Skip Navigation


Human Molecular Genetics Advance Access originally published online on February 8, 2006
Human Molecular Genetics 2006 15(6):953-964; doi:10.1093/hmg/ddl012
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Material
Right arrow All Versions of this Article:
15/6/953    most recent
ddl012v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (17)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Pondarré, C.
Right arrow Articles by Fleming, M. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pondarré, C.
Right arrow Articles by Fleming, M. D.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The mitochondrial ATP-binding cassette transporter Abcb7 is essential in mice and participates in cytosolic iron–sulfur cluster biogenesis

Corinne Pondarré1, Brendan B. Antiochos1,{dagger}, Dean R. Campagna1,{dagger}, Stephen L. Clarke2, Eric L. Greer1, Kathryn M. Deck2, Alice McDonald3, An-Ping Han1, Amy Medlock4, Jeffery L. Kutok5, Sheila A. Anderson2, Richard S. Eisenstein2 and Mark D. Fleming1,*

1Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA 02115, USA, 2Department of Nutritional Sciences, University of Wisconsin, Madison, WI 53706, USA, 3Millennium Pharmaceuticals, Cambridge, MA 01239, USA, 4Department of Biochemistry and Molecular Biology, The Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA and 5Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA

* To whom correspondence should be addressed at: Department of Pathology, Children's Hospital and Harvard Medical School, Enders 1116.1, 320 Longwood Avenue, Boston, MA 02115, USA. Email: mark.fleming{at}childrens.harvard.edu

Received December 7, 2005; Accepted February 1, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Proteins with iron–sulfur (Fe–S) clusters participate in multiple metabolic pathways throughout the cell. The mitochondrial ABC half-transporter Abcb7, which is mutated in X-linked sideroblastic anemia with ataxia in humans, is a functional ortholog of yeast Atm1p and is predicted to export a mitochondrially derived metabolite required for cytosolic Fe–S cluster assembly. Using an inducible Cre/loxP system to delete exons 9 and 10 of the Abcb7 gene, we examined the phenotype of mice deficient in Abcb7. We found that Abcb7 was essential in extra-embryonic tissues early in gestation and that the mutant allele exhibits an X-linked parent-of-origin lethality effect. Furthermore, using X-chromosome inactivation assays and tissue-specific deletions, Abcb7 was found to be essential for the development and function of numerous other cell types and tissues. A notable exception to this was liver, where loss of Abcb7 impaired cytosolic Fe–S cluster assembly but was not lethal. In this situation, control of iron regulatory protein 1, a key cytosolic modulator of iron metabolism, which is responsive to the availability of cytosolic Fe–S clusters, was impaired and contributed to the dysregulation of hepatocyte iron metabolism. Altogether, these studies demonstrate the essential nature of Abcb7 in mammals and further substantiate a central role for mitochondria in the biogenesis of cytosolic Fe–S proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mitochondria play a central role in cellular iron metabolism. Not only do the initial and final steps of heme biosynthesis occur in mitochondria, so does the biosynthesis of iron–sulfur (Fe–S) clusters, which perform essential structural and catalytic roles in many mitochondrial enzymes (1Go–3Go). Studies in yeast have shown that the mitochondrial Fe–S cluster synthesis machinery also has an essential function in providing a component needed for extramitochondrial Fe–S protein assembly (4Go,5Go). In fact, the yeast requirement for mitochondria depends not on their function in energy metabolism, but instead is due to their necessity in forming an essential cytosolic Fe–S protein, Rli1p, involved in ribosome biogenesis (6Go,7Go). Although yeast is a genetically flexible system for modeling mammalian mitochondrial Fe–S cluster metabolism, sparingly little of this work has been directly validated in mammalian cells. The primary exception being the study of Friedreich ataxia, which is a neurodegenerative disorder due to mutations in a mitochondrial protein, frataxin, that influences mitochondrial Fe–S cluster synthesis (8Go,9Go). Nonetheless, the particular role and the extent to which extramitochondrial Fe–S clusters contribute to this or other human diseases has, for the large part, not been investigated.

Studies in yeast also indicate that there is a complex relationship between Fe–S cluster assembly pathways and iron metabolism as a whole. For instance, disruption of Fe–S cluster biogenesis in mitochondria impairs heme formation in yeast by inhibiting the activity of ferrochelatase (10Go). Furthermore, Aft1p and Aft2p, iron-regulated transcription factors controlling yeast iron homeostasis, respond not to cytosolic iron, but to the rate of mitochondrial Fe–S cluster synthesis (11Go–13Go). Similarly, in mammals, the action of iron regulatory protein 1 (IRP1), a cytosolic modulator of iron homeostasis, is controlled in part by an Fe–S cluster-dependent mechanism (14Go,15Go). However, how perturbations in cytosolic Fe–S cluster assembly alone directly affect IRP1 function and influence cellular and systemic mammalian iron metabolism have not been explored.

In yeast, Atm1p, an ATP-binding cassette (ABC) transporter of the inner mitochondrial membrane, is required for maturation of cytosolic Fe–S apo-proteins to their holo-forms (4Go). Consequently, Atm1p links the mitochondrial and cytosolic pathways for Fe–S cluster assembly, presumably by mediating the transport of a component required for cytosolic Fe–S cluster assembly from the mitochondria to the cytosol. Yeast with chromosomal deletions in ATM1 ({Delta}atm1) develops mitochondrial iron overload, which can be fully rescued by the human ortholog ABCB7 (16Go–19Go). Mitochondrial iron overload resembling the {Delta}atm1 phenotype is a feature of several human diseases, most notably sideroblastic anemias (20Go). An unusual form of X-linked sideroblastic anemia, syndromically associated with ataxia and severe developmental hypoplasia of the cerebellum [X-linked sideroblastic anemia with ataxia (XLSA/A)] has been described in four families and is due to mutations in ABCB7 (16Go,21Go–25Go). Each of the three-recorded disease-causing alleles is a missense mutation in exon 9 or 10 of the gene. In order to further explore the function of mammalian Abcb7 and the link between mitochondrial and cytosolic Fe–S cluster biogenesis and cellular iron metabolism, we have created a conditionally targeted allele of the gene in mice. Here, we report that Abcb7 is an essential gene in mice and, like its yeast counterpart, participates in the maturation of cytosolic Fe–S cluster proteins in mammals.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Abcb7 is an essential gene in mice associated with X-linked parent of origin lethality
We targeted a loxP site and a loxP-flanked neomycin-resistance cassette (NeoR) into introns 8 and 10 of Abcb7 of embryonic stem (ES) cells to create a conditionally targeted allele flanked by loxP sites (‘floxed’ allele, Fig. 1A and B). Transient transfection of correctly targeted ES clones harboring the floxed allele (including the NeoR cassette, Fig. 1C) with a plasmid encoding Cre-recombinase readily yielded neomycin-sensitive subclones, but none of these >50 colonies contained a null allele in which exons 9 and 10 were also deleted (Fig. 1D). As the ES cells are karyotypically male (i.e. 40, XY and hemizygous for the X-linked Abcb7gene), and we were able to obtain deletion of exons 9 and 10 in vivo (Fig. 1E and discussed subsequently), we conclude that Abcb7 is an essential gene in ES cells cultured in vitro.


Figure 0121
View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. Abcb7 gene targeting. (A) The endogenous wild-type Abcb7 locus. (B) ES cells were transfected with a targeting construct that introduces a neomycin-resistance cassette flanked by loxP sites into intron 10 and a solitary loxP site into intron 8. (C) Neomycin resistant clones were analyzed for homologous recombination by Southern blot using an external 5' (exon 5) probe and BglII and a 3' probe (exon 14) and KpnI. (D) Transient transfection with a Cre recombinase expression plasmid yielded subclones lacking the neomycin resistant cassette. (E) Rearrangement to the null allele lacking exons 9 and 10 could be obtained in vivo in female animals carrying a Cre transgene.

 
Several independently targeted ES cell clones were injected into blastocysts and gave rise to chimeric males that transmitted the modified Abcb7 allele through the germline. Mice hemi- and homozygous for the NeoR-less floxed allele (Fig. 1D, Abcb7fl/Y and Abcb7fl/fl, respectively) on mixed 129S substrain, and 129S6/SvEvTac (N4) and C57BL/6J (N8) congenic backgrounds, were viable and fertile and had no grossly visible neurological phenotype or measurable hematological abnormality (data not shown). In order to obtain a germline null allele, we crossed chimeric 129S4/SvJae Abcb7fl/Y males to a Cre-recombinase transgenic (Tg) line, FVB-Tg(Gata1-Cre), which typically behaves as a generalized deleter strain (26Go), to obtain [FVBx129]F1 females nominally heterozygous for an Abcb7 null allele (Abcb7+/–, Supplementary Material, Fig. S1A). Breeding these females to wild-type males yielded neither live born male nor female pups with a germline null allele (Table 1). Similarly, the reciprocal intercross mating, in which an Abcb7fl/fl female was crossed to a Gata1-Cre male produced no animals with a germline null allele (Supplementary Material, Fig. S1B; Table 1). In both crosses, several females and males bearing the conditional allele with no or partial somatic deletion were born alive; in these cases, rearrangement was essentially limited to the bone marrow of Cre-positive female animals, consistent with the preferential bone marrow deletion in animals escaping early embryonic deletion previously reported with this Cre transgene (26Go). Embryonic dissections indicated that both male and female Cre-positive embryos died at or prior to E6.5–E7.5 (Table 2). Histological examination revealed slightly growth-retarded, inviable embryos, typically with fibrin and hemorrhage in the region of the ectoplacental cone, but without other distinctive histological features or histochemical evidence of excess iron deposition (Fig. 2 and data not shown). Notably relevant to XLSA/A, yolk sac hematopoietic progenitors could be identified histologically (Fig. 2C).


View this table:
[in this window]
[in a new window]
  		Table 1. Global deletion of Abcb7 leads to X-linked parent of origin lethality: weaned animal analysis
 

View this table:
[in this window]
[in a new window]
  		Table 2. Global deletion of Abcb7 leads to X-linked parent of origin lethality: embryonic dissection analysis
 

Figure 0122
View larger version (149K):
[in this window]
[in a new window]
  		Figure 2. Abcb7 deficiency leads to early-mid-gestational death. H&E stained tissue sections of [Abcb7fl/flxGata1-CreTg]F1 embryos, without (A) and with (B) Gata1-Cre. (C) Higher magnification of (B). Black arrowhead indicates embryo, gray arrowhead yolk sac hematopoietic progenitors and asterisk hemorrhage and fibrin in the area of the ectoplacental cone.

 
These data indicate that Abcb7 is essential for mouse development and provided evidence for a parent of origin effect of expression of the gene; inheritance of either a germline null allele from the female or a conditional allele and a Gata1-Cre transgene from the female and male parents, respectively, resulted in death whether the embryo was a hemizygous Abcb7fl/Y male or a heterozygous Abcb7fl/+ female. Furthermore, this specifically implicated an abnormality in the extra-embryonic tissues, as the female-derived X-chromosome is preferentially active in the extra-embryonic tissues of eutherian mammals (27Go).

To further explore the likelihood of an extra-embryonic defect, we crossed Abcb7fl/fl females to males hemizygous for a Sox2-Cre (Supplementary Material, Fig. S1C) transgene, which drives Cre expression only in the embryonic epiblast, sparing many of the extra-embryonic tissues (28Go); we also performed a cross with males hemizygous for a Villin-Cre transgene (Supplementary Material, Fig. S1D), which is selectively expressed in ciliated cells in the extra-embryonic visceral endoderm (29Go,30Go). In toto, these Cre transgenic lines produce nearly complementary, non-overlapping patterns of deletion in the embryo and extra-embryonic tissues. Consistent with our hypothesis, using the Sox2-Cre line, we were able to obtain healthy live-born females that appeared to have fully rearranged the single conditional allele inherited from their mother (Table 3 and data not shown). As with the Gata1-Cre transgene, these nominally Abcb7+/– females were unable to transmit the null allele to live-born progeny (data not shown). Dissection of embryos from Abcb7fl/flxSox2-Cre pairings revealed that transgene-positive Abcb7fl/Y males died at E7.5–8.5, slightly later than in the Gata1-Cre crosses, indicating that sparing the extra-embryonic defect is succeeded by a lethal Abcb7-dependent embryonic abnormality in early-mid gestation. Furthermore, and supportive of the notion that the extra-embryonic tissues are the cause of the early lethality, we observed no live born Villin-Cre positive males or females in crosses between Abcb7fl/fl females and Villin-Cre males (Supplementary Material, Fig. S1D; Table 3). Taken together, these data demonstrate that Abcb7 is an essential gene in mice because of a requirement for Abcb7 in the extra-embryonic tissues early in gestation.


View this table:
[in this window]
[in a new window]
  		Table 3. Abcb7 is required in the extra-embryonic tissues
 
Abcb7 is required for the development or maintenance of multiple adult cell lineages
In order to evaluate the role of Abcb7 at later stages in development, we bred the Abcb7fl allele to several tissue-specific or inducible Cre transgenic lines. We were particularly interested in the development of the central nervous system (CNS) and hematopoietic system, as these tissues are clinically affected in the human disorder XLSA/A. Inducible deletion in the bone marrow with MX1-Cre (31Go) led to bone marrow failure (C. Pondarré and M.D. Fleming, unpublished data). Nestin-Cre mediated CNS deletion (32Go) resulted in no gross abnormalities in brain development, but proved to be lethal in the immediate perinatal period (Table 4). As cerebellar development occurs primarily after birth in mice, the nature of the cerebellar dysgenesis seen in XLSA/A patients could not be evaluated.


View this table:
[in this window]
[in a new window]
  		Table 4. Abcb7 is essential in the central nervous system
 
Given the failure of the targeted, tissue-specific approach to produce viable animals in which we could study a biochemical phenotype, we sought to simultaneously evaluate the requirement for Abcb7 in multiple tissues. In order to do so, we examined X-inactivation patterns in female mice harboring a paternally inherited Abcb7fl allele in cis with a ‘ubiquitously’ expressed X-linked green fluorescent protein (GFP) transgene (33Go) in the presence or absence of a maternally derived Gata1-Cre transgene. In female animals without Cre, X-inactivation should be stochastic, and GFP should be expressed in a variegated pattern in any tissue in which the promoter is active. In contrast, in the presence of Cre, GFP should not be expressed tissues in which Abcb7 is essential at any point in development or for maintenance of that cell lineage. We found that in heterozygous females lacking a Cre transgene, GFP protein was expressed in a variegated pattern, consistent with random X-inactivation, in most tissues (Fig. 3A). Notably, however, GFP was not expressed in most bone marrow cells, as well as lymphoid cells in the thymus and spleen, and present in only a minor subset of hepatocytes; the inactivity of the promoter precluded X-inactivation analysis in these tissues. However, in the presence of Cre, essentially all organs lacked GFP-positive parenchymal cells, providing evidence that Abcb7 is essential for the development and/or maintenance of numerous cell types (Fig. 3B and C). This result is congruous with the widespread expression of Abcb7 in embryonic and adult murine tissues (Fig. 3F and G). Remarkably, capillary endothelial cells in many tissues, including glomerular tufts in the kidney, retained the variegated pattern of expression regardless of Cre status (Fig. 3D and E), suggesting that Abcb7 is not essential in this lineage.


Figure 0123
View larger version (67K):
[in this window]
[in a new window]
  		Figure 3. X-inactivation and RNA expression patterns indicate that Abcb7 is essential in multiple tissues. (A) GFP immunostaining in pancreas: [Gata1-CreTg/+xAbcb7fl-GFP/Y]F1 females without Cre show random inactivation of the GFP-tagged Abcb7fl chromosome as demonstrated by a mixture of cells positive and negative for GFP marking an active conditional allele. (B) [Gata1-CreTg/+xAbcb7fl-GFP/Y]F1 females with Cre showed no staining in pancreatic parenchymal cells. (C) Table summarizing GFP immunohistochemistry X-inactivation assay results. Values reflect number of animals positive for GFP in that tissue/total number examined. (Asterisk) A small subset of hepatocytes was positive in four of six Cre-positive animals analyzed. Glomerular tufts from (D) Gata1-Cre negative and (E) Gata1-Cre positive Abcb7fl-GFP/+ females showing persistence of GFP positivity and presumptive activity of the null allele in endothelial cells even in Cre-positive animals. (F) Northern blot of Abcb7 in adult tissues. (G) In situ hybridization of E15.5 mouse embryo demonstrating widespread expression of Abcb7 with high-level expression present in the neuroectoderm of the forebrain and the fetal liver.

 
Hepatocyte-specific Abcb7 deletion disturbs cellular and systemic iron metabolism
Although the GFP transgene was expressed only in a minor subset of hepatocytes in animals lacking Cre, in the presence of Cre, GFP staining persisted in a fraction of these cells (data not shown). This suggested that the active X-chromosome in these cells expressed the modified Abcb7 allele. In order to investigate this more thoroughly, we bred the conditional deletion allele to a hepatocyte-specific Cre-transgenic line: B6.Cg-Tg(Alb-Cre)21Mgn/J [Alb-Cre]. The albumin promoter driving Cre expression in this line is generally not substantially active until after birth, with complete hepatocyte deletion not occurring until 4–6 weeks after birth (34Go and data not shown). Male animals carrying the Abcb7fl allele and Alb-Cre will be subsequently referred to as Abcb7lv/Y to reflect the hepatocyte-specific deletion.

Abnormal iron metabolism in XLSA/A and in yeast deficient in Atm1p ({Delta}atm1) lead us first to examine systemic iron parameters in Abcb7lv/Y mice. We found that the serum iron and total iron binding capacity (TIBC) were unchanged, however, the transferrin saturation was significantly increased in knockout animals (Fig. 4A). In order to investigate the cause of this difference, we examined liver iron parameters and found that total liver iron was increased by 76% in the mutants (Fig. 4B). As XLSA/A and {Delta}atm1yeast are specifically associated with increased mitochondrial iron, it was somewhat surprising that the increase in liver iron was not associated with mitochondrial iron overload (Fig. 4B). Liver iron loading was accompanied by a trend (P=0.07) toward a slight increase in ferritin protein and a co-existent, apparently paradoxical, rise in transferrin receptor-l (TfR1) protein (Fig. 4C and discussed subsequently). Perl's Prussian blue iron stain showed a small subset of hepatocytes, typically located adjacent to a portal triad or central vein, that contained abundant, coarsely granular cytosolic iron deposits, often with a central clearing, and that were morphologically distinct from ferritin (Fig. 4D). Electron microscopy showed that these structures were electron-dense rings with a homogenous center morphologically typical of neutral lipid (Fig. 5B and D). We also observed numerous cytosolic lipid droplets and pale, swollen mitochondria suggestive of metabolic and/or mitochondrial injury (Fig. 5B and D); mitochondria with electron-dense deposits typical of iron were not present. Consistent with the impression of mild hepatocellular injury, Abcb7lv/Y livers also showed mild hepatic architectural disarray and hepatocellular multinucleation on routine hematoxylin and eosin (H&E) tissue sections (data not shown). Limited, ongoing liver damage was further substantiated by a 2-fold increase in serum levels of liver-derived transaminases (data not shown).


Figure 0124
View larger version (51K):
[in this window]
[in a new window]
  		Figure 4. Abnormal iron metabolism in Abcb7lv/Y animals. (A) Liver total and mitochondrial iron (n=5), (B) serum iron, TIBC, and transferrin saturation (n=5) and (C) tissue ferritin and transferrin levels (n=5) were determined in wild-type (open bars) and Abcb7lv/Y mice (black bars). A significant (P<0.05) difference between groups is indicated with an asterisk. P=0.07 for ferritin. (D) Perl's Prussian blue stain of Abcb7lv/Y animal showing periportal hepatocellular iron deposition with characteristic round structures with central clearing (arrow).

 

Figure 0125
View larger version (182K):
[in this window]
[in a new window]
  		Figure 5. Electron microscopy of Abcb7 liver-specific deletion. (A and C) Wild-type and (B and D) Abcb7lv/Y livers. Pale, swollen mitochondria, lipid vacuoles and cytoplasmic, electron-dense circular inclusions are present in the mutant.

 
Hepatocellular deletion of Abcb7 impairs the activity of cytosolic but not mitochondrial Fe–S proteins
Because the yeast ortholog of Abcb7, Atm1p, is required for formation of Fe–S clusters in the cytosol, but not in the mitochondria (4Go), we determined the activity of Fe–S enzymes in wild-type and Abcb7lv/Y mice. Ferrochelatase is a mitochondrial Fe–S enzyme in the heme biosynthetic pathway. We found that its activity was not different between wild-type and Abcb7lv/Y mice (Fig. 6A). The activity of mitochondrial aconitase, a Fe–S enzyme in the tricarboxylic acid cycle, was slightly increased in the mutant animals (Fig. 6A). Activity of succinate dehydrogenase (SDH), another mitochondrial Fe–S enzyme that participates in the electron transport chain (complex II), was reduced by 20% in mutants when normalized to the activity of cytochrome C oxidase (CCO) (complex IV) (35Go). As in liver, the activity of complex I is one-half of complex II, the minor decrease in complex II activity observed here is not likely to limit electron transport chain flux, at least when NADH is the source of electrons (36Go). Overall, deletion of Abcb7 had little impact on hepatocyte mitochondrial Fe–S enzyme activity. In contrast, the activity of the cytosolic Fe–S protein xanthine oxidase (XO), which also contains a molybdenum cofactor prosthetic group, whose biosynthesis also appears to be dependent on Fe–S proteins (37Go), was reduced by 50% (Fig. 7A). As disruption of Atm1p in yeast resulted in a loss in the activity of the cytosolic Fe–S protein Leu1p, without affecting its protein level (4Go), we determined the abundance of XO protein. XO protein level was not affected by the loss of Abcb7 (Fig. 7A). In sum, these data support a role for Abcb7 in the assembly of cytosolic, but not mitochondrial, Fe–S clusters in the liver.


Figure 0126
View larger version (14K):
[in this window]
[in a new window]
  		Figure 6. Mitochondrial Fe–S enzyme activities in Abcb7ly/Y liver. (A) Activity of ferrochelatase and mitochondrial aconitase (n=3) and (B) SDH, CCO and CS (n=7) were determined in isolated mitochondria. Enzyme activities determined in wild-type (open bars) and Abcb7ly/Y mice (black bars) are shown. A significant (P<0.05) difference between groups is indicated with an asterisk.

 

Figure 0127
View larger version (20K):
[in this window]
[in a new window]
  		Figure 7. Activity and abundance of XO, IRP1 and IRP2 in Abcb7ly/Y liver. (A) Liver cytosolic XO activity was quantified enzymatically (n=8) and abundance of XO protein (n=5) was determined by western blotting. (B) IRP1 RNA binding activity was determined in liver cytosol by quantitative gel shift assay (n=5). The enzymatic activity of cytosolic aconitase was determined in liver cytosol (n=3). IRP1 protein level was determined in liver cytosol by western blotting (n=5). (C) IRP2 RNA binding activity was determined in liver cytosol by quantitative gel shift assay (n=5). IRP2 protein level was determined in liver cytosol by western blotting (n=3). Activities determined in wild-type (open bars) and Abcb7ly/Y mice (black bars) are shown. A significant (P<0.05) difference between groups is indicated with an asterisk.

 
Activation and altered regulation of IRP1 and IRP2 in Abcb7lv/Y mice
As the function of IRP1 (aconitate hydratase 1), a cytosolic regulator of mammalian iron metabolism, is controlled through gain and loss of its Fe–S cluster, we sought to determine whether it too was influenced by the loss of Abcb7. In iron deficiency, IRP1 lacks its [4Fe–4S] cluster and controls the synthesis of ferritin and TfR1 by binding iron responsive elements in their mRNAs (14Go,15Go). When iron levels increase, assembly of the Fe–S cluster in IRP1 inactivates RNA binding, thereby maintaining iron homeostasis; in the holo-form, IRP1 is the cytosolic isoform of aconitase (c-acon). Similar to XO, c-acon activity declined significantly (~90%) in the liver of Abcb7lv/Y mice relative to wild-type animals (Fig. 7B). This was accompanied by the expected reciprocal rise in IRP1 RNA binding activity, which increased by nearly 6-fold (Fig. 7B). This provides a likely explanation for the 60% increase in TfR1 protein in Abcb7lv/Y liver (Fig. 4C). Unexpectedly, and in marked contrast to XO, IRP1 protein level declined by nearly 60% in Abcb7lv/Y liver, suggesting regulation of IRP1 at the protein level. We have subsequently gone on to show that this effect is likely a consequence of the unique role that IRP1 plays in iron metabolism and is, in large part, due to iron-dependent, cluster-independent mechanisms of regulating IRP1 protein (38Go).

We also determined the activity of IRP2, an RNA binding protein functionally homologous to IRP1 whose activity is controlled through iron-dependent changes in protein turnover. In Abcb7lv/Y liver, we found that IRP2 RNA binding activity was increased 50% and its protein level was increased by 24% (Fig. 7C). This effect is contrary to the apparent cellular iron overload and suggests that the iron sensing mechanism is reacting as if the cell is iron deficient (4Go,13Go). As recent evidence suggests that IRP2 protein turnover can be directly dependent on cytosolic heme levels (39Go), it is possible that these cells are heme deficient, yet iron overloaded.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Animal models of rare human disorders can provide unique insights into the pathogenesis of disease and the normal function of the mutated proteins. Our studies of Abcb7 have afforded an opportunity to examine the function of the protein in a mammalian system. We found that Abcb7 is an essential gene in mice because of an early requirement for the protein in the extra-embryonic tissues and have provided additional genetic evidence that Abcb7 is indeed functionally orthologous to Atm1p, playing a role in the formation of cytosolic Fe–S proteins. Furthermore, we demonstrate that in mammals, as in yeast, there is a complex interplay between the mitochondria and cytosol in sensing and controlling the iron status of the cell.

That ES cells and male mice deficient in Abcb7 were inviable was not surprising, given the requirement for a similar protein in lower eukaryotes. Unexpected, however, was the fact that a maternally inherited modified Abcb7 allele was lethal to female embryos. By breeding to a series of Cre-transgenic lines, we were able to demonstrate that the lethality was specifically due to a defect in the extra-embryonic visceral endoderm, which like all of the extra-embryonic tissues preferentially maintains the female X-chromosome as the active allele. This so-called X-linked parent of origin effect has been reported for several other null or severely hypomorphic alleles of X-linked genes, including several modeling hematological diseases in man (e.g. glucose-6-phosphate dehydrogenase deficiency and X-linked dyskeratosis congenita) (40Go,41Go). In each case, embryonic death can be attributed to morphologically distinctive developmental defects in the extra-embryonic tissues. However, in most other examples, lethality occurs somewhat later in embryogenesis and at a point where male and female embryos can be distinguished by genotyping. In the case of Abcb7, we were unable to determine the sex of the embryos because of their early demise and cannot infer whether males were more severely affected than females. If male embryos did indeed die earlier than female embryos, this would imply that Abcb7 was essential for an embryonic function prior to the requirement in the extra-embryonic tissues. However, the observation that male embryos in which deletion occurs only in the epiblast (using Sox2-Cre) die at a slightly later time would suggest that the immediate cause of death is truly due to a defect in the extra-embryonic visceral endoderm.

Abcb7 mRNA is widely distributed and tissue-specific deletions in the CNS and bone marrow are lethal (Table 1 and Fig. 5) (data not shown). This confirms the suggestion that XLSA/A in man is likely due to ABCB7 partial loss of function mutations, as a complete loss of function allele would almost certainly be lethal because of its effects on the extra-embryonic tissues, CNS and/or bone marrow at a minimum. Furthermore, X-inactivation analysis indicated that Abcb7 was essential in nearly all tissues. The primary exception to this is the observation that hepatocytes and endothelial cells can apparently survive in the absence of the protein. Why hepatocytes, in particular, are spared from the lethal effects of Abcb7 is unclear. Three possibilities include (i) that the Alb-Cre achieves only partial deletion of the conditional allele, (ii) that Abcb7 is redundant in certain cell types and (iii) that certain cell types are more or less sensitive to or dependent on the downstream effects of loss of Abcb7. Genotyping of Abcb7lv/Y livers by Southern blot or PCR demonstrates that 80–90% of the cells have rearranged the Abcb7fl conditional allele to the null allele (data not shown). The small fraction of cells with a residual, unrearranged conditional allele is not inconsistent with the number of Kupffer, vascular and other accessory cells present in the liver in which Alb-Cre is inactive, suggesting that there is a complete or near-complete deletion in the hepatocyte lineage. These cells undoubtedly contribute to the residual enzymatic activities (such as XO) measured in these samples and consequently mitigate the effects on bulk tissue enzyme assays. Furthermore, there are at least four other mitochondrial ABC half transporters in mammals (42Go). One of these, Abcb6 (mtAbc3 and UMAT1), has previously been shown to partially complement the {Delta}atm1 yeast phenotype (43Go–45Go). Furthermore, the expression of Abcb6 and Abcb7 are largely overlapping in adult tissues, with Abcb6 being expressed at particularly high levels in the liver (Supplementary Material, Fig. S2). Consequently, functional redundancy could both play a role in the survival of Abcb7 null hepatocytes and contribute to the incomplete loss of Fe–S protein activity. Alternatively, the hepatocyte may be less dependent on cytoplasmic Fe–S proteins such as the mammalian homolog of Rli1p. Furthermore, given the hepatocyte's pivotal role in systemic iron metabolism, it may be uniquely adapted to accommodate metabolic dysregulation brought on by Abcb7 deficiency. In this regard, the surprising decrease in IRP1 protein level in liver of Abcb7 mice suggests the presence of a compensatory mechanism to prevent excessive accumulation of RNA binding activity when Fe–S cluster-insertion is impaired and thereby limit hepatocyte damage (38Go).

The localization of Abcb7 to the mitochondrial inner membrane with its ABC domain facing the matrix places the protein at the gateway between the mitochondria and the cytosol and predicts that Abcb7 exports a mitochondrially generated metabolite to the cytosol (2Go,4Go). Interestingly, and in some contrast to yeast cells and erythroid precursors in XLSA/A, Abcb7 deficiency in the liver yields a modest mitochondrial phenotype; to the extent we examined mitochondrial function, we observed only mild morphologic and enzymatic defects. The apparent discrepancy in mitochondrial iron overload and dysfunction between yeast and XLSA/A patient erythroid cells and Abcb7-deficient hepatocytes may, among other possibilities, be due to functionally redundant proteins, tissue-specific effects resulting from a relatively high requirement for mitochondrial heme biosynthesis in erythroid cells, secondary, compensatory effects unique to mammalian cells that are dependent on the persistence of at least some Abcb7 activity or differences in iron requirements in proliferating yeast cells relative to mature hepatocytes.

More apparent than the mitochondrial effects, however, were the cytosolic effects of Abcb7 deletion, particularly cytosolic iron inclusions. Although the inclusions have some resemblance in size and shape to mitochondria, we were unable to identify intermediate structures, indicating that it is unlikely that the inclusions were derived from mitochondria. Furthermore, in no case were the inclusions seen to be associated with a membrane, indicating that they are not derived from any another organelle. Lastly, review of electron micrographs of murine neurons lacking frataxin also shows cytosolic lipid droplets with a similar, but somewhat eccentric, electron-dense rim (46Go). These structures, it would appear, may not be unique to Abcb7-deficient liver and may instead be a more generalizable effect of cytosolic Fe–S cluster deficiency.

Fe–S cluster deficiency was accompanied by dysregulated activation of IRP1 and IRP2, which likely contributed to increased TfR1 expression and iron uptake in Abcb7lv/Y animals; the slight, but not statistically significant, simultaneous rise in ferritin expression may reflect iron-regulation of ferritin gene transcription or protein stability (47Go,48Go). In the end, however, the increased liver iron appears to not be appropriately sensed by IRP and suggests that like yeast Aft1p and Aft2p, IRP RNA binding activity may respond more to the flux of iron through specific metabolic pathways (i.e. Fe–S cluster assembly) than to the absolute level of cellular iron. Similarly, recent observations in zebrafish with mutations in the mitochondrial Fe–S cluster biogenesis protein glutaredoxin 5 indicate that deficiency of Fe–S clusters may lead to inappropriate activation of IRP1 and death not as a consequence of mitochondrial deficiency of Fe–S clusters per se, but rather because of inappropriate regulation of downstream targets of cytosolic IRP1 (49Go). Hence, although the targeted deletion of IRP1 alone may not have a substantial phenotype (50Go,51Go), dysregulation of IRP1 by alterations in Fe–S metabolism may well indeed contribute to the pathogenesis of Fe–S cluster disorders, such as Friedreich ataxia and XLSA/A.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Animals
J1 ES (129S4/SvJae lineage) cells were transfected with the gene targeting construct shown in Figure 1, selected for G418 resistance and ganciclovir sensitivity, and analyzed by Southern blot for homologous recombination using standard techniques (52Go). The NeoR cassette was excised in vitro by transient transfection of a Cre recombinase plasmid (Dr Charles Roberts, Dana Farber Cancer Institute, Boston, MA, USA) and G418 sensitive subclones analyzed for recombination of the locus by Southern blot using external probes 5' and 3' of the targeting construct (Fig. 1). Chimeric male mice were generated from individual recombinants with and without the NeoR cassette, and the modified Abcb7 allele transmitted through the germline. In many instances, experiments were performed with and without the NeoR cassette in place; the results did not differ between these groups. C57BL/6J-Albumin-, Nestin- and Sox2-Cre-recombinase transgenic lines were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). FVB/J-Tg(Gata1-Cre) and outbred Villin-Cre lines were obtained from Drs Stuart Orkin (Children's Hospital, Boston, MA, USA) and Sylvie Robine (Paris, FR), respectively. Embryo dissections were performed using routine techniques with the morning that the post-coital plug was observed defined as E0.5. All experiments with Alb-Cre were performed in Abcb7fl/Y males at 6 or 8 weeks of age. All animal procedures were reviewed and approved by the Animal Care and Use Committee, Children's Hospital Boston.

Gene expression and phenotypic and X-inactivation analyses
An adult mouse multitissue poly-A selected northern blot (OriGene, Rockville, MD, USA) was probed with murine Abcb7, Abcb6 and ß-actin ORF probes. In situ hybridization of murine embryos was performed as previously described (53Go), using a 35S-labeled riboprobe corresponding to nucleotides 308–691 of the murine Abcb7 cDNA (Ensembl transcript ID ENSMUST00000033695). Serum iron parameters were determined using the serum iron/UIBC kit from Sigma Diagnostics (Sigma-Aldrich, St Louis, MO, USA). Tissue and mitochondrial iron were measured as previously described (54Go). All histological and immunohistochemical analyses were performed on routine formalin-fixed, paraffin-embedded sections. GFP immunohistochemistry was performed as previously described (55Go). Electron microscopy was performed using routine osmium tetroxide treated, uranyl/lead acetate stained thin sections in the Electron Microscopy Facility in the Department of Pathology, Children's Hospital Boston.

IRE RNA binding activity
IRP1 and IRP2 RNA binding assays were determined using a quantitative RNA binding assay with rat L-ferritin RNA as described previously (57Go). After binding of [32P]IRE to protein, heparin was added and bound and free RNA separated by electrophoretic mobility shift assay as described (57Go). Results were quantified by phosphorimaging including the use of an RNA standard curve (58Go).

Liver subcellular fractionation and enzyme assays
Cytosol and mitochondria were obtained as described (56Go). Aconitase assays were performed at 25°C for 10 min as described (59Go). For XO activity, a kit from Molecular Probes, Inc. (A-22182, Eugene, OR, USA) was used. Ferrochelatase (60Go), citrate synthase (CS) (61Go), SDH (62Go) and CCO (Sigma-Aldrich, St Louis, MO, USA) assays were performed as described. Protein levels were quantified using the ImageQuant software supplied with the BioRad ChemiDoc gel documentation system (Hercules, CA, USA).

Immunoblotting and antibodies
For determination of protein levels, either whole-tissue homogenate or cytosol was used. The IRP1 blotting was performed as described (57Go). Other antibodies included rabbit anti-bovine XO (cat. no. ab6194, Abcam, Cambridge, MA, USA), Ferritin (63Go) and monoclonal rat anti-TfR1 (rat anti-CD71, cat. no. MCA1033G, Accurate Chemical & Scientific Corp., Westbury, NY, USA).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH DK62474, the Pew Biomedical Scholars Program, the Wilkes Fund of the Children's Hospital, Department of Pathology (M.D.F.), NIH RO1 DK47219 (R.S.E.), L'Asssociation Française Contre le Cancer (C.P.) and NIH training grant T32 DK07665, which partially supported S.L.C. Transgenic core facilities were provided by the Mental Retardation Research Center (MRRC) at Children's Hospital, supported by NIH P30-HD 18655. Howard Mulhern and James Edwards of the Children's Hospital, Department of Pathology, Electron Microscopy Facility, and Histology Laboratory, respectively, provided expert technical assistance. Ronald Parsons, Kavita Sharma and Katherine Shea are acknowledged for technical assistance early in the project. Members of the Fleming, Eisenstein and Andrews laboratories, particularly Lance Lee and Jeremy Goforth, are acknowledged for ongoing criticism of the project and review of the manuscript.

Conflicts of Interest statement. The authors have no conflicts of interest to declare.


   FOOTNOTES
 
{dagger} These authors contributed equally. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

  1. Beinert, H., Holm, R.H. and Munck, E. (1997) Iron–sulfur clusters—nature's modular, multipurpose structures. Science, 277, 653–659.[CrossRef][ISI][Medline]

  2. Lill, R. and Muhlenhoff, U. (2005) Iron–sulfur-protein biogenesis in eukaryotes. Trends Biochem. Sci., 30, 133–141.[CrossRef][ISI][Medline]

  3. Rouault, T.A. and Tong, W.H. (2005) Iron–sulphur cluster biogenesis and mitochondrial iron homeostasis. Nat. Rev. Mol. Cell Biol., 6, 345–351.[CrossRef][ISI][Medline]

  4. Kispal, G., Csere, P., Prohl, C. and Lill, R. (1999) The mitochondrial proteins Atm1p and Nfs1p are essential for biogenesis of cytosolic Fe/S proteins. EMBO J., 18, 3981–3989.[CrossRef][ISI][Medline]

  5. Leighton, J. and Schatz, G. (1995) An ABC transporter in the mitochondrial inner membrane is required for normal growth of yeast. EMBO J., 14, 188–195.[ISI][Medline]

  6. Kispal, G., Sipos, K., Lange, H., Fekete, Z., Bedekovics, T., Janaky, T., Bassler, J., Netz-Aguilar, D.J., Balk, J., Rotte, C. et al. (2005) Biogenesis of cytosolic ribosomes requires the essential iron–sulphur protein Rli1p and mitochondria. EMBO J., 24, 589–598.[CrossRef][ISI][Medline]

  7. Yarunin, A., Pause, V.G., Petfalski, E., Dez, C., Tollervey, D. and Hurt, E.C. (2005) Functional link between ribosome formation and biogenesis of iron–sulfur proteins. EMBO J., 24, 580–588.[CrossRef][ISI][Medline]

  8. Stehling, O., Elasser, H.P., Bruckel, B., Muhlenhoff, U. and Lill, R. (2004) Iron–sulfur protein maturation in human cells: evidence for a function of frataxin. Hum. Mol. Genet., 13, 3007–3015.[Abstract/Free Full Text]

  9. Seznec, H., Simon, D., Bouton, C., Reutenauer, L., Hertzog, A., Golik, P., Procaccio, V., Patel, M., Drapier, J.C., Koenig, M. et al. (2005) Friedreich ataxia: the oxidative stress paradox. Hum. Mol. Genet., 14, 463–474.[Abstract/Free Full Text]

  10. Lange, H., Muhlenhoff, U., Denzel, M., Kispal, G. and Lill, R. (2004) The heme synthesis defect of mutants impaired in mitochondrial iron–sulfur protein biogenesis is caused by reversible inhibition of ferrochelatase. J. Biol. Chem., 279, 29101–29108.[Abstract/Free Full Text]

  11. Chen, O., Crisp, R.J., Valachovic, M., Bard, M., Winge, D.R. and Kaplan, J. (2004) Transcription of the yeast iron regulon does not directly respond to iron but rather to iron–sulfur cluster biosynthesis. J. Biol. Chem., 279, 29513–29518.[Abstract/Free Full Text]

  12. Li, J., Kogan, M., Knight, S.A.B., Pain, D. and Dancis, A. (1999) Yeast mitochondrial protein, Nfs1p, coordinately regulates iron–sulfur cluster proteins, cellular iron uptake, and iron distribution. J. Biol. Chem., 274, 33025–33034.[Abstract/Free Full Text]

  13. Rutherford, J.C., Ojeda, L., Balk, J., Muhlenhoff, U., Lill, R. and Winge, D.R. (2005) Activation of the iron regulon by yeast Aft1/Aft2 transcription factors depends on mitochondria but not cytosolic iron–sulfur protein biogenesis. J. Biol. Chem., 280, 10135–10140.[Abstract/Free Full Text]

  14. Eisenstein, R.S. (2000) Iron regulatory proteins and the molecular control of mammalian iron metabolism. Annu. Rev. Nutr., 20, 627–662.[CrossRef][ISI][Medline]

  15. Hentze, M.W., Muckenthaler, M.U. and Andrews, N.C. (2004) Balancing acts: molecular control of mammalian iron metabolism. Cell, 117, 285–297.[CrossRef][ISI][Medline]

  16. Allikmets, R., Raskind, W.H., Hutchinson, A., Schueck, N.D., Dean, M. and Koeller, D.M. (1999) Mutation of a putative mitochondrial iron transporter gene (ABC7) in X-linked sideroblastic anemia and ataxia (XLSA/A). Hum. Mol. Genet., 8, 743–749.[Abstract/Free Full Text]

  17. Csere, P., Lill, R. and Kispal, G. (1998) Identification of a human mitochondrial ABC transporter, the functional orthologue of yeast Atm1p. FEBS Lett., 441, 266–270.[CrossRef][ISI][Medline]

  18. Kispal, G., Csere, P., Guiard, B. and Lill, R. (1997) The ABC transporter Atm1P is required for mitochondrial iron homeostasis. FEBS Lett., 418, 426–436.

  19. Senbongi, H., Ling, F. and Shibata, T. (1999) A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA. Mol. Gen. Genet., 262, 426–436.[CrossRef][ISI][Medline]

  20. Fleming, M.D. (2002) The genetics of inherited sideroblastic anemias. Semin. Hematol., 39, 270–281.[CrossRef][ISI][Medline]

  21. Bekri, S., Kispal, G., Lange, H., Fitzsimmons, E., Tolmie, J., Lill, R. and Bishop, D.F. (2000) Human ABC7 transporter: gene structure and mutation causing X-linked sideroblastic anemia with ataxia with disruption of cytosolic iron–sulfur protein maturation. Blood, 96, 3256–3264.[Abstract/Free Full Text]

  22. Hellier, K.D., Hatchwell, E., Duncombe, A.S., Kew, J. and Hammans, S.R. (2001) X-linked sideroblastic anaemia with ataxia: another mitochondrial disease? J. Neurol. Neurosurg. Psychiatry, 70, 65–69.[Abstract/Free Full Text]

  23. Maguire, A., Hellier, K., Hammans, S. and May, A. (2001) X-linked cerebellar ataxia and sideroblastic anaemia associated with a missense mutation in the Abcb7 gene predicting V411L. Brit. J. Haematol., 115, 910–917.[CrossRef][ISI][Medline]

  24. Pagon, R.A., Bird, T.D., Detter, J.C. and Pierce, I. (1985) Hereditary sideroblastic anaemia and ataxia: an X-linked recessive disorder. J. Med. Genet., 22, 267–273.[Abstract]

  25. Raskind, W.H., Wijsman, E., Pagon, R.A., Cox, T.C., Bawden, M.J., May, B.K. and Bird, T.D. (1991) X-linked sideroblastic anemia and ataxia: linkage to phosphoglycerate kinase at Xq 13. Am. J. Hum. Genet., 48, 335–341.[ISI][Medline]

  26. Jasinski, M., Keller, P., Fujiwara, Y., Orkin, S.H. and Bessler, M. (2001) GATA1-Cre mediates Piga gene inactivation in the erythroid/megakaryocytic lineage and leads to circulating red cells and a partial deficiency in glycosyl phosphatidylinositol-linked proteins (paroxysmal nocturnal hemoglobinuria type II cell. Blood, 98, 2248–2255.[Abstract/Free Full Text]

  27. Huynh, K.D. and Lee, J.T. (2003) Inheritance of a pre-inactivated paternal X chromosome in early mouse embryos. Nature, 426, 857–862.[CrossRef][Medline]

  28. Hayashi, S., Lewis, P., Pevny, L. and McMahon, A.P. (2002) Efficient gene modulation in mouse epiblast using a Sox2Cre transgenic mouse strain. Mech. Dev., 119, S97–S101.[CrossRef][Medline]

  29. Donovan, A., Lima, C.A., Pinkus, J.L., Pinkus, G.S., Zon, L.I., Robine, S. and Andrews, N.C. (2005) The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis. Cell Metab., 1, 191–200.[CrossRef][ISI][Medline]

  30. El-Marjou, F., Janssen, K.P., Chang, B.H., Li, M., Hindie, V., Chan, L., Louvard, D., Chambon, P., Metzger, D. and Robine, S. (2004) Tissue-specific and inducible Cre-mediate recombination in the gut epithelium. Genesis, 39, 186–193.[CrossRef][ISI][Medline]

  31. Kühn, R., Schwenk, F., Aguet, M. and Rajewsky, K. (1995) Inducible gene targeting in mice. Science, 269, 1427–1429.[Abstract/Free Full Text]

  32. Tronche, F., Kellendonk, C., Kretz, O., Gass, P., Anlag, K., Orban, P.C., Bock, R., Klein, R. and Schutz, G. (1999) Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nature Genet., 23, 99–103.[CrossRef][ISI][Medline]

  33. Hadjantonakis, A.K., Cox, L.L., Tam, P.P. and Nagy, A. (2001) A X-linked GFP transgene reveals unexpected paternal X-chromosome activity in trophoblast giant cells of mouse placenta. Genesis, 29, 133–140.[CrossRef][ISI][Medline]

  34. Postic, C. and Magnuson, M.A. (2000) DNA excision in liver by an albumin-Cre transgene occurs progressively with age. Genesis, 26, 149–150.[CrossRef][ISI][Medline]

  35. Rustin, P., Chretien, D., Bourgeron, T., Gerard, B., Rotig, A., Saudubray, J.M. and Munnich, A. (1994) Biochemical and molecular investigations in respiratory chain deficiencies. Clin. Chim. Acta, 228, 35–51.[CrossRef][ISI][Medline]

  36. Masini, A., Salvioli, G., Cremonesi, P., Botti, B., Gallesi, D. and Ceccarelli, D. (1994) Dietary iron deficiency in the rat. I. Abnormalities in energy metabolism of the hepatic tissue. Biochim. Biophys. Acta, 1188, 46–52.[Medline]

  37. Hänzelmann, P., Hernández, H.K., Menzel, C., Garcia-Serres, R., Huynh, B.H., Johnson, M.K., Mendel, R.R. and Schindelin, H. (2004) Characterization of MOCS1A, an oxygen-sensitive iron–sulfur protein involved in human molybdenum cofactor biosynthesis. J. Biol. Chem., 279, 34721–34732.[Abstract/Free Full Text]

  38. Clarke, S.L., Vasanthakumar, A., Anderson, S.A., Pondarré, C., Koh, C.M., Deck, K.M., Pitula, J.S., Epstein, C.J., Fleming, M.D. and Eisenstein, R.S. (2005) Iron-responsive degradation of iron regulatory protein 1 does not require the Fe–S cluster. EMBO J., 25, 544–553.[CrossRef][ISI]

  39. Ishikawa, H., Kato, M., Hori, H., Ishimori, K., Kirisako, T., Tokunaga, F. and Iwai, K. (2005) Involvement of the heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2. Mol. Cell, 19, 171–181.[CrossRef][ISI][Medline]

  40. He, J., Navarrete, S., Jasinski, M., Vulliamy, T., Dokal, I., Bessler, M. and Mason, P.J. (2002) Targeted disruption of Dkc1, the gene mutated in X-linked dyskeratosis congenita, causes embryonic lethality in mice. Oncogene, 21, 7740–7744.[CrossRef][ISI][Medline]

  41. Longo, L., Venegas, O.C., Patel, M., Rosti, V., Li, H., Waka, J., Merghoub, T., Pandolfi, P.P., Notaro, R., Manova, K. et al. (2002) Maternally transmitted severe glucose 6-phosphate dehydrogenase deficiency is an embryonic lethal. EMBO J., 21, 4229–4239.[CrossRef][ISI][Medline]

  42. Lill, R. and Kispal, G. (2001) Mitochondrial ABC transporters. Res. Microbiol., 152, 331–340.[Medline]

  43. Emadi-Konjin, H.P., Zhang, H., Anandan, V., Sun, D., Schuetz, J. and Furuya, K.N. (2002) Isolation of a genomic clone containing the promoter region of the human ATP binding cassette (ABC) transporter, ABCB6. Biochim. Biophys. Acta, 1574, 117–130.[Medline]

  44. Hirsch-Ernst, K.L., Gaini-Rahimi, S., Ernst, B.P., Schmitz-Salue, C., Blume, S. and Kahl, G.F. (1998) Molecular cDNA cloning and tissue distribution of mRNA encoding a novel ATP-binding cassette (ABC) half-transporter. Biochem. Biophys. Res. Commun., 249, 151–155.[CrossRef][ISI][Medline]

  45. Mitsuhashi, N., Miki, T., Senbongi, H., Yokoi, N., Yano, H., Miyazaki, M., Nakajima, N., Iwanaga, T., Yokoyama, Y., Shibata, T. et al. (2000) MTABC3, a novel mitochondrial ATP-binding cassette protein involved in iron homeostasis. J. Biol. Chem., 275, 17536–17540.[Abstract/Free Full Text]

  46. Puccio, H., Simon, D., Cossee, M., Filipe-Criqui, P., Tiziano, F., Melki, J., Hindelang, C., Matyas, R., Rustin, P. and Koenig, M. (2001) Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe–S enzyme deficiency followed by intramitochondrial iron deposits. Nature Genet., 27, 181–186.[CrossRef][ISI][Medline]

  47. Truty, J., Malpe, R. and Linder, M.C. (2001) Iron prevents ferritin turnover in hepatic cells. J. Biol. Chem., 276, 48775–48780.[Abstract/Free Full Text]

  48. White, K. and Munro, H.N. (1988) Induction of ferritin subunit synthesis by iron is regulated at both the transcriptional and translational levels. J. Biol. Chem., 263, 8938–8942.[Abstract/Free Full Text]

  49. Wingert, R.A., Galloway, J.L., Barut, B., Foott, H., Fraenkel, P., Axe, J.L., Weber, G.J., Dooley, K., Davidson, A.J., Schmidt, B. et al. (2005) Deficiency of glutaredoxin 5 reveals Fe–S clusters are required for vertebrate haem synthesis. Nature, 436, 1035–1039.[CrossRef][Medline]