Associate editor: R.A. Prough
The role of transporters in cellular heme and porphyrin homeostasis

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Abstract

Heme, a complex of iron and protoporphyrin IX (PPIX), senses and utilizes oxygen in nearly all living cells. It is an essential component of various hemoproteins, including those involved in oxygen transport and storage (hemoglobin, myoglobin), electron transfer, drug and steroid metabolism (cytochromes), and signal transduction (nitric oxide synthases, guanylate cyclases). The movement of heme into and within cells was thought to occur by diffusion. However, the chemical properties of heme make diffusion too slow to keep pace with biological processes, and accumulation of heme and its pre-cursor porphyrins in membranes can be deleterious. Due to pro-oxidant effects, heme may cause damage to DNA, proteins, the cytoskeleton and membrane lipids.

The intracellular localization and concentrations of protoporphyrins and heme are tightly regulated, and elevated levels are linked to pathologic conditions (e.g., anemia, lead poisoning, thalassemias) associated with the formation of membrane lipid-damaging, reactive oxygen species. Until recently a mechanism to transport heme and protoporphyrins into organelles of mammalian cells had not been identified.

In this review, we focus on the roles of the recently identified heme/porphyrin transport proteins heme carrier protein 1 (HCP1), FLVCR, Abcg2 and Abcb6 and discuss how these transporters contribute to intracellular heme and porphyrin homeostasis.

Introduction

Heme and porphyrins have fundamental roles in many cellular processes. Heme, the iron and protoporphyrin IX (PPIX) complex, is an important cofactor in processes such as oxygen transport and storage (hemoglobin, myoglobin), mitochondrial electron transport (complexes II–IV), drug and steroid metabolism (cytochromes), signal transduction (nitric oxide synthases, soluble guanylate cyclases), transcription (N-PAS2, Bach I) and regulation of antioxidant-defense enzymes (Ponka, 1999). Heme is also a regulatory molecule; its intracellular localization (cytosolic vs. nuclear) and concentration affects gene transcription and translation (Ponka, 1999); thus, the concentration of heme must be tightly regulated.

Porphyrins give rise to the “pigments of life”: chlorophyll, heme, and vitamin B12. Their unique tetrapyrrole structure enables them to function in an array of reactions, as electron carrier, catalyst for redox reactions, and sensor of gases (e.g., NO, CO). These pigments attach as prosthetic groups to enzymes participating in metabolic processes of cellular respiration, steroid reactions, and metabolism of endogenous compounds and drugs. However, an excess of heme, porphyrins, or both is deleterious to cells. The damaging effect of excess heme is, at least in part, due to its iron-induced prooxidant effect on DNA, proteins, membrane lipids, and the cytoskeleton (Maines and Kappas, 1975, Aft and Mueller, 1983, Aft and Mueller, 1984, Bian et al., 2003). This effect is caused by iron catalyzing the Fenton reaction. An elevated level of non-iron protoporphyrins has been linked to numerous diseases and pathologic conditions (e.g., anemia, lead poisoning, thalassemias; Koenig et al., 1975, Piomelli et al., 1976, Kuross et al., 1988, Sassa and Kappas, 2000, Deacon and Elder, 2001, Magness et al., 2002, Nordmann and Puy, 2002, Gonzalez-Michaca et al., 2004, Atamna and Frey, 2004). The damaging effect of excess protoporphyrins is caused by their ability to absorb energy leading to photosensitization. Photosensitizers, such as protoporphyrins, accumulate in membranes and upon exposure to light (430–635 nm) cause the release of a singlet oxygen which causes cellular damage. Therefore, to prevent porphyrin over accumulation in cells and organelles, transporters are needed to regulate not only the cellular concentrations of these molecules but also their location within the cells.

Transporters move substances across biological membranes and can do so in either one direction or bidirectionally. In general, the energy to initiate transport can be derived from either electrochemical or osmotic gradients or the hydrolysis of ATP. Plasma membrane transporters can either efflux substances to prevent them from entering cells or facilitate their uptake. In addition, efflux transporters also export intracellular substances. Similarly, intracellular organelles have transporters to either extract molecules from the cytosol or release them into the cytosol.

In this review, we describe plasma membrane transporters involved in heme/porphyrin transport and a recently identified mitochondrial heme/porphyrin transporter. The role (s) of these transporters in cellular heme and porphyrin homeostasis will be discussed.

Section snippets

Chemical properties of heme and porphyrin and their movement across membranes

Iron is an essential element for all forms of life and is mostly acquired via the diet. Iron is absorbed in 1 of 2 forms: inorganic (or nonheme iron), which is primarily from nonanimal sources, and organic, which is typically heme and acquired mostly from hemoglobin and myoglobin in animal products. In eukaryotes, dietary heme is more readily absorbed than inorganic iron and contributes significantly to body iron stores. In most cases, dietary heme is the preferred treatment for iron deficiency.

Heme carrier protein 1 transports heme and porphyrin across cell membranes

Several studies have suggested that uptake of heme in the intestine is mediated by proteins on the apical surface that transport heme across biological membranes. Indeed, such a transport protein, heme carrier protein 1 (HCP1), was recently identified in the duodenum (Shayeghi et al., 2005). The duodenal mucosa is the primary site of absorption of dietary heme and iron in mammals. In animal models with congenital hypotransferrinemia (and consequently iron deficiency), anemia results from low

Heme efflux and cellular protection from heme toxicity

The presence of excess heme in cells is highly toxic. Most absorbed heme is probably broken down by heme oxygenase to yield ferrous iron in some cell types such as enterocytes, where there is little or no efflux of intact heme into the circulation. However, in intestinal cell line models, the existence of heme efflux pathways has been described. We speculate that the physiological significance of such secretory systems is to protect cells from the accumulation of excess heme, which might reduce

Intracellular heme biosynthesis and the necessity of a heme/porphyrin carrier

Although the transport processes that mediate the uptake and efflux of heme and porphyrin have been described, whether movement of these molecules in and out of the mitochondria requires a transporter remains unknown. There are biochemical and physiochemical requirements that indicate the need for a transporter. Transmembrane movement of heme and porphyrins is complicated by the unfavorable energetics of moving the porphyrin carboxylate side chains through the lipid bilayer (Fig. 1).

Peripheral benzodiazepine receptor

The peripheral-type benzodiazepine receptor (PBR) was initially identified as a benzodiazepine (diazepam)-binding receptor located in the outer mitochondrial membrane (Braestrup et al., 1977, Gavish et al., 1999, Lacapere and Papadopoulos, 2003). To determine if an endogenous ligand existed tissue extracts were prepared and assayed for their ability to displace radiolabeled benzodiazepines. Analysis of the activity that displaced radiolabeled benzodiazepines revealed that porphyrins were potent

Abcb6, a mitochondrial ATP-binding cassette transporter

Cells from higher eukaryotes express four mitochondrial ABC transporters: Abcb6, Abcb7, Abcb8, and Abcb10. Abcb7 regulates mitochondrial iron homeostasis (Bekri et al., 2000), perhaps by forming iron–sulfur clusters. Abcb10 has a putative role in heme export (Shirihai et al., 2000). The initial cloning and characterization of the mitochondrial half transporter Abcb6, initially termed PRP (Furuya et al., 1997, Emadi-Konjin et al., 2002), UMAT (Hirsch-Ernst et al., 1998), or MTABC3 (Mitsuhashi et

ALAS1 and heme biosynthesis

Heme biosynthesis is regulated by the rate limiting enzyme ALAS which catalyzes the first step in heme biosynthesis (Sassa, 1988)(Fig 3). Two forms of ALAS (1 and 2) from distinct genes exist. ALAS1 is ubiquitously expressed, located in the mitochondrial matrix and modulates the constitutive level of heme required for basic cellular functions (Roberts & Elder, 2001). ALAS1 is also regulated by heme, the end product of heme biosynthesis (Sassa, 1988) and can be induced by treatment with certain

Pharmacological and toxicological relevance:

The biosynthesis and the intracellular concentration of porphyrins and heme are tightly regulated. Disturbance in cellular heme synthesis or metabolism are associated with porphyrias which represent an elevation of toxic heme precursors (protoporphyrins). In many cases the porphyria remains latent until clinical manifestations (phototoxicity, neural or visceral symptoms, elevation in urinary porphyrins) occur. The acute porphyrias occur after pharmocological exposure to chemicals or alcohol

Conclusions

Heme and porphyrins are vital for cell survival, and the movement of heme and porphyrins across intracellular membranes is essential for completing hemoprotein synthesis. Recent discoveries of heme/porphyrin transporters in the cell's membrane and mitochondria have provided insight into the mechanisms conveying heme across membranes (Quigley et al., 2004, Shayeghi et al., 2005, Krishnamurthy et al., 2006). HCP1 is localized on the apical membrane of duodenal epithelial cells and functions as a

Acknowledgments

We thank Angela McArthur for editorial assistance and Julie Groff for preparation of the illustrations. This work was supported by NIH grants CA-21765, CA-77545, and ES-058571 and American Lebanese Syrian Associated Charities (ALSAC).

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