Control of Iron Homeostasis by an Iron-Regulated Ubiquitin Ligase

Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Science (Impact Factor: 33.61). 09/2009; 326(5953):718-21. DOI: 10.1126/science.1176333
Source: PubMed


Eukaryotic cells require iron for survival and have developed regulatory mechanisms for maintaining appropriate intracellular iron concentrations. The degradation of iron regulatory protein 2 (IRP2) in iron-replete cells is a key event in this pathway, but the E3 ubiquitin ligase responsible for its proteolysis has remained elusive. We found that a SKP1-CUL1-FBXL5 ubiquitin ligase protein complex associates with and promotes the iron-dependent ubiquitination and degradation of IRP2. The F-box substrate adaptor protein FBXL5 was degraded upon iron and oxygen depletion in a process that required an iron-binding hemerythrin-like domain in its N terminus. Thus, iron homeostasis is regulated by a proteolytic pathway that couples IRP2 degradation to intracellular iron levels through the stability and activity of FBXL5.

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    • "In vitro analyses suggest that BTS interacts with (Fig. 3) and restricts the accumulation of iron-responsive bHLH transcription factors ILR3 and bHLH115 through its E3 ligase activity and can mediate proteasomal degradation of these targets even in the absence of HHE domains (Fig. 5, A–C). Notably, the mammalian protein FBXL5, unlike BTS, does not contain a RING domain and thus lacks the ability to directly ubiquitinate its targets for degradation via the 26S proteasome pathway (Salahudeen et al., 2009; Vashisht et al., 2009; Thompson et al., 2012). FBXL5 does, however, provide the specificity to the SCF FBXL5 E3 ligase complex to regulate iron homeostasis. "
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    ABSTRACT: Iron uptake and metabolism are tightly regulated in both plants and animals. In Arabidopsis thaliana, BRUTUS (BTS), which contains three hemerythrin (HHE) domains and a Really Interesting New Gene (RING) domain, interacts with bHLH transcription factors that are capable of forming heterodimers with POPEYE, a positive regulator of the iron deficiency response. BTS has been shown to have E3 ligase capacity and to play a role in root growth, rhizosphere acidification, and iron reductase activity in response to iron deprivation. To further characterize the function of this protein we examined the expression pattern of recombinant ProBTS::GUS and found that it is expressed in developing embryos and other reproductive tissues, corresponding with its apparent role in reproductive growth and development. Our findings also indicate that the interactions between BTS and PYE-like (PYEL) bHLH transcription factors occur within the nucleus and are dependent on the presence of the RING domain. We provide evidence that BTS facilitates 26S proteasome-mediated degradation of PYEL proteins in the absence of iron. We also determined that upon binding iron at the HHE domains, BTS is destabilized, and that this destabilization relies on specific residues within the HHE domains. This study reveals an important and novel mechanism for plant iron homeostasis whereby an E3 ubiquitin ligase may post-translationally control components of the transcriptional regulatory network involved in the iron deficiency response. Copyright © 2014, American Society of Plant Biologists.
    Plant physiology 12/2014; 167(1). DOI:10.1104/pp.114.250837 · 6.84 Impact Factor
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    • "This allows proteasomal degradation of FBXL5, which leads to concomitant accumulation of IRP2. Interestingly, FBXL5 can also promote the ubiquitination and degradation of IRP1 mutants that cannot form a 4Fe-4S cluster (Salahudeen et al., 2009; Vashisht et al., 2009). Proteasomal degradation of wild type apo-IRP1 under conditions of impairment of the iron-sulfur cluster assembly pathway has been proposed to operate as a reserve mechanism to control the IRE-binding activity of IRP1 (Clarke et al., 2006; Wang et al., 2007). "
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    ABSTRACT: Iron regulatory proteins 1 and 2 (IRP1 and IRP2) post-transcriptionally control the expression of several mRNAs encoding proteins of iron, oxygen and energy metabolism. The mechanism involves their binding to iron responsive elements (IREs) in the untranslated regions of target mRNAs, thereby controlling mRNA translation or stability. Whereas IRP2 functions solely as an RNA-binding protein, IRP1 operates as either an RNA-binding protein or a cytosolic aconitase. Early experiments in cultured cells established a crucial role of IRPs in regulation of cellular iron metabolism. More recently, studies in mouse models with global or localized Irp1 and/or Irp2 deficiencies uncovered new physiological functions of IRPs in the context of systemic iron homeostasis. Thus, IRP1 emerged as a key regulator of erythropoiesis and iron absorption by controlling hypoxia inducible factor 2α (HIF2α) mRNA translation, while IRP2 appears to dominate the control of iron uptake and heme biosynthesis in erythroid progenitor cells by regulating the expression of transferrin receptor 1 (TfR1) and 5-aminolevulinic acid synthase 2 (ALAS2) mRNAs, respectively. Targeted disruption of either Irp1 or Irp2 in mice is associated with distinct phenotypic abnormalities. Thus, Irp1(-/-) mice develop polycythemia and pulmonary hypertension, while Irp2(-/-) mice present with microcytic anemia, iron overload in the intestine and the liver, and neurologic defects. Combined disruption of both Irp1 and Irp2 is incombatible with life and leads to early embryonic lethality. Mice with intestinal- or liver-specific disruption of both Irps are viable at birth but die later on due to malabsorption or liver failure, respectively. Adult mice lacking both Irps in the intestine exhibit a profound defect in dietary iron absorption due to a "mucosal block" that is caused by the de-repression of ferritin mRNA translation. Herein, we discuss the physiological function of the IRE/IRP regulatory system.
    Frontiers in Pharmacology 07/2014; 5:176. DOI:10.3389/fphar.2014.00176 · 3.80 Impact Factor
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    • "This expansion of the LIP is reflected in lowered levels of iron regulatory protein 2 (IRP2), which undergoes ubiquitin-mediated degradation in the presence of increased labile iron (Guo et al., 1995). Whether the degradation associated with PCBP depletion is dependent on the recently described ubiquitin ligase FBXL5 has not been determined (Salahudeen et al., 2009; Vashisht et al., 2009). HEK 293T cells depleted of PCBPs exhibit reduced activity of cytosolic (c-) aconitase (Frey et al., 2014), an enzyme that carries a labile [4Fe-4S] cluster in its active site. "
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    ABSTRACT: Eukaryotic cells contain hundreds of proteins that require iron cofactors for activity. These iron enzymes are located in essentially every subcellular compartment; thus, iron cofactors must travel to every compartment in the cell. Iron cofactors exist in three basic forms: Heme, iron-sulfur clusters, and simple iron ions (also called non-heme iron). Iron ions taken up by the cell initially enter a kinetically labile, exchangeable pool that is referred to as the labile iron pool. The majority of the iron in this pool is delivered to mitochondria, where it is incorporated into heme and iron-sulfur clusters, as well as non-heme iron enzymes. These cofactors must then be distributed to nascent proteins in the mitochondria, cytosol, and membrane-bound organelles. Emerging evidence suggests that specific systems exist for the distribution of iron cofactors within the cell. These systems include membrane transporters, protein chaperones, specialized carriers, and small molecules. This review focuses on the distribution of iron ions in the cytosol and will highlight differences between the iron distribution systems of simple eukaryotes and mammalian cells.
    Frontiers in Pharmacology 07/2014; 5:173. DOI:10.3389/fphar.2014.00173 · 3.80 Impact Factor
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