reversible reaction of mammalian ferrochelataseMasayoshi Sakaino1, Mutsumi Ishigaki1, Yoshiko Ohgari1, Sakihito Kitajima1, Ryuichi Masaki2, Akitsugu Yamamoto3and Shigeru Taketani1,4 1 De
Trang 1reversible reaction of mammalian ferrochelatase
Masayoshi Sakaino1, Mutsumi Ishigaki1, Yoshiko Ohgari1, Sakihito Kitajima1, Ryuichi Masaki2, Akitsugu Yamamoto3and Shigeru Taketani1,4
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: taketani@kit.ac.jp
(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 ) by cosedimentation 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 ) by cosedimenta-tion through density gradients ( MI:0029 )
l MINT-7233195 : ATP synthase (uniprotkb: Q50DL5 ) and Ferrochelatase (uniprotkb: P22315 ) colocalize ( MI:0403 ) by fluorescence 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.
Trang 2In 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 presdiffer-ent 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
Trang 3ferrochelatase [1,16] Next, mitochondria were purified
from the crude mitochondrial fraction, and intact
mitochondria were treated with trypsin or Na2CO3 As
shown in Fig 2A, an immunoblot analysis revealed
that the amount of NADH-cytochrome b5 reductase
(b5-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 Na2CO3) 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
Merged
0 0.2 0.4 0.6 0.8
1 1.2
Mitochondria Cytosol
MDH Cytochrome oxidase Zinc insertion Iron removal
1
Supernatants
Pellets
43
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 centricentri-fugation 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.
Trang 4sera 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
0.6
1
0
1.2
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
Ferrochelatase
COXIV
ABCB7
B5-reductase
None Trypsin Na 2 CO 3
Mitochondria
Whole Inner membrane Outer membrane
Ferrochelatase
COXIV
ABCB7
membrane
Outer membrane
–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
Fig 2 Submitochondrial location of ferrochelatase (A) Trypsin or alkaline treatment Purified mitochondria were treated with trypsin (150 lgÆmL)1) or 0.1 M Na2CO3for 30 min on ice Immunoblotting was carried out with antibodies for ferrochelatase, Cox IV, ABCB7 and b5 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 b5-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.
Trang 5was 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
Ferrochelatase
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.
Trang 6Lysates Control IgG Anti-HA
HA
Immunoprecipitation
Immunoblots
P- Serine
P- Serine P- Serine
P- Serine
Ferrochelatase
Mitochondria
Outer Membrane
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- Blue + Red
100
50
0
/ S303A S303A S330A S130A
S130A Wt
Ferrochelatase
Zn-mesoporphyrin formed (µ
–1 protein·h
–1 )
Protoporphyrin formed (pmol·mg
–1 protein·h
–1 )
0 0.5 1 1.5 2 2.5 3
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) Ferrochelaanti-ferrochela-tases 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.
Trang 7The 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 b5-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
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.
Trang 8The 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 ferrochelaferrochela-tase 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 b5-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 37C 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
Trang 9the inner membrane, the mitochondria pellet was
resus-pended in 20 mm potassium phosphate⁄ 0.2% defatted
BSA⁄ 1 mm NaVO4 (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 MgCl2were 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 4C at 22 550 g and the pellet was
gently resuspended in 50 mL of 20 mm potassium
phos-phate⁄ 0.2% defatted BSA ⁄ 1 mm NaVO4 (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 4C 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⁄ 1 mm
NaVO4(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 4C for 16 h [35,36] The
35–40% sucrose fraction was collected and diluted diluted
10-fold with 20 mm potassium phosphate⁄ 1 mm NaVO4
(pH 7.2) The inner membrane was recovered by
centrifuga-tion at 22 550 g at 4C for 1 h The cytosolic fraction was
obtained from the post-mitochondrial supernatant by
centrifugation at 105 000 g at 4C 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 Na2CO3 for 30 min on ice,
and the membrane fraction was collected by centrifugation
at 9000 g at 4C 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⁄ Pi(+) (NaCl⁄ Pi contain-ing 1 mm CaCl2and 0.5 mm MgCl2), fixed with 4% parafor-maldehyde for 20 min, and permeabilized in 0.1% Triton X-100 in NaCl⁄ Pi(+) for 1 h After blocking with 2% fetal bovine serum in NaCl⁄ Pi(+), 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
Trang 10hemin-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
45C 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
FECH, FECH S130A, FECH S303A,
pET-FECH S330A and pET-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-b5 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
References
1 Taketani S (1994) Molecular and genetic
characteriza-tion of ferrochelatase In Regulacharacteriza-tion of Heme Protein
Synthesis(Fujita H, ed), pp 41–54 Alpha Med Press, Dayton, OH
2 Ferreira GC (1999) Ferrochelatase Int J Biochem Cell Biol 31, 995–1000
3 Jones MS & Jones OTG (1969) The structural organiza-tion of haem synthesis of rat liver mitochondria Biochem J 113, 507–514
4 Taketani S & Tokunaga R (1982) Purification and substrate specificity of bovine liver-ferrochelatase Eur J Biochem 127, 443–447
5 Dailey HA, Dailey TA, Wu CK, Medlock AE, Wang
KF, Rose JP & Wang BC (2000) Ferrochelatase at the millennium: structures, mechanisms and [2Fe-2S] clus-ters Cell Mol Life Sci 57, 1909–1926
6 Taketani S (2005) Aquisition, mobilization and utiliza-tion of cellular iron and heme: endless findings and growing evidence of tight regulation Tohoku J Exp Med 205, 297–318
7 Taketani S, Ishigaki M, Mizutani A, Uebayashi M, Numata M, Ohgari Y & Kitajima S (2007) Heme synthase (ferrochelatase) catalyzes the removal of iron from heme and demetallation of metalloporphyrin Biochemistry 46, 15054–15061
8 Loeb MR (1995) Ferrochelatase activity and protopor-phyrin IX utilization in Haemophilus influenzae J Bac-teriol 177, 3613–3615
9 Rodriguez-Sinovas A, Boengler K, Cabestrero A, Gres
P, Morente M, Ruiz-Meana M, Konietzka I, Miro´ E, Totzeck A, Heusch G et al (2006) Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection Circ Res 99, 93–101
10 Karniely S & Pines O (2005) Single translation–dual destination: mechanisms of dual protein targeting in eukaryotes EMBO Rep 6, 420–425
11 Pagliarini DJ & Dixon JE (2006) Mitochondrial modu-lation: reversible phosphorylation takes center stage? Trends Biochem Sci 31, 26–34
12 Ogbi M & Johnson JA (2006) Protein kinase Cepsilon interacts with cytochrome c oxidase subunit IV and enhances cytochrome c oxidase activity in neonatal car-diac myocyte preconditioning Biochem J 393, 191–199
13 De Rasmo D, Panell D, Sardanelli AM & Papa S (2008) cAMP-dependent protein kinase regulates the mitochondrial import of the nuclear encoded NDUFS4 subunit of complex I Cell Signal 20, 989–997
14 Raha S, Myint AT, Johnstone L & Robinson BH (2002) Control of oxygen free radical formation from mitochon-drial complex I: roles for protein kinase A and pyruvate dehydrogenase kinase Free Radic Biol Med 32, 421–430
15 Taketan S & Tokunaga R (1981) Rat liver ferrochela-tase Purification, properties and activation by fatty acids J Biol Chem 256, 12748–12753