Of Two Cytosolic Aconitases Expressed in Drosophila, Only One Functions as an Iron-regulatory Protein

Abstract

In mammalian cells, iron homeostasis is largely regulated by post-transcriptional control of gene expression through the binding of iron-regulatory proteins (IRP1 and IRP2) to iron-responsive elements (IREs) contained in the untranslated regions of target mRNAs. IRP2 is the dominant iron sensor in mammalian cells under normoxia, but IRP1 is the more ancient protein in evolutionary terms and has an additional function as a cytosolic aconitase. The Caenorhabditis elegans genome does not contain an IRP2 homolog or identifiable IREs; its IRP1 homolog has aconitase activity but does not bind to mammalian IREs. The Drosophila genome offers an evolutionary intermediate containing two IRP1-like proteins (IRP-1A and IRP-1B) and target genes with IREs. Here, we used purified recombinant IRP-1A and IRP-1B from Drosophila melanogaster and showed that only IRP-1A can bind to IREs, although both proteins possess aconitase activity. These results were also corroborated in whole-fly homogenates from transgenic flies that overexpress IRP-1A and IRP-1B in their fat bodies. Ubiquitous and muscle-specific overexpression of IRP-1A, but not of IRP-1B, resulted in pre-adult lethality, underscoring the importance of the biochemical difference between the two proteins. Domain-swap experiments showed that multiple amino acid substitutions scattered throughout the IRP1 domains are synergistically required for conferring IRE binding activity. Our data suggest that as a first step during the evolution of the IRP/IRE system, the ancient cytosolic aconitase was duplicated in insects with one variant acquiring IRE-specific binding.Iron is required for aerobic metabolism and is actively sensed, sequestered, and regulated by organisms, including hosts and pathogens, that often compete for metal acquisition (1). In multicellular organisms, signals exist for systemic control of iron metabolism (2). One of the central regulators of cellular iron metabolism is the IRP/IRE system (3). In brief, absence of iron is sensed by IRP1 and IRP2, which then bind to ironresponsive elements (IREs) 4 in the 5Ј-untranslated regions of mRNAs encoding several storage or consumer iron proteins, inhibiting their translation. IRP1 and IRP2 also bind to IREs in the 3Ј-untranslated regions of mRNAs encoding proteins that function in cellular iron import, stabilizing the transcripts and enhancing iron sequestration. There is much interest in defining how IRP1 and IRP2 sense iron levels. IRP1 interconverts between an iron-sulfur protein with aconitase activity and the apoprotein that binds to the IRE (4). IRP2 has an additional 73-amino acid domain inserted in the protein and has no aconitase activity (5, 6). Extensive analysis of knock-out mice has led to the conclusion that IRP2 dominates mammalian cellular iron metabolism (7). IRP1 also contributes to the regulation of cellular iron metabolism, albeit to a lesser extent, because its iron-sulfur cluster is efficiently repaired and the protein functions mostly as an aconitase (8 -10). It is also well established that b...

Heme as a Magnificent Molecule with Multiple Missions: Heme Determines Its Own Fate and Governs Cellular Homeostasis

“…9). It is notable that the expression of each mRNA encoding Bach1 (Igarashi and Sun 2006), HRI (Chen 2007) or IRP2 (Lind et al 2006) is restricted in higher eukaryotes. While CP motifs in the presequence of ALAS are conserved throughout yeast to human except some higher plants, there is no evidence that heme inhibits mitochondrial transport of yeast ALAS precursor either in vivo or in vitro (Duncan et al 1999).…”

Homeostatic Mechanisms for Iron Storage Revealed by Genetic Manipulations and Live Imaging of Drosophila Ferritin

“…Intron/exon donor and acceptor splice sites flank the GFP sequence on the P element; consequently, all Fer1HCH G188 mRNA types (IRE 1/À) are predicted to contain the GFP exon, which should encode GFP in frame with the Fer1HCH protein ( Figure 2D). Therefore, post-transcriptional regulation of GFP-Fer1HCH mediated by the IRP-1A (Lind et al 2006) would remain unaffected by the GFP exon. GFP is inserted two amino acids downstream of the predicted cleavage site for the Fer1HCH signal peptide (Charlesworth et al 1997), which is encoded from the second exon of the gene ( Figure 2D).…”

“…In contrast, no IRE is present in Fer2LCH mRNA . IRP homologs are expressed in the fly (Muckenthaler et al 1998), and one homolog (IRP-1A) has been shown to bind to IREs from both Drosophila and mammals (Lind et al 2006).…”

Iron depletion in the intestines ofMalvoliomutant flies does not occur in the absence of a multicopper oxidase

“…As a consequence, some aspects of systemic iron homeostasis, defined as all movement of iron in the organism, and regulatory mechanisms that control this movement maintaining an overall stable condition (Hentze et al, 2004;Nichol et al, 2002), may differ between invertebrates and vertebrates. Nevertheless, key proteins involved in iron regulation have conserved functions as exemplified by the ferritins Missirlis et al, 2007;Pham and Winzerling, 2010), the iron regulatory proteins (Lind et al, 2006) and the transferrins Tiklová et al, 2010). We became interested in the DMT1 homologue in Drosophila melanogaster, encoded by the Mvl gene, originally identified in a mutagenesis screen designed to identify genes affecting taste behaviour in flies (Rodrigues et al, 1995).…”