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Corinne Pondarré,
Brendan B Antiochos,
Dean R Campagna, Stephen L Clarke,
Eric L Greer,
Kathryn M Deck,
Alice McDonald,
An-Ping Han,
Amy Medlock,
Jeffery L Kutok,
Sheila A Anderson,
Richard S Eisenstein,
Mark D Fleming
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ABSTRACT: 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.
Human Molecular Genetics 04/2006; 15(6):953-64. · 7.64 Impact Factor
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ABSTRACT: The generally accepted role of iron-regulatory protein 1 (IRP1) in orchestrating the fate of iron-regulated mRNAs depends on the interconversion of its cytosolic aconitase and RNA-binding forms through assembly/disassembly of its Fe-S cluster, without altering protein abundance. Here, we show that IRP1 protein abundance can be iron-regulated. Modulation of IRP1 abundance by iron did not require assembly of the Fe-S cluster, since a mutant with all cluster-ligating cysteines mutated to serine underwent iron-induced protein degradation. Phosphorylation of IRP1 at S138 favored the RNA-binding form and promoted iron-dependent degradation. However, phosphorylation at S138 was not required for degradation. Further, degradation of an S138 phosphomimetic mutant was not blocked by mutation of cluster-ligating cysteines. These findings were confirmed in mouse models with genetic defects in cytosolic Fe-S cluster assembly/disassembly. IRP1 RNA-binding activity was primarily regulated by IRP1 degradation in these animals. Our results reveal a mechanism for regulating IRP1 action relevant to the control of iron homeostasis during cell proliferation, inflammation, and in response to diseases altering cytosolic Fe-S cluster assembly or disassembly.
The EMBO Journal 03/2006; 25(3):544-53. · 9.20 Impact Factor
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ABSTRACT: Iron-regulatory protein 1 (IRP1) is a dual-function protein with mutually exclusive roles as a posttranscriptional regulator of animal-cell iron metabolism or as the cytosolic isoform of the iron-sulfur enzyme aconitase (c-acon). Much effort has focused on the role of IRP1 in posttranscriptional gene regulation and in factors that influence its interconversion with c-acon, but little is known about the metabolic function and regulation of c-acon. The role of PKC-dependent phosphorylation of S711 on IRP1/c-acon function was examined. Phosphorylation state-specific antibodies revealed that S711 is phosphorylated by PKC in vitro and in human embryonic kidney cells treated with a PKC activator. In aco1 yeast, the phosphomimetic mutants S711D and S711E exhibited severely impaired aconitase function, whereas S711A and S711T were unaffected relative to the WT protein. Aconitase activity in yeast extracts displayed a similar pattern when assayed for capacity to convert citrate to isocitrate: WT, S711A, and S711T were active, but S711D and S711E activity was undetectable. In contrast, when measured by the conversion of isocitrate to cis-aconitate, S711D and S711E displayed substantial activity, indicating that phosphorylation impairs the citrate but not isocitrate mode of aconitase function. This possibility was confirmed in vivo by demonstrating that S711D and S711E specifically antagonized the requirement for isocitrate in two metabolic scenarios. Iron-responsive element RNA-binding affinity was unaffected by S711 mutations. Our results show that S711 is a target of phosphorylation capable of conferring distinct effects on c-acon function potentially dictating changes in cytosolic citrate/isocitrate metabolism.
Proceedings of the National Academy of Sciences 08/2004; 101(30):10907-12. · 9.68 Impact Factor
