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: 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)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. References 1 Taketani S (1994) Molecular and genetic characteriza- tion of ferrochelatase. 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Dual mitochondrial localization and different roles of the reversible reaction of mammalian ferrochelatase Masayoshi Sakaino 1 ,. reported. The present study reports the localization of ferrochelatase in the outer and inner membranes of mitochondria and the possible regulation of its reversible