Báo cáo khoa học: Dual mitochondrial localization and different roles of the reversible reaction of mammalian ferrochelatase ppt

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

E-mail:
(Received 9 May 2009, revised 18 June
2009, accepted 25 July 2009)
doi:10.1111/j.1742-4658.2009.07248.x
Ferrochelatase catalyzes the insertion of ferrous ions into protoporphyrin
IX to produce heme. Previously, it was found that this enzyme also partici-
pates in the reverse reaction of iron removal from heme. To clarify the role
of the reverse reaction of ferrochelatase in cells, mouse liver mitochondria
were fractionated to examine the localization of ferrochelatase, and it was
found that the enzyme localizes not only to the inner membrane, but also to
the outer membrane. Observations by immunoelectron microscopy con-
firmed the dual localization of ferrochelatase in ferrochelatase-expressing
human embryonic kidney cells and mouse liver mitochondria. The conven-
tional (zinc-insertion) activities of the enzyme in the inner and outer mem-
branes were similar, whereas the iron-removal activity was high in the outer
membrane. 2D gel analysis revealed that two types of the enzyme with dif-
ferent isoelectric points were present in mitochondria, and the acidic form,
which was enriched in the outer membrane, was found to be phosphory-
lated. Mutation of human ferrochelatase showed that serine residues at
positions 130 and 303 were phosphorylated, and serine at position 130 may
be involved in the balance of the reversible catalytic reaction. When mouse
erythroleukemia cells were treated with 12-O-tetradecanoyl-phorbol 13-ace-
tate, an activator of protein kinase C, or hemin, phospho-ferrochelatase
levels increased, with a concomitant decrease in zinc-insertion activity and a
slight increase in iron-removal activity. These results suggest that ferrochela-
tase localizes to both the mitochondrial outer and inner membranes and
that the change in the equilibrium position of the forward and reverse activ-
ities may be regulated by the phosphorylation of ferrochelatase.
Structured digital abstract
l

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

its mature size of 41–42 kDa [1,2] The active site of
the mammalian enzyme faces the matrix of the mito-
chondrion [3]. The enzyme not only utilizes ferrous
ions as a substrate in vivo, but also inserts divalent
metal ions such as zinc and cobaltic ions into the por-
phyrin ring in vitro [4,5]. Thus, the enzyme is able to
synthesize metalloporphyrins in vitro, although the uti-
lization of ferrous ions to form heme in cells is strictly
controlled [6]. Recently, the reverse reaction of ferroch-
elatase, namely the removal of iron from heme, was
reported to occur both in vivo and in vitro [7]. Ferroch-
elatase in the heme-requiring pathogen Haemophilus
influenzae functions in the reverse reaction, enabling
the bacteria to obtain iron from the host [8]. The yeast
and bacterial enzymes also exhibit the reverse reaction,
although the role of the reverse reaction is unclear [7].
Because hemoproteins, including myoglobin and
hemoglobin, become substrates of the removal reaction
of ferrochelatase [7], the question arises as to how the
cytoplasmic protein myoglobin-heme is moved to the
matrix side of the inner membrane of mitochondria,
where ferrochelatase is known to be located. More-
over, how the forward and reverse reactions of
ferrochelatase are regulated in cells has not been dem-
onstrated. To clarify the utilization of heme for the
reverse reaction of ferrochelatase, the localization of
ferrochelatase in mitochondria was re-examined. It was
found that ferrochelatase is localized in the outer and
inner membranes of mitochondria. The translation
product of ferrochelatase is a single isoform, and the

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

Localization of ferrochelatase in mitochondria
To examine the localization of ferrochelatase, the
cDNA for ferrochelatase was transfected into Cos-7
cells and the localization of expressed ferrochelatase
was compared with that of an inner membrane pro-
tein, ATP synthase. Immunofluorescence analysis with
anti-ferrochelatase sera indicated that mouse ferroch-
elatase appeared predominantly in mitochondria,
which was similar to the location of ATP synthase in
Cos-7 cells (Fig. 1A). Mitochondria and cytosol from
mouse liver were fractionated and the conventional
zinc-chelating (forward) and iron-removal (reverse)
activities, corresponding to the two ferrochelatase
activities (Fig. 1B), were examined in the mitochondria
and cytosol. The activity of cytochrome c oxidase, an
inner membrane protein, was only found in the mito-
chondrial fraction, whereas the activity of malate dehy-
drogenase (MDH), a matrix enzyme of mitochondria,
was mostly found in mitochondria, although approxi-
mately 15% of the activity leaked into the cytosol.
Large parts of the zinc-chelating and iron-removal
activities were found in mitochondria, and approxi-
mately 10% of both enzyme activities were in the
cytosol. Although ferrochelatase is known to be a
membrane-bound protein [1,2], these results suggested
that some ferrochelatase had leaked to the cytosolic
fraction. To examine how the enzyme is bound to the
mitochondrial membrane, mitochondria were frozen
and thawed, and then separated from the supernatants
(Fig. 1C). Immunoblot analysis showed that ferrochela-

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

3
) treatment of mitochondria mark-
edly reduced the level of ferrochelatase, demonstrating
that the enzyme at the surface and inside of mitochon-
dria is bound peripherally to membranes. To examine
the location of ferrochelatase in detail, purified mito-
chondria were fractionated into the outer and inner
membrane fractions (Fig. 2B). Compared with the
proteins located in the outer and inner membranes,
ferrochelatase was located in both membranes of
mouse liver mitochondria, and approximately 60% of
ferrochelatase was found in the inner membrane. The
iron-removal activity in the outer membrane was
higher than that in the inner membrane, whereas the
forward (zinc-chelating) activity was similar in both
membranes (Fig. 2C–E).
Electron microscopic analysis of ferrochelatase
localization
To further examine the localization of ferrochelatase,
pcDNA-HA-FECH was transfected into human
embryonic kidney HEK293T cells, after which the cells
were fixed, and cryo-ultrathin sections of the cells were
processed for immunogold labeling. Gold particles
(10 nm) showed the presence of HA-tag ferrochelatase
bound to the outer membrane of mitochondria and
co-localized with TOM 20 (5 nm gold particles), as
well as to the inner side of mitochondria around the
inner membrane (Fig. 3A). The location of ferrochela-
tase was confirmed by co-localization with an inner
membrane protein, apoptosis inducible factor (AIF)

(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

also measured. The values are the average of three independent experiments. (C) The release of ferrochelatase from mitochondria. Isolated
miotchondria were untreated (lane 1), frozen at )30 °C and thawed twice (lane 2), and the freeze-thawed treatment was repeated (lane 3).
The treated mitochondria were separated from supernatants by centrifugation at 105 000 g for 60 min. Aliquots were withdrawn and immu-
noblotting was performed with anti-ferrochelatase serum.
M. Sakaino et al. Mitochondrial location of ferrochelatase
FEBS Journal 276 (2009) 5559–5570 ª 2009 The Authors Journal compilation ª 2009 FEBS 5561
sera was performed using cryo-ultrathin sections of
mouse liver, ferrochelatase was detected in both the
outer membrane and the inner part of mitochondria
(Fig. 3C). These observations indicated that ferrochela-
tase was localized not only in the inner membrane, but
also in the outer membrane of mitochondria.
Phosphorylation of ferrochelatase
Next, we examined whether the different locations of
ferrochelatase lead to different structural and func-
tional properties. Ferrochelatase from the inner and
outer membranes was analyzed by 2D gel electropho-
resis (Fig. 4A). Immunoblot analysis revealed that the
IEF point of ferrochelatase of size 42 kDa was differ-
ent between the inner and outer membrane forms.
Namely, the protein from the outer membrane was
more acidic than that from the inner membrane. Anti-
phosphoserine sera reacted with acidic ferrochelatase
(Fig. 4B). Immunoprecitated ferrochelatase (HA-tag)
from human embryonic kidney cells expressing HA-
ferrochelatase was phosphorylated (Fig. 4C). When the
phosphorylation of ferrochelatase was compared
between the inner and outer membranes, ferrochelatase
in the outer membrane was more heavily phosphory-
lated than that in the inner membrane (Fig. 4D). Pre-

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

0.4
0.6
0.8
1
0
1.2
1.4
Relative protein levels
None Trypsin Na
2
CO
3
0
0.2
0.4
0.6
0.8
1
1.2
Whole Inner
membrane
Outer
membrane
Ferrochelatase
COXIV
ABCB7
B5-reductase
Relative protein levels
Ferrochelatase
COXIV

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

protein·h
–1
)
0
100
200
300
400
500
600
700
A
BD
CE
Fig. 2. Submitochondrial location of ferrochelatase. (A) Trypsin or alkaline treatment. Purified mitochondria were treated with trypsin
(150 lgÆmL
)1
) or 0.1 M Na
2
CO
3
for 30 min on ice. Immunoblotting was carried out with antibodies for ferrochelatase, Cox IV, ABCB7 and b
5
reductase. (B) Densitometric quantitation of mitochondrial proteins. Values are expressed as the mean ± SD of four experiments. (C) Loca-
tion of ferrochelatase in the outer and inner membranes. Mitochondria were separated into inner and outer membranes and immunoblotting
was performed. (D) Densitometric quantitation of ferrochelatase, COX IV, ABCB7 and b
5
-reductase of the inner and outer membranes. (E)
Ferrochelatase activity in outer and inner membranes. Zinc-insertion and iron-removal activities were measured in the outer and inner
membranes. The values obtained are the mean ± SD of three experiments.

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

gel analysis. One major spot of ferrochelatase was
found at the position of the acidic site (Fig. 5A). Phos-
phoserine levels corresponding to the position of
ferrochelatase increased in hemin-treated cells (data
not shown). The forward activity of the enzyme in
TPA-treated cells was decreased, whereas the iron-
removal activity increased slightly. In hemin-treated
cells, iron-removal activity also increased, but the
insertion of zinc ions into mesoporphyrin decreased
(Fig. 5B). These results suggest that phosphorylation
of ferrochelatase in MEL cells, as mediated by PKC,
led to a decrease of the conventional ferrochelatase
activity, indicating a preference for the removal of iron
from heme.
TOM 20/Ferrochelatase
F
e
rr
oc
h
e
l
atase

AIF/Ferrochelatase
A
C
B
Fig. 3. Immunoelectron microscopic analy-
ses of the localization of ferrochelatase. (A)

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

Outer membrane
+ inner membrane
10
pH
43 kDa
43 kDa
10
pH
Ferrochelatase
pH
10
kDa
Blue
Red
43-
43-
43-
Blue + Red
100
50
0
/ S303A
S303A S330AS130AS130AWt
Ferrochelatase
Zn-mesoporphyrin formed
(µmol·mg
–1
protein·h
–1
)

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

Discussion
The present study first demonstrated that mammalian
ferrochelatase is located not only in the inner mem-
brane, but also in the outer membrane of mitochon-
dria. Immunoblot data revealed that approximately
60% of the enzyme in mouse liver mitochondria was
present in the inner membrane and the remaining
enzyme with a similar molecular mass was in the outer
membrane. Electron microscope observations con-
firmed the outer and inner membrane localization of
ferrochelatase. A previous study [7] demonstrated that
the enzyme exhibited two catalytic reactions: iron-
insertion into porphyrin and the removal of iron from
porphyrin. The reversible reaction of ferrochelatase
may be ascribed to the different location. Because the
myoglobin-heme can be utilized for the removal reac-
tion of iron from heme [7], ferrochelatase located in
the outer membrane of mitochondria is able to contact
directly with cytosolic myoglobin. Thus, the outer
membrane enzyme may demonstrate a preference for
the iron-removal reaction. b
5
-Reductase is localized
not only in the endoplasmic reticulum, but also the
outer membrane of mitochondria in various tissues
[19,20], suggesting that the ferric ions of hemoproteins,
including myoglobin and hemoglobin, are reduced by
this enzyme, and that the reduced ferrous ions can be
removed by ferrochelatase.
We previously reported [21] that mammalian fer-

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-

type enzyme, whereas the iron-removal activity of the
mutant was similar to the wild-type enzyme. Further-
more, the treatment of mitochondria with alkaline
phosphatase resulted in a decrease in the iron-removal
reaction (data not shown). Thus, the phosphorylation
of serine at 130 may contribute to a change in the
equilibrium position of the reverse reaction of the
enzyme.
It has been reported that more than 50 mitochon-
drial proteins are phosphorylated [11]. The phosphory-
lation of these proteins is mediated by various protein
kinases, including protein kinase A and PKC [11,22].
10
Untreated
Hemin
TPA
-43 kDa
-43 kDa
-43 kDa
0
40
80
120
160
200
240
280
320
Control Hemin
Protoporphyrin formed

(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

The data obtained in the present study indicated that
the phosphorylation of ferrochelatase in MEL cells
was enhanced by treatment with TPA and hemin.
Because TPA is known to be an activator of PKC, the
phosphorylation of ferrochelatase may be mainly medi-
ated by PKC. Immunoelectron microscopic observa-
tions revealed that the kinase was associated with the
inner membrane and cristae [23], and physiological
studies suggested that PKC isoforms play a direct role
in regulating mitochondrial functions. Because the acti-
vation of PKC induces apoptosis [24–26], the phos-
phorylation of mitochondrial proteins by PKC may
lead to the inhibition of mitochondrial functions. Simi-
lar to the case for heme biosynthesis, activation of
PKC repressed the expression of d-aminolevulinic syn-
thase-1, with a concomitant increase in expression of
heme oxygenase-1 [27–29]. Thus, PKC may be
involved in the decrease in the intracellular level of
heme to help depress mitochondrial functions by
reducing the production of mitochondrial hemopro-
teins. The present study demonstrated that the increase
in phosphorylated ferrochelatase in TPA- or hemin-
treated cells caused a decrease in the metal ions-
insertion reaction, indicating that phosphorylated
ferrochelatase functions in the suppression of heme
biosynthesis.
Previously, it was demonstrated that the treatment
of MEL cells with hemin for 24–48 h resulted in an
increase in the mRNA and protein levels of ferrochela-
tase [30,31]. The ferrochelatase activity in MEL cells

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

Mesoporphyrin IX was purchased from Porphyrin Products
(Logan, UT, USA). Restriction endonucleases and DNA
modifying enzymes were obtained from Takara Co.
(Tokyo, Japan) and Toyobo Co. (Tokyo, Japan). Antibod-
ies for bovine ferrochelatase and b
5
-reductase (methemoglo-
bin reductase) were produced as described previously [4,7].
Anti-ATP-synthase and anti-phosphoserine sera were
obtained from Millipore-Upstate (Tokyo, Japan) and
Zymed Laboratory (San Francisco, CA, USA), respectively.
Anti-COX IV sera was from Abcom Co. (Tokyo, Japan).
Anti-TOM 20, anti-AIF and anti-actin sera were products
of Santa Crutz Co. (Santa Crutz, CA, USA). Percoll was
obtained from Fulka Biochemika (Steinheim, Sweden).
Ferrochelatase was purified using Blue-Sepharose (GE
Healthcare Biosciences, Amersham, UK) or Red-Agarose
(Millipore Corp., Bedford, MA, USA) and the purified
enzyme was digested with trypsin, followed by peptide anal-
ysis by MALDI-TOF MS [7]. All other chemicals were of
analytical grade.
Plasmids
The full-length cDNA of mouse ferrochelatase [16] was
digested with KpnI and ligated into KpnI-digested pcDNA3
(HA) vector [33]. The resulting plasmid, pcDNA-HA-
FECH, was introduced into E. coli XL1-Blue.
Cell culture and DNA transfection
Monkey kidney Cos-7 cells, MEL cells and human embry-
onic kidney HEK293-T cells were grown in DMEM supple-
mented with 10% fetal bovine serum and antibiotics. The

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

(pH 7.2). The
mitochondrial suspension was treated with two strokes of a
tight-fitting pestle (Wheaton Industries Inc., Millville, NJ,
USA) and centrifuged at 1900 g for 15 min at 4 °C. The
supernatant was removed and centrifuged for 20 min at
22 550 g to obtain the crude outer membrane pellet. The
outer membrane was purified by centrifugation at
121 000 g using a discontinuous sucrose gradient consisting
of 17%, 25%, 35% and 60%. The 25–35% sucrose fraction
was diluted 10-fold with 20 mm potassium phosphate ⁄ 1mm
NaVO
4
(pH 7.2) and the pellet was recovered by centrifuga-
tion at 184 000 g . To isolate the inner membrane, the
1900 g pellet was loaded onto a discontinuous sucrose gra-
dient consisting of 17%, 25%, 37.5%, 50% and 61% and
centrifugation at 100 000 g at 4 °C for 16 h [35,36]. The
35–40% sucrose fraction was collected and diluted diluted
10-fold with 20 mm potassium phosphate ⁄ 1mm NaVO
4
(pH 7.2). The inner membrane was recovered by centrifuga-
tion at 22 550 g at 4 °C for 1 h. The cytosolic fraction was
obtained from the post-mitochondrial supernatant by
centrifugation at 105 000 g at 4 °C for 60 min to remove
microsomes.
Alkaline treatment and trypsin digestion of
mitochondria
Purified mitochondria were treated with trypsin
150 lgÆmL
)1

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

ATTO Image Freezer AE-6905.
Immunofluorescence microscopy
Cos-7 cells were washed with NaCl ⁄ P
i
(+) (NaCl ⁄ P
i
contain-
ing 1 mm CaCl
2
and 0.5 mm MgCl
2
), fixed with 4% parafor-
maldehyde for 20 min, and permeabilized in 0.1% Triton
X-100 in NaCl ⁄ P
i
(+) for 1 h. After blocking with 2% fetal
bovine serum in NaCl ⁄ P
i
(+), incubation with anti-HA as the
primary antibody was carried out, followed by incubation
with Cy3-conjugated goat anti-(mouse Ig) (BD Biosciences
Co.) [34]. For double staining experiments, the cells were fur-
ther incubated with anti-ATP synthase (Millipore Co.,
Tokyo, Japan), followed by Cy2-conjugated goat anti-(rabbit
Ig) (Becton-Dickinson Biosciences, Franklin Lakes, NJ,
USA). The cells were examined using a Carl Zeiss LSM 510
confocal microscope (Carl Zeiss, Oberkochen, Germany).
Immunoelectron microscopy
Cryo-ultramicrotomy and double-immunogold staining on
the cryo-ultrathin sections were performed as described pre-

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-

rescence emissions with excitation at 400 nm. The zinc-
chelating ferrochelatase activity was measured as described
previously [7,40]. The activities of cytochrome c oxidase
and MDH were measured by the methods of Yamamoto
et al. [41] and Kitto et al. [42], respectively.
Recombinant enzymes
Human wild-type ferrochelatase protein carrying a his-tag
was described previously [40]. cDNAs for mutated human
ferrochelatase S130A, S303A and S330A were prepared: in
the first round of PCR, human ferrochelatase [43] was used
as a template. Primer pairs used were primer A (5¢-AAG
AATTCGGTGCAAAACCTCAAGT-3¢) as forward pri-
mer, a mutagenic primer (5¢-GAGGCGGAGCCCCCATC
-3¢), primer B (5¢-AAAAGCTTCACAGCTGCTGGCTGG
-3¢) as reverse primer, and the mutagenic primer (5¢-GATG
GGGGCTCCGCCTC-3¢) for the substitution S130A. In
the preparation of the substitution S303A, 5¢-TGTGGC
AAGCCAAGG-3¢ and 5¢-AACCTTGGCTTGCCACA-3¢
were used as mutagenic primers. In the case of the substitu-
tion S330A, 5¢-CATTTACCGCTGCCCATA-3¢ and 5¢-TA
TGGGCAGCGGTAAATG-3¢ were used. In the second
round, the (A) and (B) primer pair was used to amplify the
full-length human ferrochelatase sequences with the muta-
tion, and the DNA fragments were purified, sequenced and
inserted into a pET vector as described above. Plasmids
pET-FECH, pET-FECH S130A, pET-FECH S303A, pET-
FECH S330A and pET-FECH S303A ⁄ S330A were intro-
duced into E. coli strain BL21. Proteins were overexpressed
and purified as described previously [40].
Acknowledgements

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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