Iron and Porphyrin Trafficking in Heme Biogenesis
2010; Elsevier BV; Volume: 285; Issue: 35 Linguagem: Inglês
10.1074/jbc.r110.119503
ISSN1083-351X
AutoresIman J. Schultz, Caiyong Chen, Barry H. Paw, Iqbal Hamza,
Tópico(s)Heme Oxygenase-1 and Carbon Monoxide
ResumoIron is an essential element for diverse biological functions. In mammals, the majority of iron is enclosed within a single prosthetic group: heme. In metazoans, heme is synthesized via a highly conserved and coordinated pathway within the mitochondria. However, iron is acquired from the environment and subsequently assimilated into various cellular pathways, including heme synthesis. Both iron and heme are toxic but essential cofactors. How is iron transported from the extracellular milieu to the mitochondria? How are heme and heme intermediates coordinated with iron transport? Although recent studies have answered some questions, several pieces of this intriguing puzzle remain unsolved. Iron is an essential element for diverse biological functions. In mammals, the majority of iron is enclosed within a single prosthetic group: heme. In metazoans, heme is synthesized via a highly conserved and coordinated pathway within the mitochondria. However, iron is acquired from the environment and subsequently assimilated into various cellular pathways, including heme synthesis. Both iron and heme are toxic but essential cofactors. How is iron transported from the extracellular milieu to the mitochondria? How are heme and heme intermediates coordinated with iron transport? Although recent studies have answered some questions, several pieces of this intriguing puzzle remain unsolved. IntroductionCellular Iron TransportIron is essential to most living organisms, which have different sophisticated ways to obtain this element from the environment. Uptake and regulation of iron in bacteria and yeast have been described in detail (1Krewulak K.D. Vogel H.J. Biochim. Biophys. Acta. 2008; 1778: 1781-1804Crossref PubMed Scopus (350) Google Scholar, 2Philpott C.C. Protchenko O. Eukaryot. Cell. 2008; 7: 20-27Crossref PubMed Scopus (177) Google Scholar). Here, we will highlight iron processing in higher eukaryotes, particularly mammals. The daily requirements for iron in mammals are high, exerted mainly by red blood cells (RBCs), 4The abbreviations used are: RBCred blood cellMΦmacrophage(s)TftransferrinTfR1Tf receptor-1IMinner membraneIREiron regulatory elementUTRuntranslated regionFECHferrochelataseMELmouse erythroleukemiaIRPiron regulatory proteinALAδ-aminolevulinic acidCPgenIIIcoproporphyrinogen IIIIMSintermembrane spaceOMouter membranePPIXprotoporphyrin IXPPOXprotoporphyrinogen oxidaseGSTglutathione S-transferaseERendoplasmic reticulumMAMmitochondria-associated membraneFLVCRfeline leukemia virus subtype C receptor. which generate vast amounts of hemoglobin. The majority of this iron is efficiently recycled from senescent RBCs by macrophages (MΦ), but a small portion needs to be extracted from the diet (3Hentze M.W. Muckenthaler M.U. Andrews N.C. Cell. 2004; 117: 285-297Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar). Dietary iron is taken up by the enterocytes in the proximal region of the small intestine. The proteins involved in intestinal iron absorption and transport have been reviewed in detail (4De Domenico I. McVey Ward D. Kaplan J. Nat. Rev. Mol. Cell Biol. 2008; 9: 72-81Crossref PubMed Scopus (343) Google Scholar, 5Simpson R.J. McKie A.T. Cell Metab. 2009; 10: 84-87Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). After a series of reduction and oxidation steps during its passage through the enterocyte, iron is released into the bloodstream by the basolateral transporter FPN1 (ferroportin 1) and then oxidized to ferric iron (Fe3+) by the ferroxidases hephaestin and ceruloplasmin. Ferric iron is bound by transferrin (Tf), which has high affinity for ferric but not ferrous (Fe2+) iron. Under normal circumstances, serum iron is bound to Tf and transported to all cells of the body.The best characterized mechanism for cellular iron uptake is uptake through Tf receptor-1 (TfR1; CD71) (Fig. 1). TfR1 binds two Tf molecules and has the highest affinity for diferric Tf. The TfR1-Tf complexes are concentrated in clathrin-coated pits on the cell surface and endocytosed. Once the complexes are internalized, a membrane-associated ATPase proton pump lowers the pH in the endosome to create an environment where ferric iron can be released from Tf. To be transported out of the endosome, iron must be reduced. In developing RBCs, this step is performed by the STEAP3 (six-transmembrane epithelial antigen of prostate 3) protein (6Ohgami R.S. Campagna D.R. Greer E.L. Antiochos B. McDonald A. Chen J. Sharp J.J. Fujiwara Y. Barker J.E. Fleming M.D. Nat. Genet. 2005; 37: 1264-1269Crossref PubMed Scopus (481) Google Scholar). Other members of the STEAP family are thought to be endosomal ferrireductases in non-erythroid cells (7Ohgami R.S. Campagna D.R. McDonald A. Fleming M.D. Blood. 2006; 108: 1388-1394Crossref PubMed Scopus (421) Google Scholar). The reduced iron is exported out of the endosome by the divalent metal transporter (DMT1/SLC11A2/NRAMP2) in erythroid and non-erythroid cells (8Fleming M.D. Romano M.A. Su M.A. Garrick L.M. Garrick M.D. Andrews N.C. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 1148-1153Crossref PubMed Scopus (801) Google Scholar, 9Fleming M.D. Trenor 3rd, C.C. Su M.A. Foernzler D. Beier D.R. Dietrich W.F. Andrews N.C. Nat. Genet. 1997; 16: 383-386Crossref PubMed Scopus (1015) Google Scholar, 10Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google Scholar). TfR1 is returned to the plasma membrane, where it can participate in another round of Tf-mediated iron uptake. A protein involved in TfR1 recycling in RBCs, Sec15l1, was identified from the hemoglobin-deficit (hbd) mouse, which shows microcytic hypochromic anemia (11Lim J.E. Jin O. Bennett C. Morgan K. Wang F. Trenor 3rd, C.C. Fleming M.D. Andrews N.C. Nat. Genet. 2005; 37: 1270-1273Crossref PubMed Scopus (80) Google Scholar). It was demonstrated that Mon1a plays a role in trafficking FPN1 to the cell surface of MΦ in mice (12Wang F. Paradkar P.N. Custodio A.O. McVey Ward D. Fleming M.D. Campagna D. Roberts K.A. Boyartchuk V. Dietrich W.F. Kaplan J. Andrews N.C. Nat. Genet. 2007; 39: 1025-1032Crossref PubMed Scopus (55) Google Scholar).The steps in cellular iron transport following release from the endosome are poorly understood. After its release, some ferrous iron will be stored in ferritin, a ubiquitously expressed iron storage protein that regulates intracellular iron availability (13Torti F.M. Torti S.V. Blood. 2002; 99: 3505-3516Crossref PubMed Scopus (849) Google Scholar). Storing iron in ferritin prevents free iron from generating toxic radicals and allows the regulated release of iron. Recent work identified a cytosolic iron chaperone, PCBP1 (poly(rC)-binding protein 1), that transports iron to ferritin (14Shi H. Bencze K.Z. Stemmler T.L. Philpott C.C. Science. 2008; 320: 1207-1210Crossref PubMed Scopus (355) Google Scholar). The human cell lines used in this study were non-erythroid; it remains to be investigated if PCBP1 or any orthologs serve this role in erythroid cells.Mitochondrial Iron Uptake and ProcessingThe majority of cellular iron is utilized in the mitochondria for the biosynthesis of both heme and FeS clusters (15Lill R. Nature. 2009; 460: 831-838Crossref PubMed Scopus (819) Google Scholar, 16Ajioka R.S. Phillips J.D. Kushner J.P. Biochim. Biophys. Acta. 2006; 1763: 723-736Crossref PubMed Scopus (355) Google Scholar). This makes the mitochondrion an important organelle in iron trafficking. Some cytosolic iron may be used for extramitochondrial FeS cluster synthesis (17Tong W.H. Jameson G.N. Huynh B.H. Rouault T.A. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 9762-9767Crossref PubMed Scopus (182) Google Scholar, 18Tong W.H. Rouault T. EMBO J. 2000; 19: 5692-5700Crossref PubMed Scopus (168) Google Scholar, 19Tong W.H. Rouault T.A. Cell Metab. 2006; 3: 199-210Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). How iron is transferred from the endosome, the cytosol, or ferritin to the mitochondria is unknown. In developing RBCs, iron may be delivered by docking of the endosome to the mitochondrion (20Sheftel A.D. Zhang A.S. Brown C. Shirihai O.S. Ponka P. Blood. 2007; 110: 125-132Crossref PubMed Scopus (206) Google Scholar). This "kiss-and-run" mechanism would provide an efficient means for delivering iron to mitochondria in RBCs, which generate vast amounts of heme. Recent evidence showed that mitochondria fuse with each other and with the endoplasmic reticulum (21Liu X. Weaver D. Shirihai O. Hajnóczky G. EMBO J. 2009; 28: 3074-3089Crossref PubMed Scopus (232) Google Scholar, 22Kornmann B. Currie E. Collins S.R. Schuldiner M. Nunnari J. Weissman J.S. Walter P. Science. 2009; 325: 477-481Crossref PubMed Scopus (889) Google Scholar). Small organic compounds or some proteins may also bind free cellular iron (23Kruszewski M. Mutat. Res. 2003; 531: 81-92Crossref PubMed Scopus (381) Google Scholar). If and how these are involved in the trafficking of iron to the mitochondrion are unknown.The vertebrate mitochondrial iron importer was discovered through studies with the anemic zebrafish mutant frascati (24Shaw G.C. Cope J.J. Li L. Corson K. Hersey C. Ackermann G.E. Gwynn B. Lambert A.J. Wingert R.A. Traver D. Trede N.S. Barut B.A. Zhou Y. Minet E. Donovan A. Brownlie A. Balzan R. Weiss M.J. Peters L.L. Kaplan J. Zon L.I. Paw B.H. Nature. 2006; 440: 96-100Crossref PubMed Scopus (433) Google Scholar). The gene responsible for the anemic phenotype, Mfrn1 (mitoferrin-1)/SLC25A37, belongs to the SLC25 (solute carrier 25) family of proteins, which are located primarily in the mitochondrial inner membrane (IM). The mouse Mfrn1 knock-out in early erythroid cells shows impaired mitochondrial iron import and reduced heme synthesis upon terminal differentiation (24Shaw G.C. Cope J.J. Li L. Corson K. Hersey C. Ackermann G.E. Gwynn B. Lambert A.J. Wingert R.A. Traver D. Trede N.S. Barut B.A. Zhou Y. Minet E. Donovan A. Brownlie A. Balzan R. Weiss M.J. Peters L.L. Kaplan J. Zon L.I. Paw B.H. Nature. 2006; 440: 96-100Crossref PubMed Scopus (433) Google Scholar). To import iron and regulate heme synthesis in RBCs, Mfrn1 must interact with the mitochondrial ATP-binding cassette transporter Abcb10 (25Chen W. Paradkar P.N. Li L. Pierce E.L. Langer N.B. Takahashi-Makise N. Hyde B.B. Shirihai O.S. Ward D.M. Kaplan J. Paw B.H. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 16263-16268Crossref PubMed Scopus (161) Google Scholar). The Mfrn1 paralog, Mfrn2 (Slc25a28), is ubiquitously expressed and was proposed to be the non-erythroid mitochondrial iron importer (24Shaw G.C. Cope J.J. Li L. Corson K. Hersey C. Ackermann G.E. Gwynn B. Lambert A.J. Wingert R.A. Traver D. Trede N.S. Barut B.A. Zhou Y. Minet E. Donovan A. Brownlie A. Balzan R. Weiss M.J. Peters L.L. Kaplan J. Zon L.I. Paw B.H. Nature. 2006; 440: 96-100Crossref PubMed Scopus (433) Google Scholar, 26Paradkar P.N. Zumbrennen K.B. Paw B.H. Ward D.M. Kaplan J. Mol. Cell. Biol. 2009; 29: 1007-1016Crossref PubMed Scopus (215) Google Scholar). How iron is delivered to Mfrn1/2 from the cytosol for mitochondrial import remains unclear.Once delivered to the mitochondria, iron is either stored in mitochondrial ferritin or utilized for the synthesis of heme and FeS clusters. FeS clusters are prosthetic groups in proteins that play an essential role in cell metabolism (15Lill R. Nature. 2009; 460: 831-838Crossref PubMed Scopus (819) Google Scholar). In developing RBCs, iron is utilized primarily for the synthesis of large quantities of heme. Non-erythroid cells also make heme, but their iron requirements are much lower. Defects in heme synthesis genes result in human hematological disorders characterized by defective cellular iron homeostasis (27Sassa S. Br. J. Haematol. 2006; 135: 281-292Crossref PubMed Scopus (150) Google Scholar). The mechanisms that regulate vertebrate heme synthesis and the accompanying iron flux are beginning to be illuminated.Heme biogenesis is closely linked to the availability of FeS clusters. The first gene in erythroid heme synthesis, ALAS2 (aminolevulinic acid synthase-2), is regulated by the FeS cluster-binding protein IRP1 (iron regulatory protein 1) (3Hentze M.W. Muckenthaler M.U. Andrews N.C. Cell. 2004; 117: 285-297Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar). A pioneering study showed that, in the anemic zebrafish mutant shiraz, defective FeS cluster synthesis resulted in IRP1 binding constitutively to the iron regulatory element (IRE) in the 5′-untranslated region (UTR) of the ALAS2 mRNA. Consequently, translation of ALAS2 and synthesis of heme were blocked (28Wingert R.A. Galloway J.L. Barut B. Foott H. Fraenkel P. Axe J.L. Weber G.J. Dooley K. Davidson A.J. Schmid B. Schmidt B. Paw B.H. Shaw G.C. Kingsley P. Palis J. Schubert H. Chen O. Kaplan J. Zon L.I. Nature. 2005; 436: 1035-1039Crossref PubMed Scopus (320) Google Scholar). Mammalian ferrochelatase (FECH), the terminal enzyme in heme synthesis, is a [2Fe-2S]-containing protein (29Dailey H.A. Finnegan M.G. Johnson M.K. Biochemistry. 1994; 33: 403-407Crossref PubMed Scopus (178) Google Scholar, 30Wu C.K. Dailey H.A. Rose J.P. Burden A. Sellers V.M. Wang B.C. Nat. Struct. Biol. 2001; 8: 156-160Crossref PubMed Scopus (198) Google Scholar). Deletion of the [2Fe-2S]-binding region at the C terminus of FECH abolished both the binding of the cluster and the enzyme activity (29Dailey H.A. Finnegan M.G. Johnson M.K. Biochemistry. 1994; 33: 403-407Crossref PubMed Scopus (178) Google Scholar). Similarly, inactivation of FeS clusters by nitric oxide significantly inhibited the activity of FECH (31Sellers V.M. Johnson M.K. Dailey H.A. Biochemistry. 1996; 35: 2699-2704Crossref PubMed Scopus (122) Google Scholar). A recent study utilized a systems biology approach to analyze ≈35,000 cDNA microarrays and to identify mitochondrial genes that were tightly coexpressed and coregulated with the eight heme biosynthesis enzymes. Five candidate genes with putative roles in heme synthesis were selected for studies in the zebrafish. Two genes were known to be involved in FeS cluster synthesis, whereas the other three were mitochondrial transporters. Morpholino knockdown of all five genes resulted in profound anemia; silencing of the transporter SLC25A39 in differentiating mouse erythroleukemia (MEL) cells strongly reduced heme synthesis (32Nilsson R. Schultz I.J. Pierce E.L. Soltis K.A. Naranuntarat A. Ward D.M. Baughman J.M. Paradkar P.N. Kingsley P.D. Culotta V.C. Kaplan J. Palis J. Paw B.H. Mootha V.K. Cell Metab. 2009; 10: 119-130Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). These newly identified genes may play important roles in mammalian mitochondrial iron homeostasis.In addition to heme biosynthesis, a significant portion of iron in the mitochondrion is utilized for the assembly of FeS clusters. This pathway is highly conserved among species, and studies in yeast have guided research on the mammalian components of the FeS cluster pathway, although additional proteins have been identified (33Rouault T.A. Tong W.H. Trends Genet. 2008; 24: 398-407Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Genetic defects in several genes involved in FeS cluster assembly are associated with human diseases in which defective mitochondrial iron homeostasis is a hallmark. Patients with mutations in GRX5 (glutaredoxin-5) suffer from microcytic anemia with accumulation of iron in mitochondria (34Camaschella C. Campanella A. De Falco L. Boschetto L. Merlini R. Silvestri L. Levi S. Iolascon A. Blood. 2007; 110: 1353-1358Crossref PubMed Scopus (265) Google Scholar). Likewise, patients with Friedreich ataxia have genetic defects in the mitochondrial FeS cluster iron chaperone frataxin, which results in mitochondrial iron overload (35Rötig A. de Lonlay P. Chretien D. Foury F. Koenig M. Sidi D. Munnich A. Rustin P. Nat. Genet. 1997; 17: 215-217Crossref PubMed Scopus (866) Google Scholar). In addition, defects in ABCB7, a protein implicated in export of mitochondrial FeS clusters, lead to anemia accompanied by mitochondrial iron deposits (36Bekri S. Kispal G. Lange H. Fitzsimons E. Tolmie J. Lill R. Bishop D.F. Blood. 2000; 96: 3256-3264Crossref PubMed Google Scholar). These examples clearly indicate that the FeS cluster pathway plays an essential role in regulating mammalian mitochondrial and cellular iron homeostasis.Cellular Iron Homeostasis: The IRE/IRP Regulatory MechanismBecause both iron overload and iron deficiency are incompatible with normal body physiology, mammals regulate their iron levels at both the systemic and cellular levels. Excellent reviews detailing advances made in the past 2 decades have been published (37Nemeth E. Ganz T. Annu. Rev. Nutr. 2006; 26: 323-342Crossref PubMed Scopus (560) Google Scholar, 38Andrews N.C. Blood. 2008; 112: 219-230Crossref PubMed Scopus (477) Google Scholar, 39Zhang A.S. Enns C.A. J. Biol. Chem. 2009; 284: 711-715Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). This minireview focuses on the regulation of cellular iron homeostasis.Many of the genes that are involved in iron transport or utilization contain one or more IREs in their mRNA. These elements are conserved RNA stem-loop structures that are recognized by IRP1 and IRP2. Depending on where the IRE is located in the mRNA, binding of an IRP leads to blocking translation or to stabilizing mRNA (reviewed in Ref. 40Muckenthaler M.U. Galy B. Hentze M.W. Annu. Rev. Nutr. 2008; 28: 197-213Crossref PubMed Scopus (468) Google Scholar). Generally, mRNAs of genes such as FPN1, ALAS2, and ferritin that reduce cellular iron levels or the availability of iron have an IRE in the 5′-UTR of their mRNA; the IRE in the mRNA of genes like TfR1 and DMT1 that increase the availability of iron is located in the 3′-UTR. Therefore, binding of IRPs to IREs results in increased iron uptake and availability and reduced iron utilization and storage. This mechanism prevents a cell from becoming iron-deficient or iron-overloaded and is essential for cellular iron homeostasis.The binding of IRP1 and IRP2 to their target mRNAs is differentially regulated. IRP1 (also known as aconitase 1) is a dual function protein that can convert citrate to isocitrate or bind IREs. IRP1 binds an FeS cluster and acts as an aconitase under iron-replete conditions when FeS cluster synthesis is normal. When cellular iron levels are low, there is a corresponding reduction in FeS cluster synthesis. Consequently, IRP1 loses its aconitase activity, binds IREs, and alters iron uptake and utilization. IRP2 does not bind an FeS cluster, and its activity is regulated by degradation by a proteasomal complex (41Salahudeen A.A. Thompson J.W. Ruiz J.C. Ma H.W. Kinch L.N. Li Q. Grishin N.V. Bruick R.K. Science. 2009; 326: 722-726Crossref PubMed Scopus (285) Google Scholar, 42Vashisht A.A. Zumbrennen K.B. Huang X. Powers D.N. Durazo A. Sun D. Bhaskaran N. Persson A. Uhlen M. Sangfelt O. Spruck C. Leibold E.A. Wohlschlegel J.A. Science. 2009; 326: 718-721Crossref PubMed Scopus (302) Google Scholar). The key component of this complex is the cytosolic protein FBXL5 (F-box and leucine-rich repeat protein 5). The FBXL5 protein contains a domain that can bind iron. When iron levels are high, FBXL5 binds iron and targets IRP2 for degradation; iron-depleted conditions result in FBXL5 degradation. IRP2 appears to be regulated directly by cytosolic iron levels, whereas IRP1 binding to IREs depends on iron utilization by the mitochondrial FeS cluster assembly machinery. Vashisht et al. (42Vashisht A.A. Zumbrennen K.B. Huang X. Powers D.N. Durazo A. Sun D. Bhaskaran N. Persson A. Uhlen M. Sangfelt O. Spruck C. Leibold E.A. Wohlschlegel J.A. Science. 2009; 326: 718-721Crossref PubMed Scopus (302) Google Scholar) showed that FBXL5 also binds IRP1 but does not target it for degradation under iron-replete conditions. The relevance of this interaction and how IRP1 escapes degradation remain to be elucidated.The IRE/IRP mechanism is particularly important in tissues that regulate iron homeostasis or have high iron demands. IRPs play an essential role in regulating iron uptake in the intestine (43Galy B. Ferring-Appel D. Kaden S. Gröne H.J. Hentze M.W. Cell Metab. 2008; 7: 79-85Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The IRE/IRP system is also important in regulating iron processing by developing RBCs, as deletion of IRP2 results in microcytic anemia (44Cooperman S.S. Meyron-Holtz E.G. Olivierre-Wilson H. Ghosh M.C. McConnell J.P. Rouault T.A. Blood. 2005; 106: 1084-1091Crossref PubMed Scopus (182) Google Scholar, 45Galy B. Ferring D. Minana B. Bell O. Janser H.G. Muckenthaler M. Schümann K. Hentze M.W. Blood. 2005; 106: 2580-2589Crossref PubMed Scopus (182) Google Scholar, 46Meyron-Holtz E.G. Ghosh M.C. Iwai K. LaVaute T. Brazzolotto X. Berger U.V. Land W. Ollivierre-Wilson H. Grinberg A. Love P. Rouault T.A. EMBO J. 2004; 23: 386-395Crossref PubMed Scopus (322) Google Scholar). The high demand for iron of immature erythroid cells may require, however, adaptations in the IRE/IRP network (47Schranzhofer M. Schifrer M. Cabrera J.A. Kopp S. Chiba P. Beug H. Müllner E.W. Blood. 2006; 107: 4159-4167Crossref PubMed Scopus (74) Google Scholar). In addition to being essential for cellular iron homeostasis, the IRE/IRP mechanism is also important in the regulation of systemic body iron levels (40Muckenthaler M.U. Galy B. Hentze M.W. Annu. Rev. Nutr. 2008; 28: 197-213Crossref PubMed Scopus (468) Google Scholar).Transport of Heme Synthesis IntermediatesWith few exceptions, metazoans synthesize heme via eight conserved, enzyme-catalyzed steps using glycine, succinyl-CoA, and ferrous iron as substrates (Fig. 2A). In the heme synthesis pathway, the first and the last three conversions take place in the mitochondria, whereas all remaining steps occur in the cytosol. The intermediates must therefore cross mitochondrial membranes for heme synthesis to progress.FIGURE 2Transport of heme synthesis intermediates and heme in metazoans. A, heme is synthesized via a conserved eight-step pathway involving both mitochondrial and cytoplasmic enzymes. The intermediates ALA, CPgenIII, and PPIX and the substrate glycine need to be transported across mitochondrial membranes for the subsequent reactions. The solute carrier protein SLC25A38 may be involved in translocating glycine into mitochondria. The ATP-binding cassette transporter ABCB6 and the peripheral benzodiazepine receptor (PBR) were proposed to facilitate the import of CPgenIII into the mitochondria, whereas the 2-oxoglutarate carrier (OGC) and the adenine nucleotide translocator (ANT) may play a role in PPIX transport. The mechanisms for the export of ALA and the shuttling of heme precursors among the cytosolic enzymes are unknown. ALAS, aminolevulinic acid synthase; CPOX, coproporphyrinogen oxidase. B, the last step of heme biosynthesis occurs in the mitochondrial matrix. The nascent heme moiety must be translocated across membranes to multiple subcellular compartments where target hemoproteins reside. Heme can also be exported out of the cell or imported into the cell. The cell-surface FLVCR and the ABC transporter ABCG2 have been implicated in heme export in erythroid cells, whereas HRG-1 was identified as a heme importer. The question marks represent the presumptive heme trafficking pathways that are currently unclear. COX, cytochrome c oxidase; Cytb5, cytochrome b5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Aminolevulinic acid synthase catalyzes the first reaction, which is the formation of δ-aminolevulinic acid (ALA) from glycine and succinyl-CoA. For this step, glycine needs to be imported into the mitochondrial matrix; the product ALA needs to be exported to the cytosol. A mitochondrial carrier family protein, SLC25A38, was proposed recently to facilitate glycine import or to exchange glycine for ALA across the IM (48Guernsey D.L. Jiang H. Campagna D.R. Evans S.C. Ferguson M. Kellogg M.D. Lachance M. Matsuoka M. Nightingale M. Rideout A. Saint-Amant L. Schmidt P.J. Orr A. Bottomley S.S. Fleming M.D. Ludman M. Dyack S. Fernandez C.V. Samuels M.E. Nat. Genet. 2009; 41: 651-653Crossref PubMed Scopus (186) Google Scholar). Patients with mutations in this gene manifest a form of nonsyndromic congenital sideroblastic anemia. Yeast lacking the SLC25A38 ortholog displayed decreased levels of ALA, possibly because of the reduced glycine levels in mitochondria. In addition, two mammalian oligopeptide transporters, PEPT1 and PEPT2, were found to transport ALA across the plasma membrane in a pH-dependent manner (49Döring F. Walter J. Will J. Föcking M. Boll M. Amasheh S. Clauss W. Daniel H. J. Clin. Invest. 1998; 101: 2761-2767Crossref PubMed Scopus (244) Google Scholar). Expression of either gene in Pichia pastoris yeast cells or Xenopus laevis oocytes significantly increased the influx of ALA. Further studies may help identify molecules on the mitochondrial membrane that transport ALA.In the cytosol, ALA is converted to coproporphyrinogen III (CPgenIII) in four enzyme-catalyzed reactions. Because the accumulation of heme precursors is toxic to cells and usually causes porphyrias, the product of each reaction has to be quickly and efficiently delivered to the next enzyme. It is unknown how this is achieved.The sixth step in heme synthesis is the oxidative decarboxylation of CPgenIII to generate protoporphyrinogen IX. This reaction is catalyzed by coproporphyrinogen oxidase. The majority of coproporphyrinogen oxidase is present in the mitochondrial intermembrane space (IMS), whereas a small fraction may be loosely attached to the IM (50Grandchamp B. Phung N. Nordmann Y. Biochem. J. 1978; 176: 97-102Crossref PubMed Scopus (73) Google Scholar). In either case, CPgenIII must be transported from the cytosol across the mitochondrial outer membrane (OM). This translocation is suggested to be mediated by the OM ATP-binding cassette transporter ABCB6 (51Krishnamurthy P.C. Du G. Fukuda Y. Sun D. Sampath J. Mercer K.E. Wang J. Sosa-Pineda B. Murti K.G. Schuetz J.D. Nature. 2006; 443: 586-589Crossref PubMed Scopus (740) Google Scholar). ABCB6 was initially identified as a mammalian ortholog of the yeast mitochondrial iron transporter Atm1p (52Mitsuhashi N. Miki T. Senbongi H. Yokoi N. Yano H. Miyazaki M. Nakajima N. Iwanaga T. Yokoyama Y. Shibata T. Seino S. J. Biol. Chem. 2000; 275: 17536-17540Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The expression profile of ABCB6 strongly correlates with that of heme synthesis genes (32Nilsson R. Schultz I.J. Pierce E.L. Soltis K.A. Naranuntarat A. Ward D.M. Baughman J.M. Paradkar P.N. Kingsley P.D. Culotta V.C. Kaplan J. Palis J. Paw B.H. Mootha V.K. Cell Metab. 2009; 10: 119-130Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Overexpression of ABCB6 in K562 cells significantly increased the uptake of 55Fe-heme by mitochondria in an energy-dependent manner (51Krishnamurthy P.C. Du G. Fukuda Y. Sun D. Sampath J. Mercer K.E. Wang J. Sosa-Pineda B. Murti K.G. Schuetz J.D. Nature. 2006; 443: 586-589Crossref PubMed Scopus (740) Google Scholar). A non-physiological CPgenIII-related compound, coproporphyrin III, inhibited both the heme-binding and heme uptake activities of ABCB6. It needs to be confirmed if CPgenIII is a bona fide ABCB6 substrate.The final two steps are the conversion of protoporphyrinogen IX into protoporphyrin IX (PPIX) and the insertion of ferrous iron. The enzymes responsible for these reactions, protoporphyrinogen oxidase (PPOX) and FECH, are associated with the IM. However, the active site of PPOX faces the IMS, whereas the majority of FECH resides in the matrix (Fig. 2A) (53Ferreira G.C. Andrew T.L. Karr S.W. Dailey H.A. J. Biol. Chem. 1988; 263: 3835-3839Abstract Full Text PDF PubMed Google Scholar, 54Harbin B.M. Dailey H.A. Biochemistry. 1985; 24: 366-370Crossref PubMed Scopus (65) Google Scholar). This poses the challenge of delivering the highly reactive PPIX from PPOX to FECH across the IM. A model of substrate channeling between PPOX and FECH has been proposed (53Ferreira G.C. Andrew T.L. Karr S.W. Dailey H.A. J. Biol. Chem. 1988; 263: 3835-3839Abstract Full Text PDF PubMed Google Scholar, 55Koch M. Breithaupt C. Kiefersauer R. Freigang J. Huber R. Messerschmidt A. EMBO J. 2004; 23: 1720-1728Crossref PubMed Scopus (188) Google Scholar). More recently, a co-immunoprecipitation experiment showed that PPOX and FECH physically interact in the cyanobacterium Thermosynechococcus elongates (56Masoumi A. Heinemann I.U. Rohde M. Koch M. Jahn M. Jahn D. Microbiology. 2008; 154: 3707-3714Crossref PubMed Scopus (25) Google Scholar). Thus, the newly produced PPIX may be rapidly transferred from PPOX to FECH through protein-protein interactions.Possible Mechanisms for Heme Transport from Mitochondria to Other OrganellesFree heme has inherent peroxidase activity and can intercalate and disrupt lipid bilayers of cell membranes, resulting in cytotoxicity. How then is heme delivered to the target hemoproteins once it is synthesized in the mitochondrial matrix (Fig. 2B)? A portion of heme may be shuttled from FECH to those hemoproteins in close proximity. For example, a heme-containing enzyme, cytochrome P450scc (P450 cholesterol side-chain cleavage), exhibits a similar localization pattern to FECH in mitochondria (57Cherradi N. Rossier M.F. Vallotton M.B. Timberg R. Friedberg I. Orly J. Wang X.J. Stocco D.M. Capponi A.M. J. Biol. Chem. 1997; 272: 7899-7907Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Therefore, P450scc may acquire heme directly from FECH through protein-pro
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