Ferritin is a symmetric, 24-subunit iron-storage complex assembled of H and L chains. H and L heteropolymers have distinct physicochemical properties, owing to the ferroxidase activity of the H subunit, which is necessary for iron uptake by the ferritin molecule, and the ability of the L subunit to facilitate iron core formation inside the protein shell. Ferritin is found in bacteria, plants and animals, and two classes of mutations in the human L-chain gene result in hereditary hyperferritinemia cataract syndrome or in neuroferritinopathy. This study examined systemic and cellular ferritin regulation and trafficking in Drosophila; ferritin H and L transcripts are co-expressed during embryogenesis and both subunits are essential for embryonic development. Ferritin overexpression impairs the survival of iron-deprived flies. In vivo expression of GFP-tagged holoferritin confirmed that iron-loaded ferritin molecules traffic through the Golgi organelle and are secreted into hemolymph. A constant ratio of ferritin H and L subunits, secured via tight post-transcriptional regulation, is characteristic of the secreted ferritin in flies. Differential cellular expression, conserved post-transcriptional regulation via the iron regulatory element, and distinct subcellular localization of the ferritin subunits prior to the assembly of holoferritin are all important steps mediating iron homeostasis. This study revealed both conserved features and insect-specific adaptations of ferritin nanocages and provides novel imaging possibilities for their in vivo characterization (Missirlis, 2007).

Iron is an essential element of aerobic life. Cells have evolved highly regulated molecular pathways that ensure iron incorporation into heme, or formation of iron-sulfur clusters. Cellular and systemic iron levels are tightly regulated to ensure bioavailability and protect from the hazards of iron overload. Ferritin, a heteropolymer composed of H and L subunits, acts as the primary iron storage molecule. The H subunit contains a ferroxidase center, which enables the mature heteropolymer to oxidize soluble ferrous iron, whereas the L chain provides the nucleation centers for deposition of the ferrihydrite mineral (Missirlis, 2007 and references therein).

In mammals, transcriptional control of the ferritin genes influences the relative ratio of H to L chains in different cell types (Torti, 2002; Pham, 2004). Translation of ferritin proteins is regulated by the binding of either of the two iron regulatory proteins (IRPs) to an iron responsive element (IRE) located on the 5í untranslated region (UTR) of the respective mRNAs (Pantopoulos, 2004; Rouault 2006). Hereditary hyperferritinemia cataract syndrome, a disease in which ferritin L-chain IRE mutations interfere with appropriate translational repression, illustrates the physiological importance of the IRP/IRE system (Cazzola, 2002; Rouault 2006). Moreover, adult mice lacking IRP2 overexpress ferritin, develop variable degrees of late-onset neurodegeneration (Lavaute, 2001; Smith, 2004; Galy, 2006) and anemia (Cooperman, 2005; Galy, 2005). Negative consequences of chronic ferritin H-chain overexpression have been verified in aging mice (Kaur, 2006). Conversely, protection from oxidative stress has been shown in young mice that overexpress ferritin H-chain, because of the iron-chelating properties of ferritin (Kaur, 2003; Wilkinson, 2006). A complete null for ferritin H-chain has been generated in mice; homozygous animals die in utero, whereas heterozygotes exhibit signs of mild iron deficiency (Thompson, 2003). Finally, mutations in the human ferritin L-chain lead to neurodegeneration in a condition described as neuroferritinopathy (Levi, 2005). Altogether, these results underscore the importance of ferritin regulation in mammalian health (Missirlis, 2007).

In Drosophila, Ferritin 1 heavy chain homolog (Fer1HCH) and Ferritin 2 light chain homolog (Fer2LCH) encode the ferritin subunits that compose the major, secreted form of ferritin (Charlesworth, 1997; Georgieva, 1999; Georgieva, 2002). The crystal structure of secreted ferritin from Trichoplusia ni revealed a symmetrical arrangement of H and L chains (Hamburger, 2005). Inter and intrasubunit disulfide bonds were shown to be important for the folding/assembly of Trichoplusia ni ferritin, and the respective cysteine residues mediating these bonds were also conserved in Drosophila melanogaster, suggesting that the ferritins of the two species share the same mode of assembly (Hamburger, 2005). The Fer1HCH amino acid residues that are required for ferroxidase activity in mammals were conserved in the insect ferritin structure, and a predicted Fer2LCH ferrihydrite nucleation site formed by the L-chains was also found (Hamburger, 2005; Missirlis, 2007 and references therein).

Intracellular localization of ferritin in many insects also differs from mammals. Ultra-structural studies, combining electron microscopy and energy electron loss spectroscopy, have revealed the presence of Calpodes ferritin in intracellular membrane compartments (Locke, 1984). Drosophila Fer1HCH and Fer2LCH subunits contain signal peptides that direct them to the endoplasmic reticulum upon translation. Fer1HCH also contains a predicted N-Glycosylation site. The two subunits are predominantly expressed in the midgut and are also abundant in hemolymph, where ferritin may transport iron for nutritional needs of Drosophila tissues (Georgieva; 2002; Missirlis, 2007 and references therein).

This study shows that mutational inactivation of either Fer1HCH or Fer2LCH in Drosophila, as well as the disruption of the ferroxidase center of Fer1HCH, results in developmental arrest and fly embryonic lethality. A novel fly strain expressing GFP-tagged Fer1HCH has been characterized: it was shown that GFP-Fer1HCH is incorporated into endogenous functional ferritin. This strain was used to study induction and trafficking of ferritin in the fly midgut, the major iron-storing organ in the insect (Missirlis, 2007).

Several novel findings on Drosophila ferritin are described in this work. Absence of either Fer1HCH or Fer2LCH results in embryonic lethality and modified Fer1HCH subunits (mutant in the ferroxidase center or GFP-tagged) cannot substitute for lack of Fer1HCH. However, if the same modified subunits are expressed in the presence of wild type subunits, they can be integrated into ferritin holomers without inducing dominant negative effects. Analysis of heterozygous loss-of-function ferritin fly mutants or flies overexpressing ferritin subunits revealed that a constant ratio of Fer1HCH and Fer2LCH is maintained, independent of their internal transcriptional expression levels. The structural cooperation of the two subunits that is secured via disulfide bonds (Hamburger, 2005) likely explains these observations. A posttranscriptional mechanism, possibly involving the degradation of subunits that are present in excess, ensures the presence of equal amounts of the two subunits. Such a mechanism can explain the absence of the IRE in Fer2LCH mRNAs in insects, since Fer1HCH translational repression by IRP-1A under iron-limiting conditions (that favor the IRE-containing transcripts) would then be sufficient to reduce levels of Fer2LCH. In contrast to results from mammalian cell or animal models (Thompson, 2003; Wilkinson, 2006), but consistent with what is known from human patients with hereditary hyperferritinemia cataract syndrome (Cazzola, 2002), experimental reduction or increase of ferritin levels through genetic manipulation in Drosophila caused only very mild alterations in the insectís iron homeostasis. These results point towards an independent regulatory system that controls iron sequestration into ferritin. The nature of this system is currently unknown, but could involve a putative iron chaperone that delivers iron to ferritin. However, the hypothesized chaperoneís function does not completely override the need for ferritin regulation, as shown by the phenotype of ferritin overexpressing flies that was lethal under low iron conditions but was rescued with iron supplementation. Alternatively, localization of ferritin in the Golgi apparatus of insect cells may prevent it from contact with the cytosolic and mitochondrial iron pools. It is currently not known how iron is delivered to the ferritin that resides in the secretory pathway of cells (Missirlis, 2007).

Evidence is provided that the ferritin genes are co-expressed during embryogenesis. It was wondered whether their genetic proximity is conserved in other Drosophila species with fully sequenced genomes. To this end, EvoPrinter, a new multigenomic DNA sequence analysis tool that facilitates the rapid identification of evolutionarily conserved sequences within the context of a single species, was used (Odenwald, 2005). In silico analysis of the Fer1HCH and Fer2LCH genomic locus by EvoPrinter produced an output of the combined mutational histories of six Drosophila species, superimposed on a reference sequence from Drosophila melanogaster. The results not only showed that clustering of the two genes is conserved, but also identified potentially shared regulatory regions. From the 17670 base pairs spanning the two genes that were analyzed, only 914 (or 5.2%) were conserved in an identical position in all six species tested. One third of these conserved sequences (306) were contained within the open reading frames. Surprisingly, over half of them (491) were not contained in the cDNA sequence, but were rather clustered in two regions: downstream of the Fer2LCH transcribed region or in the second intron of Fer1HCH. A particular stretch of ten base pairs, TTTGCACACG, was found three times in the second intron of Fer1HCH and could represent a binding site for an unidentified factor. The remaining conserved base pairs (117) were mostly concentrated in the 5í UTR of Fer1HCH, including the IRE itself. The conservation of the IRE over approximately 160 million years of collective evolutionary divergence underscores the functional significance of the IRE/IRP control system in all Drosophila species (Missirlis, 2007).

Ferritin regulation is a critical aspect of the organismís iron economy. The finding that ferritin expression in the iron region is constitutive (and remains so even under iron deficient conditions), but is inducible in the anterior midgut has a parallel with the expression of metallothioneins, which are the copper storage proteins. Interestingly, cuprophilic cells that are present anterior to the iron region show constitutive metallothionein expression, whereas cells in both the anterior and posterior midgut induce metallothionein expression and copper/metallothionein fluorescence at higher copper concentrations. Results presented in this paper combined with previous reports, lead to the following fundamental conclusion with respect to metal metabolism in the midgut of Drosophila: the insect midgut contains two sets of specialized cells, one of which constitutively expresses metallothionein and a second constitutively expresses ferritin. If either metal is present in great abundance, a third and fourth set of distinct midgut cells have the potential to induce transcription of the genes that encode the two metal storage proteins. Importantly, there are also cells in the midgut that do not respond significantly to high concentrations of these metals. Whether similar cellular populations exist in the mammalian intestine remains unknown, but cellular populations with specific metal contents have been described in plant seeds (Missirlis, 2007).

GFP-tagged ferritin revealed the complex physiologic orchestration of intestinal metal responses. In addition, it has enabled visualization of subcellular ferritin dynamics upon iron-mediated induction and should facilitate the dissection and subcellular localization of ferritin biosynthesis and trafficking. Serum ferritin is measured in clinical practice as a measure of total body iron stores (Beutler, 2002) and is an acute phase reactant (Tran, 1997) but few studies have addressed how ferritin is secreted in the circulatory system of humans (Ghosh, 2004; Renaud, 1991). Importantly, ferritin tagged with GFP could function and elucidate trafficking mechanisms in human cells and transgenic mice (Missirlis, 2007).

Ferritin was purified from iron-fed Drosophila extracts by centrifugation in a gradient of potassium bromide. On polyacrylamide gel electrophoresis, the product showed two protein bands corresponding to the ferritin monomer and dimer. Electrophoresis following dissociation with SDS and 2-mercaptoethanol revealed three strong bands of approximately 25, 26, and 28 kDa. N-terminal amino acid sequences were identical for the 25-kDa and 26-kDa subunits, but different for the 28-kDa subunit. Conserved ferritin PCR primers were used to amplify a 360-bp cDNA product, which was used to isolate a clone from a D. melanogaster cDNA library that contained the complete coding sequence for a ferritin subunit. Additional 5' sequence obtained by the RACE method revealed the presence of a putative iron regulatory element. The PCR product was also used to locate the position of the ferritin subunit gene at region 99F on the right arm of the third chromosome. The deduced amino acid sequence of the D. melanogaster ferritin subunit contained a signal sequence and resembled most closely ferritin of the mosquito Aedes aegypti (Charlesworth, 1997).

The organization of two closely clustered genes, Fer1HCH and Fer2LCH, encoding the heavy-chain homolog (HCH) and the light-chain homolog (LCH) subunits of Drosophila ferritin are reported in this study. The 5019-bp sequence of the cluster was assembled from genomic fragments obtained by polymerase chain reaction (PCR) amplification of genomic DNA and from sequences obtained from the BDGP. These genes, located at position 99F1, have different exon-intron structures (Fer1HCH has three introns and Fer2LCH has two introns) and are divergently transcribed. Computer analysis of the possibly shared promoter regions revealed the presence of putative metal regulatory elements (MREs), a finding consistent with the upregulation of these genes by iron, and putative NF-kappaB-like binding sites. The structure of two other invertebrate ferritin genes, from the nematode C. elegans (located on chromosomes I and V), was also analyzed. Both nematode genes have two introns, lack iron-responsive elements (IREs), and encode ferritin subunits similar to vertebrate H chains. These findings, along with comparisons of ferritin genes from invertebrates, vertebrates, and plants, suggest that the specialization of ferritin H and L type chains, the complex exon-intron organization of plant and vertebrate genes, and the use of the IRE/iron regulatory protein (IRP) mechanism for regulation of ferritin synthesis are recent evolutionary acquisitions (Dunkov, 1999).

Several mRNAs encoding the same ferritin subunit of Drosophila melanogaster were identified. Alternative RNA splicing and utilisation of different polyadenylation sites were found to generate the transcripts. The alternative RNA splicing results in ferritin transcripts with four unique 5' untranslated regions. Only one of them contains an iron-responsive element. The iron-responsive element was found to bind in vitro specifically to human recombinant iron regulatory protein 1. Furthermore, the ferritin subunit mRNAs are differentially expressed during development. These data provides the first molecular evidence that the presence of iron-responsive element in a ferritin mRNA is regulated by alternative RNA splicing (Lind, 1998).

The cDNA sequence of the Drosophila ferritin shares 99.4% identity at the DNA level with the recently published sequence reported by Charlesworth (1997). The deduced amino acid sequences show 100% identity and correspond to the 24 and/or the 26 kDa ferritin subunit of D. melanogaster. The resemblance between the sequences suggest that these are cDNAs corresponding to the same gene, but different alleles. Many insect ferritins are glycosylated and a putative glycosylation site (N-A-S) starting at residue 54 in the deduced amino acid sequence of the cDNA was found. The cDNA sequence reported here has a 3' UTR that is 229 nucleotide residues longer than the 3' UTR reported by Charlesworth (1997). The 3' UTR has two different putative poly(A) signals. One of the phage cDNA clones contains a poly(A) region 21 nt downstream of the first poly(A) signal. This is in the range of 11-30 nt, which are normally found downstream of the signal, and this suggests that both signals may be used. The gene encoding human ferritin H also has two alternative poly(A) signals, which are used in a tissue- and age-specific way. By analysing the 5' UTR for secondary structures, an IRE-like stem-loop structure was identified. This IRE binds to human recombinant IRP1. This binding is specific because excess of cold transcripts containing human IRE were able to reduce the binding of the radiolabelled transcript, whereas the same amount of cold transcript containing a mutated non-functional human IRE was unable to do this (Lind, 1998).

Comparison of the 5' UTR from different clones, derived from the phage library, it was found that this region differed between some clones. This diversity was further investigated by performing PCR on cDNA and cloning of the genomic sequence of the 5' flanking region of the ferritin gene. The PCR resulted in four subset of cDNAs and this suggests that ferritin subunit transcripts with at least four different 5' UTR exist in D. melanogaster. Further evidence for the presence of a set of ferritin transcripts in Drosophila was obtained from the database of expressed sequence tags (dbEST). By comparing the cDNA1-4 with the dbEST, sequences corresponding to cDNA1, cDNA3 and cDNA4. Alignment of the sequences of the PCR products and the genomic sequence reveals the existence of alternative RNA splicing of the 5' UTR of the ferritin transcript. Alternative RNA splicing is usually used to alter the open reading frame of a transcript, which will result in different translational products. In this case, the alternative splicing only affects the 5' UTR. Interestingly, only cDNA1 contains an IRE. All of the spliced cDNAs (cDNA2-4) have lost their IRE-containing intron. These transcripts will be insensitive to the iron-controlled regulation by the IRE/IRP system. As a consequence, cells containing ferritin transcripts without the IRE intron should be able to express ferritin even when the cellular iron concentration is low (Lind, 1998).

Total RNAs from different developmental stages of D. melanogaster, but also from separate parts of adult flies and from the Drosophila haemocyte cell line, mbn-2, were analysed by RT-PCR and Northern blot. The RT-PCR showed that both unspliced and spliced ferritin transcripts exist in all analysed samples. The Northern blot analysis showed that at least three different ferritin transcripts are present. The size of the largest transcript (1400 nt) is similar to the size of the cDNA reported here. The other two transcripts may be generated by alternative splicing and/or differential polyadenylation. A transcript with a 5' UTR corresponding to cDNA4 is 224 nt shorter than an unspliced transcript, and a transcript with a poly(A) tail after the first poly(A) site is 241 nt shorter than a transcript with a poly(A) tail after the second site. Therefore, the smallest transcript (900 nt) may be generated by both splicing of the 5' UTR and using the first poly(A) signal, because these two events will give a transcript with approximately the same size as the transcript detected in the Northern blot, while the middle transcript (1150 nt) may be generated by either splicing of the 5' UTR or using the first poly(A) signal. Further Northern blot analysis with probes specific for unspliced transcripts or transcript containing the very end of the 3' UTR supported these events. The Northern blot analysis suggests that the splicing event does not change drastic during development, except between the embryonic and the first instar larval stages. Instead, the splicing of the ferritin 5' UTR may be tissue-specific in Drosophila. The amount of the smallest transcript (900 nt) compared to the other transcripts varied significantly among different developmental stages. The same pattern was seen for the 1150 nt transcript in the Northern blot analysis with a cDNA1-specific probe, and this may be due to the fact that the two poly(A) signals are alternatively used in an age-specific way as in humans. If this is the case, the utilisation of the different polyadenylation sites varies significantly during the development of D. melanogaster, e.g. the first poly(A) site is only commonly used in first instar larvae and in adults, and is not or unusually used in embryos and pupae (Lind, 1998).

Eight different mRNAs encoding the same ferritin subunit seem to exist in Drosophila, and this diversity may be a valuable advantage for the organism to be able to regulate the expression of the ferritin subunit. The distribution of apo- and holo-ferritins has been studied in insects including Drosophila. The differences in the expression of ferritin in different tissues could be due to regulatory events, both at the transcriptional and at the posttranscriptional level. However, considering the findings reported in this study, it is likely that cells in ferritin-rich tissues, e.g. midgut, pericardial cells, and malpighian tubules, and/or in the fat body, which synthesised the haemolymph ferritin, contain more of the spliced transcripts without the IRE intron than the unspliced transcript, and that most of the expression of ferritin in these cells is not regulated at the translational level by the IRPs. The importance of this is still unknown, but perhaps certain insects need a continuous high amount of stored apoferritin as a buffer to be able to rapidly take care of excess iron. Another explanation may be that ferritin of insects has more functions than that of vertebrates, such as iron transport and excretion. Furthermore, the secreted ferritins of snail and Schistosoma mansoni have been shown to function as yolk storage protein and the transcripts encoding these proteins lack IREs (Lind, 1998).