonal anti-HA mouse serum and
polyclonal anti-TOM 20 or anti-AIF rabbit sera, followed by
incubation with a mixture of anti-mouse IgG coupled with
10 nm gold particles and anti-rabbit IgG coupled with 5 nm
gold particles. Frozen sections of HEK293T cells were incu-
bated with polyclonal anti-ferrochelatase rabbit serum and
then anti-rabbit IgG coupled with 10 nm gold particles.
Stained sections were negatively stained, embedded in poly-
vinyl alcohol [39], and examined using a Hitachi H7600
electron microscope (Hitachi, Tokyo, Japan).
Enzyme assay
The reaction mixture for iron-removal activity contained
25 mm potassium phosphate buffer (pH 5.7), 50 lm hemin-
M. Sakaino et al. Mitochondrial location of ferrochelatase
FEBS Journal 276 (2009) 5559–5570 ª 2009 The Authors Journal compilation ª 2009 FEBS 5567
imidazole, 2 mm EDTA, and 2 mm ascorbate, in a final
volume of 1.0 mL in a Thunberg vacuum tube. The air in
the tube was replaced with nitrogen gas and dissolved gas
was removed in vacuo [7]. The reaction was carried out at
45 °C for 1 h. After the resulting mixture was centrifuged
at 1000 g for 10 min at room temperature, fluorescence was
measured in the supernatant by scanning 550–700 nm fluo-

We thank Drs T. Ogishima, H. Otera and K. Mihara
for the kind gifts of anti-b
5
reductase and anti-TOM
20, respectively; Dr Y. Iwai for the kind gift of
pcDNA3-HA vector; Drs T. Endo and T. Kataoka for
valuable advice; and S. Gotoh and Y. Kohno for
providing excellent technical assistance. This study was
supported in part by grants from the Ministry of
Education, Science, Sports and Culture of Japan.
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