Metal Metabolism: Transport, Development and Neurodegeneration 1313 Genetic screening for novel Drosophila mutants with discrepancies in iron metabolism Anuja Mehta 1 , Abhyuday Deshpande and Fanis Missirlis School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, U.K. Abstract Ferritin, a symmetrical 24-subunit heteropolymer composed of heavy and light chains, is the primary iron-storage molecule in bacteria, plants and animals. We used a genetically engineered strain of the model organism Drosophila melanogaster which expresses a GFP (green fluorescent protein)-tagged ferritin 1 heavy chain homologue from its native chromosomal locus and incorporated it into endogenous functional ferritin, enabling in vivo visualization of the protein and permitting easy assessment of ferritin status following environmental or genetic perturbations. Random mutagenesis was induced, and individual mutagenized chromosomes were recovered by classic crossing schemes involving phenotypical markers and balancer chromosomes. In wild-type larvae, ferritin is predominantly localized in the brain, in regions of the intestine, in wreath cells and in pericardial cells. A pilot genetic screen revealed a mutant fruitfly strain expressing GFP–ferritin in the anal pads, a pair of organs located ventrally in the posterior end of the fruitfly larva, possibly involved in ion absorption and osmoregulation, which are normally devoid of ferritin. Our continuing genetic screen could reveal transcription factors involved in ferritin regulation and novel proteins important in iron metabolism, hopefully with conserved functions in evolution. Introduction Iron is a major constituent of the Earth’s core. Iron oxides are abundant on the planet’s crust and are water-insoluble. Iron is ubiquitous in biological organisms; however, it is only present at trace levels. Because of its unique chemical versatility of transitioning between its more soluble, but potentially toxic, ferrous (Fe 2+ ) state and its insoluble ferric (Fe 3+ ) state, distinct molecular pathways have evolved to maintain cellular, tissue and systemic iron homoeostasis [1]. However, much less is understood about how iron is delivered to its various subcellular locations and about how cellular and biochemical iron requirements are fulfilled and co-ordinated. In the present article, we present a strategy to uncover new genes involved in iron metabolism using Drosophila genetics. Iron storage in insects Ferritin, a symmetrical 24-subunit iron-storage protein com- posed of heavy (H) and light (L) subunits, functions in iron storage [2]. Ferritin is present in bacteria, fungi, plants, insects and vertebrates. The hollow apoprotein can surround a core of approx. 4500 iron atoms. Ferritin brings about both sequestration and mobilization of the metal from the intra- cellular labile iron pool. The H subunit acts as the ferroxidase centre and oxidizes Fe 2+ to Fe 3+ , whereas the L chain is in- volved in biomineralization and has nucleation centres for deposition of the ferrihydrite mineral [3]. In mammals, Key words: anal pad, Drosophila melanogaster, ferritin, genetic screen, iron metabolism, mutagenesis. Abbreviations used: EMS, ethyl methanesulfonate; GFP, green fluorescent protein; Fer1HCH, ferritin 1 heavy chain homologue; Fer2LCH, ferritin 2 light chain homologue; H, heavy; L, light. 1 To whom correspondence should be addressed (email a.mehta@qmul.ac.uk). depending on the tissue type and physiological status, tight transcriptional control of the ferritin genes influences the formation of multiple isoforms of assembled ferritin complexes with varying ratios of H and L subunits [4]. Mam- malian ferritin is principally a cytosolic protein. In contrast, it has been shown that, in the insects Calpodes ethlius and Drosophila melanogaster, ferritin is present in intracellular membrane-bound compartments of the vacuolar system [5,6]. In Drosophila, the secreted form of ferritin composed of H and L subunits is encoded by the Fer1HCH (ferritin 1 heavy chain homologue) and Fe2LCH (ferritin 2 light chain homologue) genes [6–9]. In vivo expression of GFP (green fluorescent protein)-tagged holoferritin in Drosophila confirmed that iron-loaded ferritin molecules traffic through the Golgi organelle and are secreted into the haemolymph [6]. The crystal structure of secreted ferritin from Trichoplusia ni was solved and revealed a symmetrical arrangement of H and L chains [10]. Inter- and intra-subunit disulfide bonds were shown to be important for the folding/assembly of T. ni ferritin, and the respective cysteine residues mediating these bonds were also conserved in D. melanogaster, sugges- ting that the ferritins of the two species share the same mode of assembly [10]. Unlike in mammalian ferritin, disulfide linkages between the H and L subunits ensure that insect ferritin consists of 12 H and 12 L chains in the 24 subunit heteropolymer. The amino acid residues required for ferroxidase activity in mammals were conserved in the insect ferritin structure, indicating similar enzymatic functions of the H chain. A predicted Fer2LCH ferrihydrite nucleation site formed by the L chains was also found [10]. Insect ferritin subunits contain signal sequences [7], predicting that the mature polypeptide translocates to the endoplasmic Biochem. Soc. Trans. (2008) 36, 1313–1316; doi:10.1042/BST0361313 C  The Authors Journal compilation C  2008 Biochemical Society Biochemical Society Transactions www.biochemsoctrans.org