Review Do Mammalian Cells Really Need to Export and Import ?

Prem Ponka,1,2,* Alex D. Sheftel,3,4 Ann M. English,5 D. Scott Bohle,6 and Daniel Garcia-Santos1,2

Heme is a cofactor that is essential to almost all forms of life. The production of Trends heme is a balancing act between the generation of the requisite levels of the Heme is essential for life, but is toxic end-product and protection of the cell and/or organism against any toxic when in excess. Two well-character- substrates, intermediates and, in this case, end-product. In this review, we ized mechanisms keep cellular heme provide an overview of our understanding of the formation and regulation of this at appropriate levels: it is synthesized in quantities that satisfy the require- metallocofactor and discuss new research on the cell biology of heme homeo- ments for the formation of heme pro- stasis, with a focus on putative transmembrane transporters now proposed teins, while the levels of ‘toxic heme’ are maintained at a minimum. to be important regulators of heme distribution. The main text is complemented by a discussion dedicated to the intricate chemistry and biochemistry of Heme toxicity can be prevented by heme, which is often overlooked when new pathways of heme transport are 1 (HO-1), the enzyme that catalyzes heme degradation. conceived. A new concept of heme ‘detoxifica- tion’, based on the hypothesis that Introduction toxic heme can be extruded from cells, Heme1[3_TD$IF]homeostasis is important not only from the point of view of basic science, but also in is emerging. In particular, it was pro- ‘ ’ clinical medicine, since both the deficiency and excess of heme can lead to disease. In this posed that a putative heme exporter on erythroid precursors is required to review, we discuss the literature concerning the prevention of heme-mediated toxicity in protect them from heme toxicity. mammals, with the aim of stimulating discussion of this topic. Heme is essential for most forms of life, but can be toxic unless appropriately shielded. According to ‘classical’ biochem- HO-1, recently shown to be expressed istry, heme toxicity can be prevented by heme oxygenases (HO), enzymes that catalyze heme in erythroid progenitors, can degrade ‘free heme’. This, together with the fact degradation. Over the past decade, a new concept of heme ‘detoxification’, based on the that heme is not detectable in normal hypothesis that toxic heme can be extruded from cells, has emerged. plasma, indicates that these and other cells (by extension) may not need to export heme. Heme, a complex of iron with protoporphyrin IX (PPIX) (Figure 1), serves as the prosthetic moiety of numerous heme that are indispensable for the function of aerobic cells. It is involved in oxygen transport (hemoglobin), oxygen storage (myoglobin), electron transfer (cytochromes), and in gas and redox sensing [1], and is an essential component of innumerable regulatory enzymes, such as soluble guanylyl cyclase and nitric oxide synthase [2], to name just 1Lady Davis Institute for Medical a few. Additionally, heme has important roles, for instance, in the regulation of heme pathway Research, Jewish General Hospital, enzymes in erythroid and nonerythroid cells [3], in neurons, and in circadian rhythms [4]. Montréal, QC, H3T 1E2, Canada 2Department of Physiology, McGill University, Montréal, QC, H3G 1Y6, Despite its numerous physiological functions, the distribution of heme in mammals is uneven Canada 3 (Table S1 in the supplemental[34_TD$IF] information online); in humans, over 95% of heme in the body is Spartan Bioscience Inc., Ottawa, ON, K2H 1B2, Canada found in hemoglobin and myoglobin [5]. Considering the relative contribution of the heme 4High Impact Editing, Ottawa, ON, K1B 3Y6, Canada 5Department of Chemistry and Biochemistry, Centre for Research in 1Heme is ferroprotoporphyrin IX; hemin and hematin are ferriprotoporphyrin IX. In this review, the term ‘heme’ is used Molecular Modeling and PROTEO, as a generic term denoting no particular iron oxidation state. Concordia University, Montréal, QC,

Trends in Biochemical Sciences, May 2017, Vol. 42, No. 5 http://dx.doi.org/10.1016/j.tibs.2017.01.006 395 © 2017 Elsevier Ltd. All rights reserved. compartments to body mass, there is approximately 97 900 mg heme/kg tissue in the H4B 1R, Canada 6Department of Chemistry, McGill hemoglobin compartment (mostly comprising erythrocytes), 120 mg heme/kg tissue in the University, Montréal, QC, H3A 0B8, myoglobin compartment, and only 27 mg heme/kg in all other compartments combined Canada (excluding bone mass from the remaining body weight) [6]. However, this skewed distribution in no way means that heme is less important in nonerythroid, non-muscle tissues [7]. *Correspondence: [email protected] (P. Ponka). Experimental Considerations when Using Heme Heme has a rich chemistry and biochemistry. Among its important reactions are axial iron coordination, aggregation, photochemistry, and reduction and/or oxidation [8]. All of these reactions apply to PPIX complexes with many metals, such as zinc [9], and to metal-free PPIX itself, apart from metal coordination.

In its most accessible form, heme is usually used as hemin chloride (Figure 2 1a) with a paramagnetic Fe(III) ferric ion and an axial chloride. Alternatively, the related hydroxide, hematin (Figure 2 1b), is occasionally used. The first problem encountered when using heme is in dissolving it to make a uniform solution. Not only are hemin and hematin poorly soluble in aqueous buffers under physiological conditions, but they also aggregate in a pH-dependent manner [8]. The most widely used method to solubilize heme is to dissolve it in 0.001–0.01 M sodium hydroxide, which creates two difficulties: (i) under these conditions, heme dimerizes to form an oxygen-bridged dimer with a particularly strong and inert Fe-O-Fe linkage [8] (Figure 2,

2); and (ii) O2 reacts with the vinyl side chains of the dissolved heme and triggers an irreversible cascade of oxidation, resulting in several by-products [8]. Therefore, it is recommended that fresh solutions be prepared for every use and that stock solutions not be prepared and stored. Heme also can be solubilized in dimethyl sulfoxide (DMSO) [10], but even trace water in the solvent will lead to the formation of 2 (Figure 2). Both 1 and 2 aggregate in neutral buffered solutions, as determined by aggregation studies using ultracentrifugation and spectrophotometry [11–13].

In the context of investigating heme in biological experiments, key lessons to be extracted from the literature are that ‘free’ heme is sparingly soluble in neutral aqueous solutions and that it aggregates, precipitates, or partitions into lipid membranes. These obstacles should not be overlooked when considering reports of putative heme transporters. The inconvenient chemistry of heme needs to be considered when attempting to determine total heme flux, localization, chemical environment, and concentration.

Heme Biosynthesis With the exception of free-living and parasitic nematodes [14,15], all animals synthesize heme. The mechanistic aspects of heme biosynthesis have been thoroughly described in numerous review articles and books [16–18]. In vertebrates, the synthesis of heme involves eight enzymes, four of which are cytoplasmic and four are mitochondrial, but all are encoded by nuclear . The first step occurs in the mitochondria and involves condensation of succinyl- CoA and glycine to form ALA, a reaction catalyzed by ALA synthase (ALA-S). The final three steps of the biosynthetic pathway, including the insertion of Fe2+ into PPIX by , occur in the mitochondria (Figure 1) [19]. Although this review is about heme b, there are several structural variants of the heme cofactor (e.g., a, c, and d) in nature [20]. Munro et al. [20] also describe how other heme types are synthesized.

Differences in the genes encoding ALA-S (Box 1) and involved in iron account for the variation in the regulation of the heme synthesis rate in erythroid cells compared with other cells in mammals. The intracellular path of iron from endosomes to mitochondrial ferrochelatase is still obscure or, at best, controversial. The prevailing opinion is that, after its export from endosomes, iron spreads into the cytosol and finds its way to mitochondria by a mechanism

396 Trends in Biochemical Sciences, May 2017, Vol. 42, No. 5 STEAP 3 ur enzym Fo e NAD(P)H s NAD(P)+ TfR e- Hb

Globin + H+ Pump Heme e- STEAP 3 ALA Copro’gen ? DMT1 ? O uter Proto’gen me mb ran ? e CPO PPO Mitoferrin 1 ?

Inne FC r me mb ran PPIX e Glycine Heme

SLC25A38 ? ALA TCA ALAS2 Succinyl–CoA Glycine Cycle

Figure 1. Schematic Representation of Endosomal and Mitochondrial Steps Involved in Iron and Heme Metabolism in Erythroid Cells. Iron is released from transferrin [which binds two atoms of Fe(III)] within endosomes by a combination of Fe3+ reduction by Steap3 and a decrease in pH (pH 5.5). Following this, Fe2+ is transported through the endosomal membrane by DMT1. The postendosomal path of iron in the developing red blood cells remains elusive or is, at best, controversial [19]. In erythroid cells, more than 90% of iron must enter mitochondria, since ferrochelatase (FC), the enzyme that inserts Fe2+ into protoporphyrin IX (PPIX), resides on the inner leaflet of the inner mitochondrial membrane. It has been proposed [18] and documented [21,22] that, in erythroid cells, a transient –endosome interaction is involved in iron translocation to its final destination. Although the identity of the ‘gate’ through which iron enters mitochondria is unknown, it is believed that Fe2+ is carried to FC by mitoferrin 1 [77]. Coproporphyrinogen III (Copro’gen) may be transported into mitochondria by peripheral-type benzodiazepine receptors [78]. Neither the mechanisms nor the regulation of heme transport from mitochondria to globin polypeptides are fully understood. Abbreviations: CPO, coproporphyrinogen oxidase; Hb, hemoglobin; PPO, protoporphyrinogen oxidase; TfR, transferrin receptor. Adapted from [19].

that has yet to be defined. An alternative view is that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires direct interaction between transferrin endosomes and mitochondria [18], a mechanism that is supported by at least some experimental evidence [21,22].

Although the exact mechanism of heme export from mitochondria is far from being fully elucidated, it has been proposed that a carrier , heme-binding protein 1 [23,24] may be involved. Recently, Tolosano’s group provided evidence that FLVCR1b, a mitochondrial isoform of the Feline leukemia virus subgroup C receptor 1 (see below), promotes heme efflux into the cytoplasm of K562 cells [25].

Importantly, the efficient release of heme from mitochondria requires not only a transporter, but also a heme acceptor. This was demonstrated by a report showing that inhibition of translation in erythroid cells led to heme accumulation in mitochondria, and that globin significantly

Trends in Biochemical Sciences, May 2017, Vol. 42, No. 5 397 CH2 HO CH3 CH3 OH N N N Fe N X O CH3 CH CH 3 CH2 2 HO HO CH CH3 O 3 pH > 9 CH N N 2 OH Fe HO CH3 CH3 N N CH Or pyridine N O 3 OH N Fe CH3 CH3 N N HO 1 O CH3 CH 3 CH2 HO 2 (A) X =CI; Hemin , Fe(PP-IX)CI (B) X =OH; Heman, Fe(PP-IX)OH

Figure 2. Structure of Ferriprotoporphyrin IX Compounds of the Heme Family. Usual starting materials are 1a (X = Cl) or 1b (X = OH); dissolution gives oxo-bridged dimer 2. The transformation is promoted by strong base or trace pyridine or chloride concentrations. enhanced heme release from such heme-laden mitochondria [26]. There is also evidence that heme attaches to nascent globin chains growing on polysomes [27].

Heme Degradation The only known physiological mechanism of heme degradation in vertebrates is that performed by heme oxygenase [28], which catalyzes the rate-limiting step in the oxidative degradation of heme and, thereby participates in the control of cellular heme levels. The terms ‘HO-1’ and ‘HO- 2’ were coined in 1986 upon discovery of HO-2 [29] (Figure 3). By far the greatest pool of heme destined for degradation in mammals comes from the hemoglobin of senescent erythrocytes that are phagocytosed by macrophages of the reticuloendothelial system. Most hemoglobin- derived iron, which is liberated from the strongly chelating PPIX ring by heme-inducible HO-1, leaves macrophages through ferroportin (SLC40A1) to the circulation, where it is immediately bound to transferrin. Under steady-state conditions, approximately 25 mg of iron is recycled daily from the hemoglobin of effete erythrocytes. This corresponds to the iron requirement to produce hemoglobin in the 2 million red[36_TD$IF] blood cells that are formed every second [6]. Despite this enormous turnover, heme is not detectable in the plasma of healthy individuals [30].

Heme Detoxification Heme Degradation by Heme Oxygenase 1 As discussed above, in humans and likely all mammals, the highest amount of heme is in the erythron (Table S1 in the supplemental[34_TD$IF] information online). Hence, it is surprising that, until recently, almost nothing was known about the expression of HO-1 in developing red blood cells. This is understandable to some extent since most investigators realized that large quantities of heme are essential for the robust synthesis of hemoglobin.

Box 1. ALA-S Regulation in Erythroid and Nonerythroid Cells Erythroid-specific ALA-S2 [70] is uniquely regulated in erythroid cells. The mRNA for ALA-S2 contains an iron- responsive element (IRE) in its 5 0 untranslated region [71] that allows for the induction of ALA-S2 protein translation by iron. Thus, in erythroid cells, the rate-limiting step in heme synthesis is not the production of ALA (as in nonerythroid cells), but the availability of iron. In developing red blood cells, heme inhibits cellular iron acquisition from transferrin (reviewed in [18]), and serves as a positive feedback regulator that maintains high transferrin receptor levels [72]. Moreover, heme inhibits neither the activity nor the synthesis of ALA-S2. Heme is also essential for both globin transcription [42] and translation [43]. By contrast, in nonerythroid cells, iron uptake is not regulated by intracellular heme and, since the ubiquitous ALA-S1 mRNA does not contain IREs, iron availability does not control the overall heme synthesis rate. Furthermore, heme represses ALA-S1 by decreasing the half-life of its mRNA and/or blocking the entry of the enzyme into mitochondria [73]. Additionally, whereas heme inhibits the import of ALA-S1 into mitochondria, it does not inhibit the mitochondrial import of erythroid-specific ALA-S2 [73].

398 Trends in Biochemical Sciences, May 2017, Vol. 42, No. 5 Heme Figure 3. Schematic Representation of Heme Degradation by heme oxy- CH 2 Heme oxy- CH3 genase 1/2 (HO-1/HO-2). genase is the first and rate-limiting enzyme of the heme degradation path- H3C way that releases iron and yields equimo- N N CH2 lar amounts of carbon monoxide (CO) and Fe biliverdin. Heme cleavage by HO is an N N oxygen-dependent reaction requiring

H3C CH3 seven electrons provided by NADPH/ Heme, NADH-cytochrome P450 reductase. Bili- oxidave stress, verdin is further metabolized to bilirubin by hypoxia, cytokines, the enzyme , which COOH COOH heat shock, can use both NAD(P)H and NADH as cofactors [79]. Iron is recycled in the O R0S,NO,cAMP, 2 organism, whereas both CO and bilirubin NADPH and oxidized lipids are eliminated. Abbreviation: ROS, reac- Cytochrome P450 reductase HO-1(inducible) tive oxygen species. HO-2(constuve) NADP+

CO Iron

Biliverdin NADPH/NADH

Biliverdin reductase

NADP+/NAD+ Bilirubin

It was recently reported [31] that HO-1 is expressed not only in erythroid progenitors, but also in more mature hemoglobin-synthesizing cells. This study demonstrated that hemoglobinization steadily increases during differentiation despite concomitantly elevating levels of HO-1 protein. However, it also showed that overexpression of HO-1 in erythroid cells impaired hemoglobin synthesis, whereas HO-1 absence enhanced hemoglobinization. Hence, it was concluded [31] that HO-1 controls the ‘regulatory’ heme pool at appropriate levels and, thus, has an important role as a coregulator of erythroid differentiation. Since the Km of HO-1 for heme is approximately 1 mM [32], this enzyme will prevent accumulation of heme at toxic levels (>1 mM) [32]. Hence, given that erythroid cells can degrade ‘uncommitted’ (‘regulatory’) heme, we question whether they need to export ‘toxic heme’.

Heme Export from Cells Given the lingering uncertainty in intracellular heme transport (Figure 1), the proposed intercel- lular transport or export of cellular heme is particularly intriguing. Given its hydrophobic nature, protein-bound heme in cells will not spontaneously transfer to the aqueous environment of the cytosol (see the section ‘Experimental Consideration in using Heme’). Additionally, in situation such as intravascular hemolysis, heme per se is not taken up by scavenging cells, but enters them via the receptor-mediated endocytosis of heme-hemopexin complexes (Figure 4, Key Figure). Here, we focus primarily on FLVCR1, the most frequently discussed, putative cellular heme exporter from mammalian cells [33,34]. Nevertheless, much of the discussion here is applicable to other proposed heme and/or transporters that have recently attracted attention in the scientific literature.

Two FLVCR homologs have been identified [35]: FLVCR1 and the FLVCR1-related protein, FLVCR2. Here, we use the term ‘FLVCR1’ in lieu of FLVCR. FLVCR1 was originally cloned as a

Trends in Biochemical Sciences, May 2017, Vol. 42, No. 5 399 Key Figure Mechanisms Involved in the Removal of ‘Toxic Heme’ from the Circulation

Fe Intesnal luman

?

Fe

HO-1

BVFe CO FPN Intesnal cell

Blood vessel

Fe Fe Fe Fe Fe Fe Fe Fe Fe Fe Fe FeFe Fe Fe Fe Haptoglobin Hemopexin Fe Hemoglobin Heme

FPN Heme exportert CD163 CD91

Fe Fe

Fe Fe HO-1 Fe BV Fe Fe HO-1 Fe BV Fe CO Intracellular heme pool? HO-1 Fe Fe Fe Fe CO Macrophage Lysosome Lysosome

Figure 4. Heme is not detectable in the plasma of healthy individuals [30]. However, it is well known that, during intravascular hemolysis, two circulating latent plasma carrier proteins, haptoglobin and hemopexin, bind hemoglobin and heme, respectively. The hemoglobin–haptoglobin complexes bind to the macrophage scavenger receptors CD163, and the heme–hemopexin complexes to the receptor dubbed low-density lipoprotein receptor-related protein/CD91, expressed on macrophages and hepatocytes [80]. This binding is followed by the internalization of both complexes, which is intimately linked to catabolism of the toxic heme moiety via the HO-1 pathway [81]. The exact localization of HO-1 is unknown, but there is a report indicating that heme of senescent erythrocytes is catabolized in erythrophagosomes [82]. Hence, HO-1 could be recruited to lysosomes. Iron derived from heme by HO-1 enters the iron storage protein ferritin or leaves macrophages through ferroportin (FPN) to the circulation, where it is immediately bound to transferrin [19]. Despite these well-documented facts, the idea that exogenous heme can be used for cellular hemoproteins in animals flourishes even in the current literature [83]. It was proposed that a putative ‘heme exporter’ on erythroid precursors is required to protect them from heme toxicity. The hypothesis that expulsion of cellular heme contributes to maintaining appropriate intracellular heme levels is intriguing, but there is no unequivocal experimental evidence supporting it. In fact, as depicted in this figure, elaborate mechanisms have evolved to remove toxic heme from the circulation.

cell surface protein that served as a receptor for feline leukemia virus, subgroup C (FeLV-C) [36]. Cats infected with FeLV-C develop anemia due to the formation of an insufficient number of erythroid precursors [37], primarily hematopoietic colony forming units (CFU-Es) [37]. FLVCR1 is closely related to the organic phosphate antiporter and anion/cation symporter subfamilies,

400 Trends in Biochemical Sciences, May 2017, Vol. 42, No. 5 both being involved in the transport of organic anions [38]. In this context, FLVCR2, a putative heme importer, is also a member of the major facilitator superfamily (MFS), a class of membrane transport proteins that catalyze the movement of small solutes, such as inorganic and organic ions, nucleosides, amino acids, and sugars, across cell membranes [39]. There is evidence, requiring further investigation, that FLVCR2 is a calcium transporter [40].

Although heme does not belong to this category of organic anions, it was hypothesized ‘ . . . that FLVCR functions as a cell membrane export channel (overflow valve), providing a safety mechanism that is uniquely important at the CFU-E stage of differentiation’ [33]. The same group [33] later proposed that heme synthesis accelerates before globin biosynthesis at the CFU-E and/or proerythroblast stage of differentiation [41]. Unfortunately, neither heme nor globin levels nor their rates of production were measured. Only mRNA levels for one of the globin chains (Hbb-bs) and the erythroid specific ALA-S2 were estimated. Several issues make this approach inappropriate: (i) there is a time lapse between the formation of mRNAs and the appearance of their protein products; (ii) mRNAs and enzymes are subject to post-transcrip- tional and post-translational regulation, which does not reflect heme or globin levels at the time of measurement; and (iii) the use of ALA-S2 levels as a surrogate for heme levels is inappro- priate since, after ALA formation, seven additional enzymatic steps occur before the molecule of heme is born.

In the original study by Quigley et al. [33], it was emphasized that ‘FLVCR is required on development of erythroid progenitors to protect them from heme toxicity’. There are at least three facts that challenge this statement: (i) in erythroid cells, heme is required for both the transcription and translation of globin [42,43]; thus, the formation of globin matches that of heme, and there is no evidence to the contrary in healthy individuals. By contrast, it is the overproduction of globin occurring under certain conditions (i.e., in thalassemia [44] or defi- ciency of heme-regulated eIF2a kinase [45]) that can damage erythroid cells; (ii) as mentioned earlier, globin is required for efficient heme release from mitochondria [26] (reviewed by Israels et al. [46]), a finding that directly undermines the hypothesis of Quigley et al. [33]. Hence, it is highly unlikely that prominent levels of heme will accumulate in the cytosol before globin is translated; and (iii) heme enhances the growth of erythroid colonies [burst forming unit-erythroid (BFU-E) colonies that comprise numerous CFU-E colonies] in vitro (e.g., [47,48]). These studies are also in conflict with the above hypothesis, since relatively high concentrations of heme are not toxic for erythroid progenitors.

The studies that suggest a heme transport function for FLVCR1 [33] are based on[37_TD$IF] the initial uptake of zinc mesoporphyrin (a fluorescent probe) or 55Fe-hemin[35_TD$IF] followed by ‘washouts’ of the probes. Despite the claim that both zinc mesoporphyrin and 55Fe-hemin were internalized, no direct evidence for this assertion was provided. It is likely that hemopexin (a plasma heme- binding protein) removes heme adhering to the cell membrane, rather than from the cell interior, as concluded in one FLVCR-related report [49]. This latter report also proposed that FLVCR can export PPIX and coproporphyrin, but there is no known physiological need for the export of these heme precursors from cells. In fact, it is coproporphyrinogen, and not coproporphyrin, that is imported into mitochondria (Figure 1).

As mentioned above, at pH 7.4, free heme hydrolyzes to give a m-oxo dimer (Fe-O-Fe; Figure 2) in which both Fe atoms are pulled out of the heme planar structure, followed by the formation of large aggregates containing approximately 500 heme units [50,51]. Additionally, heme, which has long been known to bind nonspecifically to phospholipid bilayers and to associate with membrane skeletal proteins due to its high hydrophobicity [52], can damage cells. Thus, it cannot be stressed enough that many problems occur when free heme is added to the media of cultured cells. Experiments based on this strategy are unlikely to convincingly

Trends in Biochemical Sciences, May 2017, Vol. 42, No. 5 401 demonstrate a heme-transporting function of FLVCR and other putative heme exporters or importers.

Additional reports are in conflict with the idea that FLVCR1 has a role in heme homeostasis (see Outstanding Questions). For example, Keel et al. [53] examined the expression of FLVCR1 in various tissues and demonstrated high levels of its transcript in the kidney, lung, uterus, brain, and placenta (as discussed later, hemochorial placentas of humans and mice do not transport heme). This pattern deviates from the distribution of heme in the organism (Table S1 in the supplemental information online) and is inconsistent with FLVCR1 being required for the export of excess heme.

Recently, Tolosano’s group identified an alternative transcription start site that gives rise to the FLVCR1 isoform, FLVCR1b [25]. The FLVCR1b transcript lacks the first exon of the canonical isoform (FLVCR1a) and is ubiquitously expressed in various tissues. FLVCR1a is localized at the cell membrane, whereas FLVCR1b is present in mitochondria and may be involved in heme transport from these organelles [25]. Embryonic day (E)13.5 FLVCR1a-null mouse embryos showed multifocal and extended hemorrhages that were visible in the limbs, head, and throughout the body [25]. Flow cytometric analyses of E14.5 fetal liver cells double-stained for Ter119 (an erythroid-specificantigen)andCD71(atransferrin receptor) showed normal erythropoiesis in FLVCR1a-null embryos. This study, as well as high FLVCR1a expression primarily in primitive erythroid cells (Figure S1 in the supplemental[34_TD$IF] information online), renders the heme-transporting function of this protein dubious.

ABCG2 [a.k.a. breast cancer resistance protein (BCRP)] functions as a xenobiotic transporter that confers resistance to various anticancer drugs [54]. The first potential link between BCRP/ ABCG2 and a tetrapyrrole emerged from the study by Jonker et al. [55], which reported that PPIX accumulated in the erythrocytes of Bcrp1–/–[34_TD$IF]mice. The authors suggested that this accumulation is caused by the defective export of PPIX from erythrocytes, but it is difficult to understand the need for a PPIX-transporting system. Under normal conditions, almost all PPIX generated in hemoglobin-synthesizing cells is converted into heme. Excess PPIX occurs only in pathological situations, such as ferrochelatase defects or lead poisoning [56]. Zhou et al. confirmed that Bcrp1–/– mice have increased levels of PPIX in erythroid cells [57] and proposed that BCRP/ABCG2 has a role in regulating PPIX. However, Zhou et al. treated cells with ALA to increase PPIX, a strategy that likely obscured the results. Additionally, normal human eryth- rocytes contain 27 000-times less PPIX than heme [58] since almost all synthesized PPIX is converted to heme. Inexplicably, the focus on BCRP/ABCG2 has shifted from PPIX transport to heme transport, which has received more attention in review articles [34,59] compared with the original research.

The lack of compelling experimental evidence has not dampened enthusiasm for descrip- tions of heme export. Some schemes even show FLVCR and BCRP/ABCG2 next to each other, implying a necessity for the cell to eliminate heme [34,60]. These diagrams also depict one or two cellular heme importers, such as heme carrier protein 1 (HCP1). This protein is, in fact, the proton-coupled folate transporter, PCFT [61] in intestinal cells. The concept of heme import is further confounded by the proposition that, following its transport to the cytosol, ‘heme is inserted in apo-hemoproteins to allow the formation of functional hemo- proteins’ [34]. Nothing is known of heme trafficking between proteins within mammalian cells; thus, to assume that apo-hemoproteins reside in the cytosol waiting for imported heme is mere speculation (Figure 4). Of relevance in this context is the seminal study by Rothstein and colleagues [14], who examined the heme auxotroph Caenorhabditis briggsae (Box 2).

402 Trends in Biochemical Sciences, May 2017, Vol. 42, No. 5 Box 2. Heme Transporters in Free-Living Nematodes Outstanding Questions Caenorhabditis briggsae, a free-living nematode, requires cytochrome c or hemoglobin as a heme source for growth What are the physiological substrates fi fi fi and reproduction [14]. This discovery led to the identi cation of the rst and, so far, the only clearly de ned heme and roles of FLVCR1a and FLVCR2? transporters in another heme auxotroph, Caenorhabditis elegans. These transporters are encoded by the heme responsive genes, hrg1 ([15]; the only hrg expressed in mammals), hrg2 [74], hrg3 [75], and hrg4 [15]. The Does SLC48A1 have (an) identical mammalian HRG1 gene product (SLC48A1) has been proposed to have a role in macrophage iron homeostasis, function (s) as the product of the transferring heme from the phagolysosome to the cytoplasm during erythrophagocytosis [76], but heme transport was HRG1 gene? not directly assessed. It is unknown whether SLC48A1 has an identical function as the product of the hrg1 gene (see Outstanding Questions). What is the fate of heme, and its iron, following the turnover of cytosolic and mitochondrial hemoproteins? Lastly, none of the reports that allegedly document the capacity of a cell to purge itself of ‘ ’ toxic heme consider what ALA-S isoform is used in heme synthesis. This is an important What are the mechanisms by which issue since, as pointed out in the section ‘Heme Biosynthesis’, heme feedback inhibits the defects in FLVCR1 cause PCARP? ubiquitous ALA-S1 by decreasing the half-life of its mRNA and/or blocking its entry into mitochondria. Thus, in cells containing ALA-S1, heme is synthesized in quantities that satisfy What are the mechanisms by which the requirements for the formation of heme proteins, while the levels of ‘toxic heme’ are defects in FLVCR2 cause PVHH or encephaloclastic proliferative vascul- maintained at a minimum. opathy (Fowler syndrome)?

Perspectives for Future Research The hypothesis that expulsion of cellular heme contributes to maintaining appropriate intracel- lular heme levels and protects cells from heme toxicity is intriguing, but remains unproven. In our opinion, there is only one way to directly test this hypothesis: pulse-label cells with radioactive heme precursors and demonstrate that labeled heme is released from cells into media containing a heme-binding substance. One could use radioactive iron (59Fe or 55Fe) bound to transferrin, or [2-14C]glycine or 5-amino [3H]levulinic acid. Working with the latter precursor would be technically less demanding, since 5-amino [3H]levulinic acid is incorporated only into heme. Demonstration that the inhibition of HO-1 during the pulse and ‘release’ periods enhanced heme release would strengthen the hypothesis of cellular heme export. By contrast, the presence of a heme synthesis inhibitor (e.g., 4.6-dioxoheptanoic acid) during both periods would be expected to inhibit the release of heme from cells. These experiments should be performed using cells expressing different levels of FLVCR1 or BCRP/ABCG2. The movement of metabolic intermediates and their end-products can be reliably traced only if they are radioactively labeled. This strategy cannot be substituted by the current indirect approaches that measure mRNA or protein levels of putative transporters.

Concluding Remarks There is no question that FLVCR1 is a transporter (see Outstanding Questions) but, as discussed above, its role in heme transport has not been demonstrated beyond reasonable doubt. The only known human disorder caused by defects in FLVCR1 is posterior column ataxia with retinitis pigmentosa (PCARP), which is a rare autosomal-recessive disorder char- acterized by the dysfunction of two specific sensory modalities, vision and proprioception [62]. The lack of hematological abnormalities [63], as well as the organismal heme distribution (Table S1 in the supplemental information online), do not support the view that FLVCR1 is a transporter of heme.

There is no evidence to support the assertion that heme is transported into mammalian cells for insertion into apo-hemoproteins. A vast literature shows that free hemin, being hydrophobic, passes through the cell membrane and, in concentrations as low as 5 mM, induces HO-1 [6], which will destroy it. Hence, the proposal that FLVCR2, a homolog of FLVCR1, imports heme to cells not only is implausible, but also has no solid experimental support (see Outstanding Questions). By contrast, concentrations of hemin as high as 200 mM were needed to selectively decrease the survival of FLVCR2-overexpressing cells [64], which would suggest that this putative transporter in fact retards heme transport.

Trends in Biochemical Sciences, May 2017, Vol. 42, No. 5 403 FLVCR2 (Mfsd7c) is not significantly expressed in erythroid cells, but exhibits high expression in the placenta2[38_TD$IF]4and, in particular, the retinal pigment epithelium [66]. Defects in FLVCR2 (see Outstanding Questions) cause proliferative vasculopathy and hydranencephaly-hydrocephaly (PVHH) or encephaloclastic proliferative vasculopathy, also called Fowler syndrome, which exclusively affects the central nervous system [67]. This, together with the organismal distri- bution of heme (Table S1 in the supplemental[34_TD$IF] information online), renders a heme-transporting function for FLVCR2 unlikely.

As mentioned above, one alleged heme transporter (HCP1) was later found to be a folate transporter [61]. Additionally, a close relative of FLVCR1, the cell surface entry receptor for FeLV subgroup A, is a thiamine transporter [68] (THTR1, SLC19A2). Hence, it is not unreasonable to expect that the physiological substrates for FLVCR types of transporter will be hydrophilic (see Outstanding Questions).

In mammalian cells, the membranous transport of heme is necessary during its export from mitochondria (for cytosolic hemoproteins), its import into the nucleus (for transcriptional regulation), as well as its import into duodenal epithelial cells (Figure 4). As discussed above, the molecular identities of these transporters are not known with certainty at present; hence, further research is needed to unequivocally characterize them. We hope that our review will encourage researchers to seek the bona fide, heme or nonheme substrates of putative heme transporters. This will be a formidable task.

We feel compelled to comment on one remarkable aspect of the regulation of HO-1 expression by heme. The negative transcriptional regulator Bach1 binds to heme-responsive elements in the HO-1 promoter. When in excess, heme binds to Bach1 and induces a conformational change in the regulator that decreases its DNA-binding activity, allowing Nrf2-Maf and other activating heterodimers to occupy the heme-responsive elements in the HO-1 promoter and increase transcription and expression of HO-1 [69]. This elegant regulatory mechanism con- trolling HO-1 expression prevents heme accumulation as well as its deficit, thus raising the question why heme export or import is needed to maintain appropriate intracellular heme levels. The added advantage of such transport pathways over the fine-tuning of heme concentration provided by ALA-S1 and HO-1 is not immediately obvious. Furthermore, mechanisms that control the regulation of expression of the putative heme transporters have not emerged.

In summary, our knowledge of the biology of heme in mammalian cells significantly lags behind that of its biochemistry and chemistry. Future investigations should carefully consider how phenotypic changes correlate with putative transporter defects, with the physicochemical properties of potential substrates, and with the biochemical pathways rigorously described in textbooks. Such studies will bring us closer to an understanding of the cellular and extracellular (or lack thereof) compartmentalization of heme and, hence, of heme homeostasis.

Acknowledgments The authors thank Amel Hamdi for reading the manuscript and her valuable comments and advice. The[39_TD$IF] authors are indebted to Dr. Volker Blank for helpful discussions regarding the regulation of HO-1 expression. P.P. and D.G.S. are supported by grants from the Canadian Institutes of Health Research. A.E.M. is supported by Le Fonds de recherche du Québec – Nature et technologies and The Natural Sciences and Engineering Research Council of Canada. D.S.B. and A. M.E. are supported by the Natural Sciences and Engineering Research Council of Canada and D.S.B. is additionally supported by the Canadian Research Council.

2Humans, mice, other rodents, and some other animals have a hemochorial type of placenta, in which the tropho- blast cells are bathed directly by maternal blood. This allows the direct contact of maternal plasma transferrin with trophoblasts that acquire iron by a mechanism similar to that by which most other cells in the body obtain iron [65]. Hence, heme has no role in iron delivery to the fetuses of animals with hemochorial-type placentas.

404 Trends in Biochemical Sciences, May 2017, Vol. 42, No. 5 Supplemental[340_TD$IF] information Supplemental information associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. tibs.2017.01.006.

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