Mitochondrial Iron Metabolism: Study of Mitoferrin in Drosophila - DocsLib

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List of Papers

This thesis is based on the following papers, which will be referred to in the text by their roman numerals:

I. Metzendorf, C., Wu, W., and Lind, M. I. Overexpression of Droso- phila mitoferrin in l(2)mbn cells results in dysregulation of Fer1HCH expression. Biochem. J. 2009. 421:463-471.

II. Metzendorf, C. and Lind, M. I. Drosophila mitoferrin is essential for male fertility: evidence for a role of mitochondrial iron metabolism during spermatogenesis. (under review at BMC Developmental Bio- logy)

III. Metzendorf, C., Wiesenberger, G. and Lind, M. I. Characterization of Drosophila mitoferrin and CG18317 mutants. (Manuscript)

IV. Metzendorf, C. and Lind, M. I. The role of iron in the proliferation of Drosophila l(2)mbn cells. (Manuscript submitted to Insect Molecular Biology)

Reprint(s) were made with the permission from the publisher(s).

Contents

Introduction ...... 9 Iron Homeostasis ...... 11 Vertebrate iron homeostasis ...... 11 Systemic iron metabolism and homeostasis ...... 11 Regulation of cellular iron homeostasis – IRE/IRP ...... 12 Invertebrate iron homeostasis ...... 14 Regulation of cellular iron homeostasis ...... 14 Evolution of the IRE/IRP system ...... 16 Iron storage and transport ...... 18 Invertebrate iron homeostasis: Summarizing remarks ...... 24 Mitochondrial iron metabolism ...... 25 Iron-sulfur cluster biosynthesis ...... 26 Heme biosynthesis ...... 27 Mrs3/4 and mitoferrins ...... 27 Spermatogenesis in Drosophila melanogaster ...... 30 Objectives ...... 32 Results and discussion ...... 32 Overexpression of Drosophila mitoferrin in l(2)mbn cells results in dysregulation of Fer1HCH expression. (Paper I) ...... 32 Drosophila mitoferrin is essential for male fertility: evidence for a role of the mitochondrial iron metabolism during spermatogenesis. (Paper II) ...... 35 Characterization of Drosophila mitoferrin and CG18317 mutants. (Paper III) ...... 37 The role of iron in the proliferation of Drosophila l(2)mbn cells. (Paper IV) ...... 40 Svensk sammanfattning (Summary in Swedish) ...... 41 Acknowledgments ...... 43 References ...... 46 Abbreviations

BPS bathophenanthroline disulfonate cds coding sequence of a gene

DFO deferoxamine

DMT divalent metal transporter

FAC ferric ammonium citrate

ISC iron sulfur cluster

IRP iron-regulatory protein

IRE iron-responsive element dmfrn Drosophila mitoferrin orf open reading frame

TfR transferrin receptor

UAS upstream activating sequence (from yeast)

UTR untranslated region Introduction

Iron is an essential nutrient for almost all known organisms except for a few bacilli and lactobacilli. As it can easily be reduced and oxidized, it is an ideal cofactor for oxidases, reductases as well as dehydrogenases and plays essential roles in almost all cellular functions; e.g. cellular respiration (iron is present in all respiratory chain complexes) and DNA synthesis (iron is a cofactor of ribonucleotide reductase which converts ribonucleotides to de- oxyribonucleotides). The two most common iron-containing prosthetic groups are heme and iron-sulfur clusters (ISC). Synthesis of both is either completely or in part carried out in mitochondria, which therefore play a central role in the cellular iron metabolism and influence cellular iron homeostasis. The high reactivity of free iron with hydrogen peroxide also poses a threat to living organisms. Free iron generates reactive oxygen species via the Fenton reaction (Fig. 1). These radicals can damage proteins, lipids and DNA. In mammals, chronic iron overload results in tissue damage of nerve tissue, the liver, heart and adrenal gland (iron related diseases are reviewed in (Andrews, N. C. 2008)). In Drosophila, limiting the iron load of the diet results in a longer life span (Massie, H. R. et al. 1993) and overexpression of the metal-responsive transcription factor increases life span of flies on food with high iron concentrations and improves oxidative stress tolerance (Ba- hadorani, S. et al. 2008).

Fig. 1: The Fenton reaction. Repeated cycles of oxidation and reduction of iron in the presence of hydrogen peroxide generates reactive oxygen radicals.

Regulation and handling of iron within an organism is of great importance. Therefore, a regulation and iron storage system has evolved to keep the bal- ance between iron overload and meeting the organism´s need for iron. In the following work, different aspects of iron homeostasis in vertebrates and in-

9 vertebrates will be illuminated and compared where applicable. Understand- ing iron homeostasis in vertebrates and using invertebrates to model conserved aspects of the iron metabolism can help to understand and treat human iron-related diseases. In invertebrates several different and unique strategies exist to achieve iron homeostasis. A better overview of the diversity and function of invertebrate and vertebrate iron homeostasis will un- ravel the evolutionary background of iron homeostasis and provide knowledge that is needed to choose the appropriate model organism and to inter- pret findings from model organisms (e.g. C. elegans, D. melanogaster). In the cases of blood-borne pathogens and their blood sucking invertebrate vec- tors, such knowledge is anticipated to provide new tools and possibilities to fight malaria, yellow fever and tick-borne diseases.

10 Iron Homeostasis

Vertebrate iron homeostasis Systemic iron metabolism and homeostasis The high interest in mammalian iron homeostasis, due to several severe human diseases (e.g. Friedreich´s ataxia and hemochromatosis) caused by mutations in genes that effect iron homeostasis, has led to a rather good un- derstanding of many aspects of mammalian iron homeostasis (Andrews, N. C. 2008; Dunn, L. L. et al. 2007; Hentze, M. W. et al. 2004; Muckenthaler, M. U. et al. 2008). Mammals need huge amounts of iron for heme synthesis during erythropoiesis. Most of this iron is recycled from senescent erythrocytes, but a certain fraction, which is lost from the organism through shed- ding of epithelial cells, bleeding and secretion via urine, needs to be replen- ished. At least two iron uptake mechanisms exist in the gut. One involves duodenal cytochrome b or antioxidants in the food that reduce ferric iron -which is insoluble at physiological pH - to soluble, ferrous iron. The divalent metal transporter 1 (DMT1, also known as Nramp2) can then transport iron across the cytoplasmic membrane. The other system utilizes heme, which is transported into cells by the heme carrier protein-1 and oxidized by heme oxygenase to release the iron. Iron within duodenal enterocytes is released at the basal membrane by the transmembrane protein ferroportin. After oxidation of exported iron by the ferroxidase hephaestin, ferric iron is bound by transferrin (Tf). Tf, the major iron transport molecule in vertebrates, is composed of two similar lobes, which bind one iron atom each. Erythroblasts and most other cells present the transferrin receptor (TfR) on their surface when they are in need of iron. Iron loaded Tf binds the TfR and Tf/TfR complexes are endocytosed. Acidification of the endosome causes conformational changes in Tf that result in the release of iron. Both apo-Tf and TfR are recycled to the cell surface, while endosomal iron is reduced by STREAP3 and transported across the membrane by DMT1. In erythroblasts, iron is directly transferred into mitochondria (Sheftel, A. D. et al. 2007), where it is inserted into protoporphyrin IX – the last step of heme synthesis. Senescent erythrocytes are phagocytosed by reticuloendothelial macrophages. Iron, which is released from hemoglobulin is exported via ferroportin and bound by Tf after being oxidized by ceruloplasmin. This way iron is continuously recycled in the system.

11 A small peptide, hepcidin, which is expressed in hepatic cells, regulates the system-wide iron distribution. Upon binding of hepcidin to ferroportin, ferroportin is endocytosed and targeted for proteasomal degradation. This way, systemic iron uptake through the duodenum as well as iron release from macrophages and all other cells with ferroportin, is efficiently blocked. Hep- cidin expression is regulated transcriptionally and is induced by high iron levels and during inflammation. Low iron levels, hypoxia and erythropoiesis on the other hand act negatively on hepcidin expression.

Regulation of cellular iron homeostasis – IRE/IRP Several genes involved in cellular iron homeostasis are regulated on the translational level through the interaction of iron-regulatory proteins (IRP) and iron-responsive elements (IREs). IREs are conserved stem-loop RNA structures (Fig.2) in target mRNAs. In vertebrates two different IRPs exist. Both bind the same IREs but their binding activity is regulated through different mechanisms. While IRP-1 be- comes a cytoplasmic aconitase by binding an ISC at iron replete conditions, IRP-2 is ubiquitinated and thereby targeted for degradation in the protea- some. At low iron conditions, the ISC of IRP-1 is lost and IRP-2 accumu- lates, leading to increased binding of IRPs to IRE. In vertebrates IREs have been identified and functionally characterized for several genes (Table I). A general pattern is, that IRP binding to IREs in the 5´ untranslated region (UTR) of a mRNA results in inhibition of translation. Binding of IRP to IREs in the 3´UTR of a mRNA on the other hand results in the stabilization of the transcript, allowing increased translation of that mRNA. The specific functions of the two different IRPs are not well understood. Double IRP-1/IRP-2 knockout mice are embryonic lethal, showing that the IRP/IRE system is essential for development. Deletion of either IRP gene alone results only in rather mild phenotypes, which indicates that the functions of both IRPs are overlapping enough to complement the function of the missing IRP.

12 Fig. 2: IREs from human and Drosophila melanogaster ferritin heavy chain mRNA and a putative IRE from Hydra vulgaris ferritin heavy chain mRNA (from left to right). Iron responsive elements (IREs) are conserved stem-loop structures in mRNAs that are post-transcriptionally regulated through binding of iron regulatory protein (IRP). RNA was folded on the mFOLD server (Zuker, M. 2003).

A classic example for the regulation by the IRE/IRP system is the expression of ferritin, a cellular iron storage protein, and the TfR. At low iron conditions, IRP binding to several IREs in the 3´UTR of the TfR mRNA result in its stabilization, while translation of ferritin is blocked by IRP binding to the IRE in the 5´UTR of ferritin mRNA. Increased TfR expression results in an increased uptake of iron via the Tf/TfR pathway, until iron levels are high enough to stabilize ISCs, shifting the IRP function from IRE-binding to cytoplasmic aconitase. As IRPs are lost from the IREs of TfR and ferritin mRNAs, TfR mRNA is degraded and ferritin translation is increased. This results in a decrease in cellular iron uptake via Tf/TfR and increase in iron storage within ferritin (vertebrate iron homeostasis is excessively reviewed in (Andrews, N. C. 2008; Dunn, L. L. et al. 2007; Hentze, M. W. et al. 2004; Muckenthaler, M. U. et al. 2008)).

Table I: Vertebrate genes with IREs and the effects of IRP binding. (information in this table is accumulated from the following references (Cmejla, R. et al. 2006; Mucken- thaler, M. U. et al. 2008; Sanchez, M. et al. 2006; Tchernitchko, D. et al. 2002) ). Gene Function IRE location Effects of IRP binding H-Ferritin iron storage 5´UTR Inhibition of translation L-Ferritin -“- -“- -“- Ferroportin iron export -“- -“- eALAS erythroid heme synthesis -“- -“- HIF2alpha erythropoiesis -“- -“- MAconitase TCA Cycle -“- -“-

TfR1 iron uptake 3´UTR RNA stabilization DMT1 -“- -“- unknown CDC14A cell cycle -“- RNA stabilization MRCKα cytoskeletal reorganization -“- RNA stabilization

13 Invertebrate iron homeostasis Even though, vertebrate iron homeostasis is rather well understood, the pic- ture of invertebrate iron homeostasis and iron metabolism is rather incom- plete. One reason of course, is the great variety and heterogeneity of organisms within the group of invertebrates. Often, findings from one group of invertebrates do not apply to invertebrates in general. Some general features are conserved, but their specific function or regulation may differ. For in- stance, ferritins are conserved from invertebrates to vertebrates. While many ferritins are cytoplasmic iron storage proteins, ticks and insects have ex- creted ferritins that also fulfill iron transport functions (Hajdusek, O. et al. 2009; Nichol, H. et al. 2002). Furthermore, regulation of ferritins via the IRE/IRP system shows a great deal of variability as well. It is absent in nematodes, restricted to heavy chain homologues in Diptera, present in both subunits in Lepidoptera (Table II) and not studied at all in most invertebrates. In the following sections, some of the special features of invertebrate iron homeostasis will be highlighted, to give an impression of the diversity, but also to identify common patterns.

Regulation of cellular iron homeostasis In an early IRE binding activity assay with lysates from twelve different species, yeast and plant lysates failed to produce a mobility shift of human H- ferritin IRE, whereas vertebrate (human, mouse, chicken, frog and fish), insect and annelid lysates were shown to contain IRE-binding proteins (Rothenberger, S. et al. 1990). Proteins with similarity to human IRP-1 and with IRE-binding activity have been identified in several insect species, such as Aedes aegypti, An- opheles gambiae (Zhang, D. et al. 2002), Drosophila melanogaster (Lind, M. I. et al. 2006; Muckenthaler, M. et al. 1998) and Manduca sexta (Zhang, D. et al. 2001b; Zhang, D. et al. 2001a). Other invertebrates, where a functional IRE/IRP system has been experimentally confirmed, are the tick (Haj- dusek, O. et al. 2009) and annelids (Jeong, B. R. et al. 2006; Rothenberger, S. et al. 1990). Indications for the presence of a functional IRE/IRP system in sponges (Piccinelli, P. and Samuelsson, T. 2007), Cnidaria (Piccinelli, P. and Samuelsson, T. 2007), snails (von Darl, M. et al. 1994), bivalves (Durand, J. P. et al. 2004), crustaceans (Huang, T. S. et al. 1996; Huang, T. S. et al. 1999; Qiu, G. F. et al. 2008) and the horseshoe crab (Ong, D. S. et al. 2005) come from IREs identified in the 5´UTRs of ferritin mRNAs of these organisms. The nematode, Caenorhabditis elegans, on the other hand has a cytoplasmic protein with more similarity to human IRP-1 than to mitochondrial aconitase (Muckenthaler, M. et al. 1998), but despite its iron dependent cytoplasmic aconitase activity it does not bind IREs (Gourley, B. L. et al. 2003). Neither a bioinformatic search for conserved IRE sequences, nor binding as-

14 says with recombinant human or nematode IRP to C. elegans ferritin (FTN-1 and FTN-2), succinate dehydrogenase and mitochondrial aconitase (Aco-2) mRNAs supplied any evidence for the presence of IREs in this invertebrate (Gourley, B. L. et al. 2003). It was rather shown conclusively that regulation of C. elegans ferritins is solely regulated by transcription and that it is unlikely that C. elegans has a functional IRE/IRP system (Gourley, B. L. et al. 2003). Transcriptional regulation of ferritin expression also plays a role in organisms that have the IRE/IRP system. Cloned ferritin heavy subunit from the giant fresh water prawn, Macrobrachium rosenbergerii, contains a well conserved IRE, indicating that it is likely to be regulated on the post-transcriptional level. Still, injection of iron into the hemolymph increased the transcript abundance of this ferritin in muscle, ovary and heart (Qiu, G. F. et al. 2008). In dipterans, which express ferritin heavy chain homologue mRNAs that contain an IRE, a similar transcriptional response of ferritin expression was observed in mosquito cell culture (Pham, D. Q. et al. 1999) and in fruit flies (Georgieva, T. et al. 1999). Mapping of the promoter region of the ferritin heavy chain homologue in the mosquito identified the presence of enhancer and silencer elements of which some are iron responsive (Pham, D. Q. et al. 2005). Expression of the light chain homologue subunit which has no IRE in its mRNA has also been shown to be regulated transcriptionally in the mosquito (Pham, D. Q. and Chavez, C. A. 2005). Even though it is fre- quently stated, that ferritin expression, in response to iron, is regulated via the IRE/IRP system in vertebrates, it should be noted, that both H and L ferritins are transcriptionally regulated during development (leading to tissue specific H to L ferritin ratios), inflammation and oxidative stress (Arosio, P. et al. 2008; Torti, F. M. and Torti, S. V. 2002). To-date, ten different genes that are post-transcriptionally regulated by the IRE/IRP-system have been identified in vertebrates (Table I), while in invertebrates mRNAs with IREs have been identified in only three different genes: ferritin heavy and light chain homologue subunits and succinate dehydrogenase (Table II). In invertebrates, most IREs have been found in ferritin heavy chain homologue genes, while only a small number of ferritin light chain homologues with an IRE are known. Drosophila is the only invertebrate for which a functional IRE has been reported in the mRNA for a citric acid cycle enzyme (Table II). Furthermore, alternative splicing in the 5´UTR of Drosophila melanogaster Fer1HCH pre-mRNA results in different transcripts. Some of these transcripts do not have an IRE (Lind, M. I. et al. 1998) and their abundance is increased under iron loading conditions (Georgieva, T. et al. 1999). Searching the Drosophila melanogaster 5´ and 3´UTR sequence libraries for IREs using PatSearch (Grillo, G. et al. 2003) followed by folding of can- didate mRNA sequences did not identify any new putative IREs; while both previously identified Drosophila IREs (Fer1HCH and SdhB) were dis-

15 covered and folded correctly (unpublished data). In a screen to discover new mammalian IREs, two putative new IREs in genes S6K and ADAR were lis- ted for Drosophila, but not confirmed functionally (Sanchez, M. et al. 2006). S6K was also found in our database search, but its secondary structure pre- diction did not fit the criteria for a functional IRE.

Evolution of the IRE/IRP system In D. melanogaster two IRPs (IRP-1A and IRP-1B) have been identified and they share more similarity with human IRP-1 and with each other than with human IRP-2 (Muckenthaler, M. et al. 1998). While both have aconitase activity, only IRP-1A is able to bind IREs (Lind, M. I. et al. 2006). These findings and the fact that C. elegans lacks the IRE/IRP system, were taken as evidence that during evolution an ancient gene for cytoplasmic aconitase was duplicated in the common ancestor of vertebrates and insects and one variant had acquired IRE-binding activity (Lind, M. I. et al. 2006). One functional aconitase, which localizes to the cytosol as well as to mitochondria, has been identified in the protozoan Trypanosoma brucei (Saas, J. et al. 2000), whereas the platyhelminthe Schistosoma mansoni very likely lacks an IRE-binding protein and both of its ferritins lack an IRE in their 5´UTR (Schüssler, P. et al. 1996). On the other hand, in the protozoan Plasmodium falciparum, a cytoplasmic aconitase with IRP-binding activity as well as three putative IREs associated with unidentified genes (Loyevsky, M. et al. 2003) have been reported. Whether the protozoan IRE/IRP-system is phylo- genetically related to that of metazoa is not known. In a recent review about the IRE/IRP-system, the absence of the IRE/IRP-system in plants and the presence of an IRE-binding aconitase and IREs in operons for cytochrome oxidase and iron uptake genes in Bacillus subtilis have led to the formulation of two alternative hypothesis (Leipuviene, R. and Theil, E. C. 2007): Either the IRE/IRP system is ancient and was lost in plants and other organisms that lack this system, or it has arisen independently in several groups of organisms. A thorough bioinformatic search by Piccinelli and Samuelsson identified putative IREs in ferritin mRNAs in many metazoan taxa, all the way down to Cnidaria and Porifera (Piccinelli, P. and Samuelsson, T. 2007). The only exceptions were Caenorhabditis elegans and Schistosoma mansoni, which have previously been shown to lack a functional IRE/IRP-system (Gourley, B. L. et al. 2003; Schüssler, P. et al. 1996). The high conservation of the IRE/IRP-system in the regulation of ferritin within Metazoa is in favor of a convergent evolution of IRE/IRP systems in bacteria, protozoa and the Meta- zoan stem group. From the bioinformatic study, it can be concluded, that invertebrates possess IREs only in ferritin mRNAs (with the exception of Dro- sophila, where an IRE is present in succinate hydrogenase b) and that more genes came under the control of the IRE/IRP system during the evolution of Chordata, Vertebrata and Mammalia (Piccinelli, P. and Samuelsson, T. 2007).

16 Table II: Presence and absence of IREs in invertebrate mRNAs

Organism Ferritin SdhB References

IRE ferroxidase center Annelida Periserrula leuco- yes yes (Jeong, B. R. et al. 2006) phryna

Chelicerata tick Fer1 yes yes (Mulenga, A. et al. 2004) Fer2 no yes (Hajdusek, O. et al. 2009) horseshoe crab yes yes (Ong, D. S. et al. 2005)

Cnidaria Hydra vulgaris secreted ferritin no* yes (Böttger, A. et al. 2006) cytosolic ferritin yes* yes (Böttger, A. et al. 2006) Nematostella vectensis yes unknown** (Piccinelli, P. and Samuelsson, T. 2007)

Crustacea fresh water crayfish yes yes (Huang, T. S. et al. 1996) (Huang, T. S. et al. 1999) fresh water giant prawn yes yes (Qiu, G. F. et al. 2008) Daphnia magna yes yes (Poynton, H. C. et al. 2007)

Insecta mosquitos yes yes (Dunkov, B. C. et al. 1995; Zhang, D. et al. 2002) no no fruit fly yes yes (Georgieva, T. et al. 2002; Lind, M.I. et al. 1998) no no (Georgieva, T. et al. 2002) yes (Kohler, S. A. et al. 1995) tsetse fly yes yes (Strickler-Dinglasan, P. M. et al. 2006) no no (Strickler-Dinglasan, P. M. et al. 2006) long corn beetle yes yes (Kim, S. R. et al. 2004) no no (Kim, S. R. et al. 2004) Lepidoptera yes yes (Kim, B. S. et al. 2002; Nichol, H. and Locke, M. 1999; Zhang, D. et al. 2001a) yes no (Kim, B.S. et al. 2002; Nichol, H. and Locke, M. 1999; Zhang, D. et al. 2001a) Hymenoptera yes yes (Dunkov, B. and Georgieva, T. 2006; Wang, D. et al. 2009) yes no (Dunkov, B. and Georgieva, T. 2006;)

Mollusca oyster GF1 and GF2 yes yes (Durand, J. P. et al. 2004) abalone yes yes (De Zoysa, M. and Lee, J. 2007) no no (De Zoysa, M. and Lee, J. 2007) Lymnea stagnalis soma ferritin yes yes (von Darl, M. et al. 1994) yolk ferritin no yes (von Darl, M. et al. 1994) Helix pomatia yes yes (Xie, M. et al. 2001)

17 Table II continued.

Organism Ferritin SdhB References

IRE Ferroxidase center

Nematoda

Caenorhabditis elegans FTN-1 & FTN-1 no yes no (Gourley, B. L. et al. 2003)

Platyhelminthes Schistosoma mansoni (Schüssler, P. et al. 1996) soma and yolk no yes ferritin Clonorchis sinensis yolk ferritin no (Tang, Y. et al. 2006)

Porifera Suberites domuncula FTN1 and FTN2 no? yes (Krasko, A. et al. 2002) Suberites ficus yes unknown* (Piccinelli, P. and Samuelsson, T. 2007) * three ferritin sequences from NCBI were obtained and classified as secreted/cytoplasmic by WolfPSORT (http://wolfpsort.org/). Two very similar cytoplasmic ferritins (probably gene duplication or database errors) contain an IRE-like structure. ** IREs were identified bioinformatically by Piccinelli and Samuelsson in ferritin genes of unspecified subunit type.

Iron storage and transport Ferritin Ferritin is the major iron storage protein and can be found in most organisms - eubacteria, archaea, plants and animals- except for yeast (Arosio, P. et al. 2008). Holo-ferritin is composed of 24 subunits that form a hollow sphere that stores up to 4500 iron atoms in the form of a ferric oxohydroxide core. Bacterial holo-ferritin is composed of only one subunit type, while vertebrate (Arosio, P. et al. 2008) and insect (Nichol, H. et al. 2002) holo-ferritin is a heteropolymer of heavy and light chain homologue subunits. In several other invertebrates, such as nematodes (Romney, S. J. et al. 2008), snails (von Darl, M. et al. 1994), abalone (De Zoysa, M. and Lee, J. 2007) and ticks (Hajdusek, O. et al. 2009; Mulenga, A. et al. 2004), at least two distinct ferritin subunits have been identified, even though it is not known for all of these organisms whether these subunits form hetero- or homopolymers. Heavy chain subunits and their homologues are characterized by the presence of seven conserved amino acid residues that form a ferroxidase center, needed for oxidation of ferrous iron to ferric iron. Light chain subunits lack these conserved residues, but expose basic side chains to the cavity, which promote crystallization and stabilization of the iron core. Ferritins with more

18 heavy chain subunits show increased protection from iron induced oxidative stress, while ferritins with more light chain subunits improve long term storage of iron (Arosio, P. et al. 2008). While mammalian ferritin has a tissue specific ratio of light and heavy chain subunits, the crystal structure of lepidopteran secreted ferritin revealed a fixed ratio of 12 light and 12 heavy chain subunits (Hamburger, A. E. et al. 2005). Further inspection of the structure identified another difference to vertebrate ferritins. Inter-subunit disulfide bonds, which do not occur in vertebrate ferritins, between pairs of H and L subunits are formed and it was proposed that hetero-dimers of H and L subunits assemble in a first step, before holo-ferritin is formed (Hamburger, A. E. et al. 2005). Genetic interactions between Drosophila melanogaster Ferritin Heavy (Fer1HCH) and Light (Fer2LCH) Chain Homologue subunits indicate that the ratio between both subunits is fixed in the fruit fly as well (Missirlis, F. et al. 2007). Furthermore, it was shown that overexpression of either subunit does not increase the protein level of that subunit, unless the other subunit is overexpressed as well (Missirlis, F. et al. 2007). It was argued, that due to this unknown post-translational regulation mechanism, regulation of Fer- 2LCH by the IRE/IRP-system was not needed, as the regulation of Fer1HCH by the IRE/IRP-system would in turn lead to a co-regulation of Fer2LCH (Missirlis, F. et al. 2007). This indeed could be a good explanation for the absence of an IRE in the message of Fer2LCH. Nonetheless, both H- and L- ferritin messages of Lepidoptera, where the ratio between the ferritin subunits in holo-ferritin is fixed as well, contain an IRE. Therefore, the lack of an IRE in the light chain ferritin mRNA of dipterans may have another reason. Unlike as in vertebrates, were ferritin is mainly a cytoplasmic iron storage protein, several invertebrates (insects, horseshoe crab, snails and ticks) util- ize ferritin as a combined iron storage/transport molecule. Insect (Nichol, H. et al. 2002) and other invertebrate (Hajdusek, O. et al. 2009; von Darl, M. et al. 1994) secreted ferritins, contain an N-terminal leader sequence that tar- gets them to the endoplasmatic reticulum. Vertebrate ferritins lack such signal peptides, but are present in blood plasma, cerebrospinal fluid and synovi- al fluids. Plasma levels of vertebrate ferritin are low compared to total body ferritin, but plasma ferritin serves as a clinical parameter, since its concentra- tion is increased during the acute phase of infection, oxidative stress and iron loading, while iron deficiency results in lower levels. The mechanism of ferritin secretion in vertebrates is still unknown, but most likely dissimilar to that in insects as no export signal has been detected in mammalian ferritins (reviewed in (Arosio, P. et al. 2008)). A function of ferritin as an acute phase protein in invertebrates has also been suggested. In several animals, such as Platynereis dumerilii (Annelida) (Altincicek, B. and Vilcinskas, A. 2007), the horseshoe crab (Ong, D. S. et

19 al. 2005), Drosophila melanogaster (Levy, F. et al. 2004b; Levy, F. et al. 2004a; Vierstraete, E. et al. 2004a), and Tribolium castaneum (beetle) (Altin- cicek, B. et al. 2008) changes in ferritin protein abundance in hemolymph or ferritin mRNA in hemocytes were observed after induction of the immune response. In Drosophila melanogaster 2D-gel electrophoresis indicates that ferritin is post-translationally modified after infection (Vierstraete, E. et al. 2004b). It has also been shown that iron-loading has a negative impact on fruit fly survival after fungal infection, whereas iron-starvation has a positive impact (Chamilos, G. et al. 2008). In ticks and snails, two types of ferritin subunits, that each contain conserved amino acids of the ferroxidase center and that are expressed in a tissue specific manner, have been reported (Hajdusek, O. et al. 2009; von Darl, M. et al. 1994). In Lymnea stagnalis these ferritins are referred to as somatic and yolk ferritin. Somatic ferritin mRNA contains an IRE and the deduced protein sequence lacks a secretion signal, which is in line with its cytoplasmic localization (von Darl, M. et al. 1994). Yolk ferritin is secreted (Bottke, W. 1982) and the absence of an IRE was interpreted as a sign for its transcriptional regulation during development (von Darl, M. et al. 1994). Yolk and soma specific heavy chain homologue ferritins have also been reported for the tape worm Schistosoma mansoni (Dietzel, J. et al. 1992; Schüssler, P. et al. 1995), but both lack an IRE in their 5´UTR (Schüssler, P. et al. 1996). In the sheep tick, Ixodes ricinus, Fer1 is a cytoplasmic ferritin that is ubiquitously expressed in all tissues and its translation is regulated by the IRE/IRP- system (Hajdusek, O. et al. 2009). Expression of Fer2, a secreted ferritin, is independent of the IRE/IRE-system and strongest in the gut and present at low levels in ovary and salivary glands (Hajdusek, O. et al. 2009). The study of Hajdusek et al. also demonstrated that Fer2 is very likely transporting iron from the gut to peripheral tissues, as RNAi of Fer2 resulted in decreased Fer1 protein levels in peripheric tissues of blood fed ticks compared to mock treated ticks (Hajdusek, O. et al. 2009). A third type of ferritin, which has so far been identified in mammals (Levi, S. et al. 2001) and Drosophila (Missirlis, F. et al. 2006) is a homo- polymer and specific for mitochondria. Its expression in mice is highest in tissues with high metabolic activities (e.g. testis, heart, nervous tissue) and absent from tissues that have iron storage functions (e.g. liver), which was taken as an indication that mitochondrial ferritin may not perform iron storage functions but that it instead serves primarily to protect mitochondria of these cell types from oxidative stress (Santambrogio, P. et al. 2007). In the fruit fly, mitochondrial ferritin message is also highly abundant in testis (Missirlis, F. et al. 2006). Overexpression of vertebrate mitochondrial ferritin in HeLa cells resulted in an increased mitochondrial iron retention, down regulation of cytoplasmic ferritin and upregulation of the transferrin receptor – all indicative of a shift of the iron pool into mitochondria (Corsi, B. et al. 2002). Overexpression of Drosophila melanogaster mitochondrial ferritin

20 (Fer3HCH) did neither cause dramatic changes in the expression of Fer- 1HCH or Fer2LCH, nor a change in body iron levels or iron loading of secreted ferritin (Missirlis, F. et al. 2006). Instead, Fer3HCH overexpression reduced the female life span on low iron food, indicating that at least in fe- males, mitochondrial ferritin may sequester iron from essential processes (Missirlis, F. et al. 2006).

Transferrin Transferrins are high-affinity, ferric iron binding proteins that can be ordered in different groups: serum transferrin (Tf, transferrin), melanotransferrin (mTf), lactoferrin, ovotransferrin (oTf) and nicaTransferrin. Structurally, transferrins are divided into monolobal (nicaTransferrin), bilobal (sTf, melanotransferrin, lactoferrin and oTf) and polylobal (heavy chain subunit of pacifastin) proteins. Transferrin is the major iron transport molecule in vertebrates (Andrews, N. C. 2008; Dunn, L. L. et al. 2007), the function of lactoferrin is mainly antibiotic in all secreted fluids (Farnaud, S. and Evans, R. W. 2003), whereas oTf is a serum transferrin in birds, that also occurs in egg whites (Lambert, L. A. et al. 2005). While many roles have been attrib- uted to melanotransferrin, it is still a mystery what specific role it has (Suryo, R. Y. and Richardson, D. R. 2009). Transferrins have been shown to occur in several insect species (Nichol, H. et al. 2002) and in ascidians (Martin, A. W. et al. 1984; Uppal, R. et al. 2008). The heavy chain subunit of Pacifastacus leniusculus pacifastin – an arthropod proteinase inhibitor - is a three-lobed protein with similarity to transferrins (Liang, Z. et al. 1997). Iron binding of pacifastin was observed and from analysis of the conservation of iron binding residues it was speculated that lobes two and three were likely to bind iron (Liang, Z. et al. 1997). Bilobal transferrins are composed of two highly similar lobes (N-terminal and C-terminal lobe), each of which can bind one ferric iron atom. There- fore, it was speculated that bilobal transferrins arose from a duplication of an ancient monolobal precursor in the vertebrate stem group. It was argued, that small proteins with masses less than 70 kDa are easily lost during ultrafiltra- tion in the glomuleri of vertebrates, and that bilobal transferrins with molecular masses around 80 kDa solved that problem. Experimental support to this theory was provided by the finding that oTf half-molecules from hen eggs, each with a molecular mass of ~40 kDa, were readily lost with the urine after injection into mice (Williams, J. et al. 1982). The identification of a 40 kDa ascidian plasma transferrin seemed to confirm this hypothesis (Mar- tin, A. W. et al. 1984) until bilobal transferrins were identified in insects (Bartfeld, N. S. and Law, J. H. 1990; Huebers, H. A. et al. 1988). Investiga- tion of the iron binding properties of nicaTransferrin (Uppal, R. et al. 2008) from Ciona intestinalis and comparison with iron binding properties of bilobal transferrins revealed that iron binding in bilobal proteins is cooperative

21 and with a higher affinity than in monolobal proteins (Tinoco, A. D. et al. 2008). This was seen as the selective advantage for bilobal transferrins over monolobal transferrins (Tinoco, A. D. et al. 2008) and is currently the best theory for explaining bilobal transferrins. From a large scale phylogenetic analysis of transferrin evolution, including amino acid sequences of 71 transferrins from 51 species, it was concluded that the duplication of the transferrin lobes occurred before the split of the deuterosomes and protostomes (Lambert, L. A. et al. 2005). Another striking difference between insect and mammalian transferrin became clear after the isolation and cloning of the first insect transferrin (Bart- feld, N. S. and Law, J. H. 1990). The C-lobes of major hemolymph transferrins from holometabole insects (e.g. Drosophila melanogaster (Yoshiga, T. et al. 1999), Manduca sexta (Bartfeld, N. S. and Law, J. H. 1990), as well as Apis melifera, Bombyx mori and mosquitoes (Dunkov, B. and Georgieva, T. 2006)) contain deletions and lack the conserved iron binding residues, while hemimetabole insects (Blaberus discoidalis (Jamroz, R. C. et al. 1993), Mas- totermes darwiniensis (Thompson, G. J. et al. 2003)) have retained the conserved residues for iron-binding in both transferrin lobes. In the cockroach, iron-binding to both lobes has been shown experimentally (Gasdaska, J. R. et al. 1996). In mosquitoes, transferrin is a constitutive protein of the hemolymph and upon infection the level of its mRNA is increased (Yoshiga, T. et al. 1997). As some pathogenic bacteria can express Tf-receptors that mainly interact with the C-lobe of transferrin, Yoshiga et al. proposed that the de- funct C-lobe of insect transferrin may serve as a means to avoid “iron-pirat- ing” by pathogenic bacteria (Yoshiga, T. et al. 1997). Evidence that transferrin plays a role in insect immune response comes from a multitude of reports, where transferrin transcript abundance or protein was increased after immune challenge (De Gregorio, E. et al. 2001; Kim, B. Y. et al. 2008; Le- hane, M. J. et al. 2008; Levy, F. et al. 2004b; Levy, F. et al. 2004a; Seitz, V. et al. 2003; Thompson, G. J. et al. 2003; Wang, C. et al. 2007; Yoshiga, T. et al. 1997; Yoshiga, T. et al. 1999). Therefore, it was suggested that it may fulfill a dual role in insects, that of iron transport (like serum transferrin) and as an antibiotic (like lactoferrin) (Dunkov, B. and Georgieva, T. 2006). Interestingly, from the published genome sequences of insects and an uro- chordate, so far no homologue of the mammalian transferrin receptor could be identified (Nichol, H. et al. 2002). Delivery of transferrin iron to fat body cells has been demonstrated in vitro in Manduca sexta and radioactive iron from food was bound by hemolymph ferritin and transferrin (Huebers, H. A. et al. 1988). Transferrin expression was downregulated in iron fed Aedes aegypti and Drosophila melanogaster, and it was reasoned that iron transport is not needed in an already iron loaded organism (Harizanova, N. et al. 2005; Yoshiga, T. et al. 1999). The injection of iron into hemolymph of Protaetia brevitarsis increased transferrin mRNA levels above those after PBS injection (Kim, B. Y. et al. 2008). Injection of free iron into hemolymph generates

22 oxidative stress that seems to be countered by the expression of transferrin. A vitellogenic function, that is somewhat related to iron transport, has been at- tributed to transferrin from Sarcophaga peregrina. It was shown to transfer iron from the hemolymph into oocytes (Kurama, T. et al. 1995).These findings show that transferrin is likely to act as an iron transporter in insects, but to which extend is unclear, as insects also use ferritin for iron transport. It should also be mentioned, that insect genomes revealed the presence of two (in mosquitoes three) more transferrin-like proteins (Dunkov, B. and Georgieva, T. 2006). Other, than that they are similar to melanotransferrin, which is membrane bound, they are uncharacterized. A protein which ini- tially was described as a major yolk protein in the sea urchin, was later shown to be a transferrin-like protein with in vitro iron-binding capabilities (Brooks, J. M. and Wessel, G. M. 2002), and in the end turned out to transport zinc from the digestive tract to the gonads in vivo (Unuma, T. et al. 2007). Therefore, some of the newly identified transferrin-like proteins in insects may also turn out to transport other metals than iron.

Divalent metal transporters in invertebrates Genes coding for proteins with high similarity to divalent metal transporters (DMTs) have been identified in insects and the nematode Caenorhabditis elegans. Other than that, knowledge about iron uptake mechanisms in invertebrates is scarce. Mutations in malvolio, a Drosophila melanogaster gene coding for a protein with high similarity to human Natural resistance-associated macrophage proteins (Nramp), cause abnormal taste behavior (Rodrigues, V. et al. 1995) that can be suppressed by supplementation of the food with metal ions (Mn2+ and Fe2+ but not Ca2+, Mg2+ or Zn2+) (Orgad, S. et al. 1998) or by expression of human Nramp1 (D'Souza, J. et al. 1999). Besides its ability to transport Mn2+ and Fe2+, malvolio - in analogy to its human orthologue- has also been shown to transport copper (Southon, A. et al. 2008). It is expressed in the nervous system, plasmatocytes, malpighian tubules, the alimentary canal, testis, anterior (copper region) and posterior (iron region) midgut (Folwell, J. L. et al. 2006; Rodrigues, V. et al. 1995). Expression in the gut and in plasmatocytes suggests, that malvolio might carry out a function in both immunity (as Nramp1 does in vertebrates) and metal absorption in the gut. After a blood meal, the malvolio homologue in mosquitoes, was downregulated in the midgut and upregulated in malpighian tubules (Martinez- Barnetche, J. et al. 2007). In honey bees, it was shown that Mn2+ levels and malvolio expression in the head correlated with division of worker bees in pollen foragers, nectar foragers and nurses, indicating that feeding-related genes are used in social insects to divide labor (Ben Shahar, Y. et al. 2004). Three genes (SMF-1, SMF-2, SMF-3) coding for proteins with sequence similarity to DMT have been identified in Caenorhabditis elegans. SMF-1

23 and SMF-2 can suppress EDTA sensitivity of yeast DMT-mutants, indicating a function in metal transport. During a high throughput screen for protein localization, SMF-3 was shown to localize to the gut, implying a possible function in metal absorption (reviewed in (Au, C. et al. 2008).

Invertebrate iron homeostasis: Summarizing remarks Cellular iron homeostasis in many invertebrate taxa relies at least partially on the IRE/IRP system, but transcriptional regulation is very likely to play the major role. Furthermore, apart from the presence of two heavy chain like ferritins in Porifera (Krasko, A. et al. 2002), two ferritins in Hydra vulgaris (Böttger, A. et al. 2006) and putative IREs in the messages of these ferritins (Piccinelli, P. and Samuelsson, T. 2007), little is known about iron homeostasis in basal groups of invertebrates. Nevertheless, there seems to be a trend in the usage of transcriptional regulation to post-transcriptional regulation of iron homeostasis from invertebrates to vertebrates. Most likely this trend is a product of the functional adaptation of the regulation of iron homeostasis during evolution. One cause for this shift to more post-transcriptional regulation in vertebrates may be the need for a much closer and faster regulation or a more complex integration of multiple signals. Identifying the driving force in this shift of regulation would be extremely exciting, as it would also teach us a lot about the evolution of vertebrates and the dynamics of iron homeostasis. While the function of ferritin, despite certain adaptations (e.g. iron transport and immunity), is to store iron, it is much more difficult to ascribe a clear function to transferrins in invertebrates. It binds iron, but whether for transport or as an iron scavenger to suppress microbial growth, or both, is not clear. Furthermore, insect Tf2 and Tf3 (and Tf4 in mosquitoes), which have only been annotated in insect genomes, have not been characterized in the slightest. The polylobal transferrin-like subunit of pacifastin from Paci- fastacus leniusculus is an indication of the divers roles that transferrin may play in invertebrates, whereas the lack of reports about transferrins in invertebrate groups other than crustaceans, insects and tunicates, may suggest several things. Either it was a trait developed rather close to the deuterostome/protostome split, and lower animals lack transferrins or the putatively monolobal ancestral transferrins in lower invertebrates became unrecogniz- able during the millennia. The third explanation, and simplest, would be, that no one has looked for transferrins in lower invertebrates. Clearly, the field of invertebrate transferrin research is mainly uncharted and likely to yield inter- esting discoveries. It is also rather clear, that transferrin and ferritin play some role during the immune response. To identify this role may prove to be a difficult task, as manipulations of either gene will certainly impact the iron household and lead to secondary effects, which need to be separated from direct effects on

24 the immune response. As mutants of ferritin are embryonic lethal in Droso- phila, these difficulties become even more evident.

Mitochondrial iron metabolism Currently the mitochondrial iron metabolism is mainly studied in yeast and vertebrate systems (zebrafish, mouse and human cell lines), leaving a big gap. As the mitochondrial iron metabolism is highly conserved this may not be a big problem in itself, but yeast lacks the IRP/IRE system, ferritins and transferrins, which are all present in Drosophila. Therefore, a better under- standing of Drosophila mitochondrial iron metabolism and its integration into the cellular iron metabolisms may provide better models than yeast. Mitochondria are the hot-spot of the eukaryotic iron metabolism. Here, ISCs are synthesized and iron is inserted into protoporphyrin IX – the final step in heme biosynthesis (Fig. 3). As ISCs and heme are prosthetic groups of many proteins with essential functions (Fig. 4) the disruption of their synthesis leads to severe diseases (reviewed in (Lill, R. and Mühlenhoff, U. 2008; Mense, S. M. and Zhang, L. 2006; Rouault, T. A. and Tong, W. H. 2008)).

Iron-sulfur cluster biosynthesis The ISC biosynthesis pathway is well conserved from bacteria to higher eukaryotes and about 20 different proteins are involved in the eukaryotic ISC assembly machinery. ISC biosynthesis requires four key steps: (1) abstraction of sulfur from cystein by a cystein desulfurase (NFS1p in yeast, ISCS in human), (2) iron delivery in bioavailably form (mitoferrin and probably by frataxin), (3) assembly of an ISC on a scaffold protein (ISU1p yeast, ISCU human) and (4) transfer of the ISC to an apo-ISC protein with help of the ISC-protein assembly machinery. The transfer of assembled ISCs from the scaffold proteins to mitochondrial recipient proteins is probably carried out by ATP dependent conformational changes that are facilitated by co-chaperone HSCB and heat shock 70-kDa protein 9 (HSCPA9). In yeast, mitochondrial ISC synthesis is necessary for extramitochondrial ISC-protein maturation, but it seems very unlikely that complete clusters are exported. Atm1p (ABCB7 in humans (Csere, P. et al. 1998)) has been shown to transport an eagerly sought but still unidentified compound that is both necessary for cytoplasmic ISC maturation (Kispal, G. et al. 1999; Pondarre, C. et al. 2006) and modulation of cellular iron homeostasis (Cavadini, P. et al. 2007; Pondarre, C. et al. 2007; Rutherford, J. C. et al. 2005). Both Atm1p and ABCB7 depleted cells accumulate mitochondrial iron, indicating that mitochondrial ISC synthesis is emitting a signal that controls cellular iron homeostasis. In vertebrates a direct integration of ISC synthesis into the regu-

25 lation of cellular iron homeostasis is conveyed by the IRP-1/IRE system. In yeast – which lacks the IRP/IRE system – mitochondrial ISC synthesis regulates cellular iron homeostasis via a signalling cascade containing Fra1/Fra2/Grx3/Grx4. Recently it has been shown in vitro that Grx3/Grx4 and Fra2 form heterodimeric complexes that contain an ISC (Li, H. et al. 2009) giving first mechanistic insights how ISC synthesis may be integrated into the regulation of cellular iron homeostasis in yeast. (Reviewed by (Lill, R. et al. 2006; Lill, R. and Mühlenhoff, U. 2005; Rouault, T. A. and Tong, W. H. 2008).

?frataxin? heme scaffold CAC succinyl-CoA apo

glycine ALA desulph-SSH Cys X PP IX Ala holo PPOX ferrochelatase

? ? mfrn ? ABCB7 CPOX

ALA COPP III X apo iron uptake ALAD UROD & conservation CIA holo HMBS UROS iron utilization & storage

mfrn - mitoferrin/Mrs3p/Mrs4p

iron ALA delta-aminolevulinic acid

desulph cystein desulphurase ALAS ALA synthase

frataxin ALAD ALA dehydrogenase

scaffold protein HMBS Hydroxymethyl bilane synthase

ABCB7/Atm1p UROS Uroporphyrinogen III synthase

CIA (cytoplasmic ISC assembly machinery) UROD Uroporphyrinogen III decaboxylase

ISC protein assembly machinery CPOX Coproporphyrinogen III oxidase

apo / holo ISC protein PPOX Protoporphyrin III oxidase

mitochondrial aconitase and succinate-DH ferrochelatase

CAC iron regulatory protein (IRP) Citric acid cycle cytoplasmic aconitase unidentified mitochondrial transporters RNA binding form Fig. 3: Iron-sulfur cluster and heme biosynthesis in the eukaryotic cell. Unknown or hypothetical functions are marked by “?”. “X” is an unidentified compound essential for cytoplasmic ISC synthesis and involved in the regulation of cellular iron homeostasis.

26 ISC Biosynthesis -ferredoxins (ISC)

Krebs Cycle Heme Biosynthesis -Aconitase (ISC) -ferrochelatase (ISC) -Succinate DH (ISC) Amino Acid Respiratory chain: Synthesis - complex I & II (ISC) Apoptosis -Glt1, Lys4 (ISC) - complex III (ISC & heme) cyt c (heme) -cytochrome c (heme) mitochondrial -complex IV (heme) iron homeostasis

Energy metabolism

cytosolic/nuclear ISC protein maturation

IRP1/IRE system Ribosome Assembly - ferritin DNA repair -RLi1/ABCE1 (ISC) - mammals: TfR, ferroportin... FancJ, XPD (ISC) Amino acid synthesis DNA helicase -Leu1, Ecm17 (ISC) Rad3 (ISC) Nucleotide metabolism DNA replication -GPAT, XOR (ISC) Pri2 (ISC) t-RNA modifications cellular iron -Elp3, Tyw1 (ISC) homeostasis Receptor signaling -SPROUTY (ISC)

Fig. 4: Iron-sulfur clusters and heme are prosthetic groups of enzymes that needed in processes that are essential for life of eukaryotes (Lill, R. and Mühlenhoff, U. 2008; Rouault, T. A. and Tong, W. H. 2008).

Heme biosynthesis The biosynthesis of heme comprises eight steps, of which the very first and the last three are carried out in mitochondria, whereas the other four steps take place in the cytoplasm (Fig. 3). Amino-levulinic acid synthase converts succinly-CoA – a metabolite from the citric acid cycle – and glycine, into d- Amino-levulinic acid (dALA), which is exported to the cytoplasm where it is converted in four steps to coproporphyrinogen III (CPP III). Two oxidation steps inside mitochondria turn CPP III into protoporphyrin IX. Finally the ISC-protein Ferrochelatase inserts an iron ion into protoporhyrin IX to produce heme (Fig. 3).

27 Mrs3/4 and mitoferrins Mitochondria as the central sub-cellular site of the iron metabolism are surrounded by two lipid bilayers. The inner membrane is an efficient diffusion barrier for ions that needs to be overcome with the help of carrier proteins. Mitochondrial carrier proteins are a family of structurally related proteins, which translocate a broad spectrum of solutes between the mitochondrial in- termembrane space and the matrix. The unifying feature of mitochondrial carrier proteins is their tripartite structure, composed of three about 100 amino acid residues long repeats. Each repeat contains two transmembrane helices, one matrix side helix and the signature sequence P-X-[D/E]-X-X-[R/ K] (reviewed in (Kunji, E. R. 2004)). The mitochondrial carrier proteins Mrs3p and Mrs4p (Mrs3/4p) (Wiesen- berger, G. et al. 1991) have been shown to play a role in iron transport across the inner mitochondrial membrane (Froschauer, E. M. et al. 2009). Mutants of MRS3/4 grow poorly on low iron medium (Foury, F. and Roganti, T. 2002) and at 36 °C (Li, F. Y. et al. 2001). Both, the deletion or overexpression of MRS3/4 cause the activation of the iron regulon (Mühlenhoff, U. et al. 2003), a group of genes transcriptionally activated during low-iron conditions. MRS4 itself is a target of the iron regulon and is preferentially regulated by Aft2p (Rutherford, J. C. et al. 2003). On isolated mitochondria from iron starved MRS3/4 double mutants and MRS3/4 overexpressing yeast cells, it was shown that mitochondrial iron accumulation correlated with the level of Mrs3/4p expression (Mühlenhoff, U. et al. 2003). A similar correlation was observed for the formation of heme (Foury, F. and Roganti, T. 2002) and the maturation of ISC-proteins, and together these findings were interpreted as indirect evidence that Mrs3/4p are involved in mitochondrial iron transport (Foury, F. and Roganti, T. 2002; Mühlenhoff, U. et al. 2003). Mutations of MRS3/4 suppress mitochondrial iron overload in a yeast frataxin-deficiency strain (Foury, F. and Roganti, T. 2002) and MRS3/4 together with yeast frataxin homologue (YFH1) have been shown to be involved in iron delivery to ISC and heme synthesis in yeast (Zhang, Y. et al. 2005; Zhang, Y. et al. 2006). Orthologues of MRS3/4 have been reported in Dictyostelium discoideum (Satre, M. et al. 2007), Cryptococcus neoformans (Nyhus, K. J. et al. 2002), zebrafish (Shaw, G. C. et al. 2006), mice (Li, F. Y. et al. 2002; Shaw, G. C. et al. 2006) and humans (Li, F. Y. et al. 2001; Shaw, P. J. et al. 2002) and are also referred to as mitoferrins. The frascati mutations in the zebrafish mito- ferrin1 gene, result in decreased heme synthesis, which in turn causes a severe defect in erythropoiesis leading to embryonic death (Shaw, G. C. et al. 2006). Mitoferrin1 is expressed mainly in erythropoietic tissues, while its paralogue mitoferrin2 is ubiquitously expressed. Interestingly, expression of mitoferrin2 cannot rescue frascati mutants, although both mitoferrins do rescue the yeast Mrs3/4 double mutant. A study in cell cultures showed, that the

28 half-life of mitoferrin1 was specifically increased in mitochondria of erythroid cells, while the half-life of mitoferrin2 was unchanged (Paradkar, P. N. et al. 2008). More recently it was shown that mitoferrin1 physically interacts with the uncharacterized ABC transporter ABCB10, which is expressed at increased levels during erythroid maturation and causes mitofer- rin1 stabilization (Chen, W. et al. 2009). The difference in turnover rates of mitoferrin1 and mitoferrin2 is very likely responsible for the inability of mi- toferrin2 to rescue mitoferrin1 mutants. A function of mitoferrin2 on the or- ganismal level has so far not been described or studied.

29 Spermatogenesis in Drosophila melanogaster

Spermatogenesis in Drosophila begins in the wandering third instar larvae and progresses throughout metamorphosis, allowing male flies to mate shortly after eclosion. Each male has two testes that each contain a seminal vesicle and open into the ejaculatory duct. Drosophila testes are terminally blind tubes that contain all stages of spermatogenesis (Fig. 5 A). At the tip of a testis, the hub contains the germ line stem cells and the cyst progenitor cells. After asymmetric stem cell division, the parental stem cells remain at- tached to the hub, while the daughter cells detach. A single spermatogonium is encapsuled by two cyst cells and undergoes development to 64 mature sperm that are finally released from the cyst. The spermatogonium first undergoes four rounds of mitosis, producing 16 syncytial (interconnected by cytoplasmic bridges called ring canals) early spermatocytes which grow in size and accumulate transcripts needed during later steps of spermatogenesis. Late primary spermatocytes undergo meiosis, resulting in 64 round spermatids. The mitochondria of spermatids grow in mass and fuse to form the giant round Nebenkern of onion stage spermatids. The name onion stage refers to the morphology of the mitochondrial mem- branes as observed in the transmission electron microscope at this stage, which looks like a section through an onion. Spermatids then elongate to a length of 1.8 millimeters, with one axoneme and two mitochondrial derivatives extending along the complete length. In elongated spermatids, nuclei are needle shaped and form a parallelly organized pack at the base of the testis with the spermatid tails extending apically. An individualization complex forms at the position of the nuclei and moves as a parallelly organized complex along all 64 spermatids, removing all organelles and most of the cytoplasm, except for the major and the minor mitochondrial derivative as well as the axoneme. Through this process each spermatid is wrapped in its own membrane (Fig. 5 B1-4). At the site of individualization, a cytoplasmic bulge forms and cones of filamentous actin (actin cones) can be visualized with rhodamine stains. When the individualization complex reaches the end of the spermatids, the removed material is cast off as a structure referred to as waste bags, the mature sperm curl and are transferred from the cyst into the seminal vesicle. The major mitochondrial derivative of post-individualization spermatids is characterized by the accumulation of an uranophilic parac- rystalline structure of unknown origin and function. (the above summary about Drosophila spermatogenesis is based on these references: (Castrillon,

30 D. H. et al. 1993; Fabrizio, J. J. et al. 1998; Tokuyasu, K. T. et al. 1972; To- kuyasu, K. T. 1975)).

A. Spermatogenesis in Drosophila melanogaster elongated spermatids individualized spermatids

transfer into seminal vesicle elongation 64 onion stage spermatids meiosis individualization 16 spermatocytes before meiosis growth MATURE 16 primary spermatocytes SPERM 4 x mitosis Gonial cell surrounded by two cyst cells asymetric stem cell divisions Germline and somatic stem cells

B. Spermatid individualization cyst cell 1

nucleus pre-individualized spermatid 2

actin cone cytoplasmic bulge 3

individualized spermatid 4

waste bag

Fig. 5: Spermatogenesis in Drosophila melanogaster. (A) Drosophila testes are blind tubes with the stemcells at the apical end. Spermatogenesis proceeds toward the basal end and mature sperm are stored in the seminal vesicle. Only few germcells per cyst are depicted and only one cyst of each stage is shown. The cystcells are not shown for elongated and individualized spermatids/sperm (adapted from (Castrillon, D. H. et al. 1993)). (B) Spermatid individualization is a coordinated process that takes occurs in all 64 elongated spermatids at the same time. In the image, only three spermatids are shown for sake of simplicity (adapted from (Fabrizio, J. J. et al. 1998))

31 Objectives

Drosophila melanogaster offers a large pool of mutants and with the UAS/Gal4 system it also offers the possibility of tissue specific overexpression and RNAi of a great many genes, readily available from stock collections. The mitochondrial iron metabolism is highly conserved from yeast to vertebrates and a homologue of frataxin, the gene that causes Friedreich ataxia, was identified in Drosophila (Canizares, J. et al. 2000). It was shown that frataxin-depletion by RNAi (Anderson, P. R. et al. 2005) causes oxidative stress (Llorens, J. V. et al. 2007) and can be partially suppressed by hydrogen peroxide scavenging through the overexpression of catalase (Anderson, P. R. et al. 2008). These reports show that Drosophila can be used to elucidate the causes underlying human iron metabolism related diseases, even though Drosophila lacks erythropoiesis. At the beginning of this work, zebrafish mitoferrins were still unknown and all work on mitochondrial iron import via Mrs3/4 had been done in yeast. Work on the two mitoferrins in zebrafish mainly focused on the function of mitoferrin1, while the role of the ubiquitously expressed mitoferrin2 is still unclear. We were interested in the function of mitoferrin in metazoan organisms and wanted to identify Mrs3/4 homologues in the Drosophila genome, characterize their role in cellular iron homeostasis and study their role in whole flies.

Results and discussion Overexpression of Drosophila mitoferrin in l(2)mbn cells results in dysregulation of Fer1HCH expression. (Paper I) In this article we describe the identification of a Drosophila mitoferrin and its involvement in cellular iron homeostasis in a Drosophila cell line system. BLAST search of the Drosophila genome for mitoferrin candidates gave only one likely hit, the previously uncharacterized gene CG4963. We found that CG4963 protein localizes to mitochondria and rescues the growth defect of ΔMRS3/4 double mutants on low iron medium, confirming CG4963 as a mitoferrin. To avoid confusion of CG4963 with the erythropoiesis specific vertebrate mitoferrin1, which is sometimes referred to as mitoferrin, we chose to name it Drosophila mitoferrin (dmfrn).

32 Yeast has two mitoferrin genes, MRS3 and MRS4. It was therefore rather unexpected to find only one mitoferrin gene in Drosophila. Further BLAST analysis of genome databases of several invertebrates and vertebrates revealed, that the presence of one mitoferrin gene is common in invertebrates, while vertebrates possess two mitoferrin orthologues. As mitoferrin1 has a specific role in vertebrate erythropoiesis (Shaw, G. C. et al. 2006), the most compelling reason for one mitoferrin gene in invertebrates is that they lack erythropoiesis. The reason for two mitoferrin genes in yeast (MRS3 and MRS4) is not clear, but could have to do with fine tuning of regulation. Only MRS4, but not MRS3, is regulated by the iron dependent transcriptional regu- lator Aft2p (Rutherford, J. C. et al. 2001) and only MRS3/4 double deletion causes a phenotype. Overexpression of dmfrn in the l(2)mbn cell line (mbn-dmfrn cells) during normal growth conditions resulted in decreased IRP/IRE binding, increased cytoplasmic aconitase activity and an increase in Fer1HCH mRNA and protein. This is indicative for iron loaded cells, but to our surprise, iron levels in mbn-dmfrn were not increased, but slightly decreased, indicating that these cells may overestimate iron levels. The difference in cellular iron content and IRE/IRP binding disappeared in iron loaded cells, where iron levels were increased and IRE/IRP binding decreased in control and mbn-dmfrn cells alike. Interestingly, the increase in Fer1HCH mRNA and protein was further enhanced after iron loading of mbn-dmfrn cells. Atm1p in yeast and its homologue ABCB7 in humans (Csere, P. et al. 1998) transport an unknown compound derived from the mitochondrial ISC synthesis pathway (Kuhnke, G. et al. 2006; Lill, R. and Mühlenhoff, U. 2008). This compound is necessary for cytoplasmic ISC maturation (Kispal, G. et al. 1999; Pondarre, C. et al. 2006) and plays a role in regulation of cellular iron homeostasis in yeast an mammals (Cavadini, P. et al. 2007; Pon- darre, C. et al. 2007; Rutherford, J. C. et al. 2005). As Fer1HCH transcript was increased in mbn-dmfrn cells, we wanted to find out if this was connected to the unknown compound emitted by mitochondria. We identified a putative ABCB7 gene in Drosophila, and found that decreasing its expression by RNAi resulted in Fer1HCH expression in mbn-dmfrn cells that was similar to control cells. From these findings we deduce the model depicted in Fig.6. In mbn-dmfrn cells, iron uptake by mitochondria is increased compared to control cells, resulting in increased ISC synthesis. Consequently more of the unknown compound is emitted by mitochondria, which positively effects cytoplasmic ISC generation, reflected by the decrease in IRE-binding activity of IRP, increased cytoplasmic aconitase activity and increased Fer1HCH mRNA levels. RNAi of putative dABCB7 counters these effects by limiting the amount of the unknown compound released by mitochondria. This in turn would decrease cytoplasmic ISC synthesis and Fer1HCH mRNA levels.

33 These results show that a mechanism exists in Drosophila that regulates the expression of Fer1HCH on the transcript level in an ISC-synthesis dependent way. The integration of ISC synthesis and transcriptional regulation in yeast was not understood at the time we performed our experiments. Re- cent results suggest that heterodimerization and binding of ISC by compon- ents of the Fra1/Fra2/Grx3/Grx4 signaling cascade could bridge that gap (Li, H. et al. 2009). A similar system could be at work in regulating iron homeostasis (e.g. Fer1HCH expression) on the transcriptional level in Drosophila.

control mbn-2 cell mbn-2 cell overexpressing dmfrn

ISC apo ISC apo synthesis X synthesis X X holo X holo X

dmfrn dABCB7 dmfrn dABCB7

X X X X X

apo IRP binding IRP binding apo CIA CIA holo holo aconitase aconitase IRP IRP activity activity Fer1HCH transcript Fer1HCH transcript

mbn-2 cell overexpressing dmfrn and RNAi of dABCB7

mfrn - mitoferrin/Mrs3p/Mrs4p

iron ISC apo X synthesis X X holo ABCB7/Atm1p X CIA (cytoplasmic ISC assembly machinery) dABCB7 dmfrn RNAi apo / holo ISC protein

X iron regulatory protein (IRP) IRP binding cytoplasmic aconitase form apo CIA holo RNA binding form

aconitase IRP activity Fer1HCH transcript

Fig. 6: Model for the dmfrn overexpression phenotype in Drosophila l(2)mbn cells and the effects of RNA interference (RNAi) of putative Drosophila ABCB7 (dABCB7). To simplify the illustration, dmfrn is depicted as an iron transporter, even though it is not clear if mitoferrins transport iron or modulate the function of an iron transporter.

34 Drosophila mitoferrin is essential for male fertility: evidence for a role of the mitochondrial iron metabolism during spermatogenesis. (Paper II) After studying the effects of dmfrn overexpression on iron homeostasis in l(2)mbn cells we started to study the role of this mitoferrin2 homologue in the whole organism and found that dmfrn plays a role in spermatogenesis and development. We obtained dmfrn mutant flies that were available at public fly centers. Three mutant alleles are caused by P-element (a transposon used in Droso- phila genetics) insertions into the 5´ untranslated region (UTR) of dmfrn. The P-element insertions of alleles dmfrnBG00456 and dmfrnEY01302 are closest to the putative transcriptional start site of dmfrn (25 bp and 40 bp downstream respectively) whereas dmfrnSH115 is located furthest downstream of the transcriptional start (254 bp) and closest to the translational start (446 bp) of dmfrn. A fourth allele of dmfrn is the deletion (deficiency, Df) Df(3R)ED6277 which deletes ~10 kbp of the genome at cytogenic map position 98B6, including the genes dmfrn and CG5514, as well as parts of the 5´UTRs of Mes-4 and Gp93 and can be used to uncover alleles in dmfrn. The insertions closest to the transcriptional start site did not cause any annotated phenotype by themselves, but dmfrnEY01302 can be used to drive the expression of dmfrn using the Gal4/UAS system. Gal4 is a DNA binding protein that binds UAS sites and thereby activates transcription of downstream regions. In a genetic screen, in which the expression of genes was driven in the pattern of devel- oping muscle apodemes in Drosophila embryos using the Gal4/UAS system, it was shown that dmfrn overexpression causes muscle misdevelopment (Staudt, N. et al. 2005). The allele dmfrnSH115 was isolated in a screen for essential genes (Oh, S. W. et al. 2003) and annotated at FlyBase (http://flybase.org) to cause recessive lethality. P-elements mobilization, which is used in many screens to generate new mutants, can result in mutations during the excision process. These mutations are no longer associated with the P-element and can result in phenotypes that are not related to the gene of interest. To remove such unmarked background mutations, female flies carrying the marked P-element (red eyes) can be outcrossed for several generations to male flies of a suitable strain (e.g. w1118 white eyed flies with a genetic background widely used) to allow homologous recombination during female gametogenesis (there is no homologous recombination in male flies). After only three generations of outcrossing, we found that dmfrnSH115 flies were no longer recessive lethal but that male flies were recessive sterile instead. To ascertain that the P-element insertion itself was causing male sterility, and not a nearby background mutation, which would be difficult to remove by outcrossing (the probability of homologous recombination to occur between proximate genomic regions is lower than in distal regions), we re-

35 mobilized the P-element and selected for flies that had lost the P-element marker (red eyes) and most likely the P-element as well. All flies that had lost the P-element were not sterile, showing that indeed, the P-element of dmfrnSH115 caused recessive male sterility. During this so called hop-out assay, we obtained the deletion allele dm- frnDf13 where ~700bp of dmfrn are removed, including the 5´UTR from the insertion site of the P-element P{lacW}dmfrnSH115, the translational start site and a small part of the first intron. These flies showed a certain degree of recessive lethality and male escapers were recessive sterile. To finally show that the sterility phenotype caused by dmfrnSH115 was associated with the dmfrn gene, and not caused by genetic interactions between the P-element and another gene in its proximity, we made transgenic flies with a genomic construct of dmfrn. We also included a fluorescense tag in the form of the coding sequence for venus to the 3´ end of dmfrn to allow the visualization of dmfrn protein expression and localization. The genomic construct (dmfrnvenus) rescued male fertility of homozygous dmfrnSH115 flies and various combinations of alleles dmfrnSH115, dmfrnDf13 and Df(3R)ED6277, but not homozygous Df(3R)ED6277. The gene CG5514, which is deleted together with dmfrn, is expressed more abundantly in testes than in other tissues. It is most likely that CG5514 is essential for male fertility as well. Neverthe- less, dmfrnvenus rescued viability of dmfrnDf13 and Df(3R)ED6277 flies, showing that dmfrn-venus protein is biologically active and the rescue of male sterility of dmfrnSH115 flies shows that dmfrn is essential for male fertility in Drosophila. Male fertility can result from behavioral, morphological or spermatogenesis defects (Castrillon, D. H. et al. 1993). We found that testes of dmfrnSH115 flies showed a spectrum of defects. They were either smaller than wild type testis and had very few elongated spermatids or could contain many elongated spermatids and look almost as WT testes. But all testes we analyzed lacked mature sperm. Spermatids form mutant testes showed abnormalities in the morphology of their mitochondrial derivatives, had signs of delayed elongation and often accumulated unidentifiable masses of spherical structure. At the ultramicroscopic level we observed defects in mitochondrial derivatives as well as signs for spermatid individualization defects. Confocal laser scanning microscopy revealed that the normal organization of the nuclei of elongated spermatids in parallel bundles was disturbed in dmfrnSH115 testes. As the individualization complex forms at the nuclei, it is possible that individualization defects observed in dmfrnSH115 testes resulted from defects during elongation. On the other hand, some nuclei bundles did look more or less intact, and therefore it can not be ruled out that dmfrn plays a role in spermatid individualization. Furthermore, spermatid individualization in Drosophila depends on an apoptosis-like process that requires the testis specific cytochrome c cyt-c-d gene. Cytochromes are heme proteins, and

36 with the involvement of dmfrn in the mitochondrial iron metabolism, a role of dmfrn during spermatid individualization could be possible. The P-element P{lacW} of allele dmfrnSH115 contains the coding sequence of the β-galactosidase gene in the same orientation as dmfrn and thereby allows to monitor expression of dmfrn from upstream of the insertion site. X- Gal staining of whole mount testis as well as the expression pattern of dmfrn-venus showed that dmfrn is expressed in spermatids. During spermatogenesis, dmfrn-venus accumulated in the mitochondrial derivatives and a large fraction of it is removed during individualization and ends up in waste bags. Since mitoferrins are involved in the mitochondrial iron metabolism, and yeast MRS3/4 mutants develop a strong phenotype only on low iron medium, we wanted to know if sterility of dmfrn mutants was also effected by iron loading. Using the Df(3R)ED6277 genetic background, we quantified male fertility (fertile male flies in %) of flies with either of the three alleles dmfrnBG00456, dmfrnEY01302 and dmfrnSH115 cultured on low iron food, normal food and high iron food. While the insertions most upstream of the translational start caused sterility phenotypes that were stronger on low iron food, dmfrnSH115 males were completely sterile on all food types. Sterility of the hypomorphs on low iron food was accompanied by small testes with few and incompletely elongated spermatids. The weaker mutant alleles may sustain spermatogenesis through low level expression of dmfrn, but in flies with the strong mutant allele, expression might be too low to allow spermatogenesis, even under iron loading conditions. The elongation of spermatids to a length of almost two millimeters is likely to consume large amounts of energy. Many enzymes of the energy metabolism need prosthetic iron groups to function (Fig. 4). Defects in the expression of dmfrn are likely to cause defects in the mitochondrial iron metabolism and thereby hamper the energy metabolism. This work shows for the first time, that spermatogenesis depends on the mitochondrial iron metabolism and that dmfrn plays an essential role within this process. Mammalian and Drosophila spermatogenesis share several sim- ilarities, one of which is that testes are the tissue with highest expression levels of mitochondrial ferritin, indicating that our findings might be applicable in mammals as well.

Characterization of Drosophila mitoferrin and CG18317 mutants. (Paper III) During the study of P-element insertions in the Drosophila mitoferrin (dmfrn) gene we recovered a mutant fly strain with a small deletion in dmfrn (paper II). In flies with this deletion (dmfrnDf13) the start of the coding sequence and a small part of the first intron are deleted. In the Df(3R)ED6277

37 line which was made by the DrosDel project (Ryder, E. et al. 2004; Ryder, E. et al. 2007), the genes dmfrn and CG5514 are completely removed and the genes Mes-4 and Gp93 are truncated in their 5´UTR region (Metzendorf, C and Lind, MI, paper II). Previously we found that deletion of dmfrn, (homozygous dmfrnDf13 or dm- frnDf13/Df(3R)ED6277 or homozygous Df(3R)ED6277) causes lethality on normal food (Metzendorf, C and Lind, MI, paper II). In the current study we analyzed the lethality phenotype in further detail. As fertility of the hypo- morph dmfrn P-element mutant strains depended on food iron availability (Metzendorf, C and Lind, MI, paper II), we were interested if the deletion mutants might show a similar dependence. We found that iron supplementation of the food increased the number of adult escapers, whereas lethality was absolute if food iron availability during development was reduced by the iron chelator bathophenanthrolinedisulfonic acid (BPS). As the deletions of the parental strains that we used for the crosses were heterozygous to a balancer chromosome with the dominant marker mutation Tubby, which results in short and tubby looking larvae and pupae, we were able to quantify the number of pupae with or without the balancer. On low iron food we detected no pupae with unbalanced dmfrn deletions, indicating that low iron conditions enhance the lethality phenotype. The presence of third instar larvae without the balancer indicates, that dmfrn deletion might not cause larval lethality, but may either slow down development or cause developmental arrest. Introduction of the genomic construct dmfrnvenusB32 (Metzendorf, C and Lind, MI, paper II) rescued dmfrn deletion flies to pupal stage on low iron food, and on normal and high iron food it increased the fraction of adults with the dmfrn deletion alleles. Knocking down dmfrn expression by Gal4/UAS driven RNA interference (RNAi) with an ubiquitous driver, resulted in a phenotype similar to that of the dmfrn deletion strains. However, RNAi of dmfrn caused a weaker phenotype: only low iron food caused notable lethality. Interestingly strong overexpression of dmfrn, using a transgenic UAS-dmfrn construct, caused lethality on low iron medium as well, and no lethality on normal or high iron food. It has been reported that MRS3/4 overexpressing yeast showed signs of iron deficiency and it was suggested that this was due do the depletion of the cytosol of iron (Mühlenhoff, U. et al. 2002). This could also be the case in dmfrn overexpressing flies. On low iron food, cytoplasmic iron levels may not be repleted by the uptake of more food iron, whereas this would be possible on iron rich food. The presence of ISC-proteins and heme-proteins in the electron transport chain make the mitochondrial iron metabolism essential. As MRS3/4 deletions do not result in lethality, it was proposed that they either are not the only iron transporters or that they modulate iron transport of another mitochondrial transport protein (Mühlenhoff, U. et al. 2003). The survival of homozygous Df(3R)ED6277 flies to adulthood indicates that the function of

38 dmfrn is either similarly substituted by another low affinity iron carrier, or that dmfrn is not the actual iron transporter. The mitochondrial pyrimidine transporter Rim2p/Mrs12p (Marobbio, C. M. et al. 2006), which belongs to the mitochondrial carrier protein family (Van Dyck, E. et al. 1995), has recently been found to mediate iron transport into mitochondria and to be a multi-copy suppressor of the MRS3/4 deletion phenotype (G. Wiesenberger, unpublished data). As a putative homologue of Rim2p/Mrs12p in Drosophila we identified the protein encoded by the gene CG18317 on the second chromosome. We found that CG18317 protein localizes to mitochondria and that its transcript is ubiquitously present in all adult male fly tissues tested. We also confirmed the presence of two alternative splice variants, one of which has a 21 bp longer second exon. In yeast, RIM2/MRS12 deletion causes loss of mitochondrial DNA and results in the slow growth phenotype on glucose medium (Van Dyck, E. et al. 1995). If CG18317 has a similar function as yeast Rim2/Mrs12p its dys- function should cause lethality, as mitochondrial DNA is essential for respiration, and therefore for life. To analyze CG18317 we obtained two PiggyBac transposon mutants and two large deletions, which delete several genes in the CG18317 gene region. Both deletions cause recessive lethality, and the combination of the two different deletions also causes lethality. This is to be expected, as a the genomic region covered by both deletions contains genes with potential essential function (genes for a rRNA processing protein and a ribosomal protein). One of the PiggyBac insertions (allele CG18317e01575) is in the first intron of CG18317, while the other (allele CG18317e00411) is located at the end of the 3´UTR of the gene. While allele CG18317e00411 did not cause any detectable lethality in combination with either of the two deletions, CG18317e01575 resulted in partial lethality in combination with either of the deletions when flies were grown on low iron food. On normal food, allele CG18317e01575 in combination with one of the deletions caused partial lethality, while it did not with the other. This indicates, that there might be some genetic interaction between the allele CG18317e01575 and one of the genes that is deleted by only one of deletions. To find out if CG18317 is essential, smaller deletions, which do not effect other genes, have to be made. In summary these results allow several approaches for further research. The characterization of the dmfrn deletion strains makes it possible to use these in screens for genes that are involved in iron homeostasis or interact in other ways with dmfrn. Our initial study of CG18317 provides some evidence that this gene might be involved in the mitochondrial iron transport in the fruit fly as well and using these mutants together with our dmfrn mutants we can now investigate their genetic interaction.

39 The role of iron in the proliferation of Drosophila l(2)mbn cells. (Paper IV) When we established the parameters for the cell culture work with the l(2)mbn cell line, we also explored iron chelation to reduce iron levels of the cell culture and mimic low iron conditions. In mammalian systems it has been shown, that iron chelation of tumorous cell lines blocks cell cycle pro- gression (Richardson, D. R. et al. 1995). To test whether insect cells would behave similarly, we monitored cell viability at different concentrations of the iron chelator deferoxamine (DFO) at 24 h intervals using the MTT-assay (Mosmann, T. 1983; Tada, H. et al. 1986) and noticed that cell viability decreased at concentrations above 10 μM DFO. To determine the cause of the decreased cell viability, we manually counted live and dead cells at control conditions and conditions that decrease cell viability (25-100 μM DFO). While the number of control cells doubled every 2-3 days, the number of cells that were treated with 25-100 μM DFO decreased slightly. The fraction of dead cells in the control group did not increase (~5%), while it was a bit increased under growth inhibited conditions (10-15%), which may be attrib- uted to the accumulation of dead cells while no new cells are added by cell divisions. Furthermore, restoration of the iron levels of the growth medium of DFO treated cells, was sufficient to restore cell viability in cells that were treated 48 h with the iron chelator. Fluorescence assisted cell sorting (FACS) of cells stained with a nucleotide specific dye, allows to determine the fractions of cells that have not duplicated their genome (G0-phase, G1-phase and early S-phase), that are du- plicating their genome (S-phase) and cells that have duplicated their genome

(G2-phase, late S-phase and beginning of mitosis). FACS of cells treated with

25 μM DFO showed that the cell cycle was arrested in G1, while there were only very few cells that were in S-phase or G2-phase. Expression of cyclinE, a cyclin needed for G1/S-phase transition was decreased in cells after 48 h of

25 μM DFO treatment, indicating that the cell cycle arrested before G1/S- phase transition. This is the first report that the cell cycle arrest after iron chelation is conserved from insects to mammals. Mammalian genetic systems are often highly redundant, with several isoforms that substitute each others function, which makes it more difficult to study a process or the function of a gene. In Drosophila often only one isoform exists, reducing redundancy and the over- all complexity. Therefore, this study may lay the foundation for further research in the field of iron chelation and cell cycle arrest in Drosophila. Previously we noted an increase of dmfrn transcript in DFO treated cells (Metzendorf, C. et al. 2009), and interpreted it as a way of the cells to cope with low iron levels. In retrospect, we have to add the possibility that dmfrn expression may be dependent on the cell cycle, directly or indirectly.

40 Svensk sammanfattning (Summary in Swedish)

Järn är ett spårämne som är nödvändigt för nybildande och överlevnad av celler och därmed hela organismens välbefinnande. Dess oundgänglighet beror på att det deltar i många processer, till exempel syretransport och bild- ning av arvsmassa. Trots dess oundgänglighet kan järn i större mängd vara giftigt, eftersom det kan bilda s.k. fria syreradikaler som ger upphov till far- liga metaboliter. Därför kan överskott av järn orsaka allvariga cell- och vävnadsskador. Varje organism, till och med varje enskild cell i organismen, måste nog- grant reglera upptag, lagring och utnyttjande av järn för att hålla mängden på en måttlig nivå. Denna reglering sker genom att organismen kan förändra an- talet proteiner som är direkt eller indirekt involverade i järnmetabolismen. Mekanismerna som styr hur järnet regleras i kroppen är evolutionärt sett ganska konserverade. Av detta följer att järnmetabolismen hos lägre djur som t ex insekter sker, i princip, på samma sätt som hos människan. Därför finns stora möjligheter att i dessa djur studera järnmetabolismens basala mekanis- mer. I denna avhandling har modelldjuret bananfluga (Drosophila melanogaster) används. Bananflugan har många fördelar. Exempelvis är de billigare och etiskt mindre problematiska att arbeta med än t ex däggdjur. Det är enkelt att skapa mutantflugor, d v s flugor med kända defekter, vilket är en stor fördel vid forskningsstudier. Dessutom kan mutanter användas för att påverka mängden av utvalda proteiner i hela flugan eller i ett visst organ vid en speciell tidpunkt i flugans utveckling. Till fördel i avhandlingens studier är att bananflugan inte har några röda blodkroppar. Djur som behöver röda blodkroppar för sin syretransport är mycket känsliga för förändringar i järnbalansen eftersom det lätt resulterar i blodbrist. Därför kan det vara problematiskt att studera reglering av vissa delar av järnmetabolismen, speciellt mitokondriell järnmetabolism eftersom det är där som det syrebildande järnämnet (häm) hos röda blodkroppar bildas. Studierna i avhandlingen behandlar framförallt järnmetabolismen hos mi- tokondrier, vilka är cellens kraftverk. Studierna är fokuserade på ett mi- tokondriellt protein, mitoferrin, som är involverad i transport av järn in i mi- tokondrierna. Här behövs järnet för bl. a energiutvinning och är därför viktigt för cellens överlevnad.

41 Första studien visar att mitoferrin är evolutionärt mycket konserverat eftersom mitoferrin från bananflugan kan ersätta mitoferrins motsvarighet i jästsvampar. Vidare visar studien att om mängden mitoferrin ökas artificiellt så tror cellen att mängden järn har ökats och cellen börjar producera järnlag- ringsproteinet, ferritin, för att ta hand om järnet. Detta bevisar att den mitokondriella järnmetabolismen även är viktig för reglering av den cellulära järnmetabolismen. Till hjälp i denna studie användes en kultur av tumör- celler som härstammar från bananflugans blodceller. Denna cellkultur an- vändes också för att visa att dess tumöregenskap, att växa ohämmat, kan stoppas genom att tillsätta en kelator som binder upp järn och därmed redu- cera mängden tillgängligt järn för cellerna. Denna blockering var reversibel eftersom tillsättning av nytt järn återskapade tumöregenskapen. Med hjälp av olika mitoferrinmutantflugor och manipulering av mängden järn i flugornas föda kunde vi visa att mitoferrin har betydelse för flugans utveckling till vuxna flugor. Mitoferrin är framför allt nödvändigt då flugorna äter järnfattig kost: Vid nedreglering av funktionellt mitoferrin så utveck- lades inte flugorna till vuxna flugor utan stannar på larvstadiet om de odlades på föda med låg järntillgänglighet. Våra resultat visar också att mitoferrin behövs för spermatogenesen, d v s bildningen av spermier. I mutantflugor hos vilka produktionen av funktionellt mitoferrin var nedreglerat så var hanarna sterila. Dessa resultat visar för första gången en direkt koppling mellan spermatogenes och mitokondriell järnmetabolism och att mitoferrin har en avgörande roll i denna process. Bildningen av spermier hos högre djur som människa och andra däggdjur delar många likheter med spermatogenesen i bananflugan. Mitoferrinmutantflugorna presenterade här till- handahåller ett viktigt verktyg att studera denna process ytterligare. Resultaten visar tydligt att mitoferrin är viktig för en funktionell järnmeta- bolism, men eftersom några av mitoferrinmutantflugorna trots allt kunde överleva till larvstadiet eller längre då de fick järnrik kost indikerar att det finns ytterligare proteiner som är involverade i transport av järn in i mi- tokondrier. Vi föreslår att bananflugemotsvarigheten till det mitokondriella proteinet Rim2p/Mrs12 hos jäst kan fungera som en järntransportör. Resultaten i denna avhandling kommer att öka förståelse för hur mitokondriell järnmetabolism fungerar och påverkar olika utvecklingsprocesser och kan komma att användas vid utveckling av metoder för att kunna behandla sterilitet hos män.

42 Acknowledgments

I would like to thank:

My supervisor Maria Lind Karlberg to have me as her first PhD student and introducing me to iron homeostasis. You said in the beginning that you preferred a less authorative way of supervision and I think we fared well with this. Actually it was to my best. I learned to work and develop my projects indepentently. I also liked it a lot, that I could always drop by to discuss small matters or new ideas or just develop a new idea. I deeply appreciate the trust that you had in me.

My assistant supervisor Prof. Kenneth Söderhäll for having faith in me and for recommending me to Maria. I like your straight forward way and hope I picked up some of it myself. Also, thank you for the opportunity to visit the Kawabata Lab.

My opponent Prof. Martina Muckenthaler and the members of the committee for taking interest in my findings.

Mitch Dushay for teaching me how to work with the fruit fly and for setting up the fly-lab.

Fanis Missirlis. Your open way to talk to people impressed me when I first met you on the BioIron meeting in Kyoto. Luckily it rubbed off a bit and I think it had a certain impact on my development as a scientist. I am very grateful for the email conversations and for the invitation to be a speaker at the first IronFly meeting.

Karin Johansson, my first supervisor from back when I was an ERASMUS student at Uppsala University. During the project work with you I made the transition from student to going-to-be scientist.

All former and current members of Comarative Physiology: Irene (many many primers...), Ragnar (the “lab police”, I really enjoyed our early morn- ing conversations), Olof Tottmar and Lage Cerenius. (lab-teaching “Cellen i Centrum” was great and thank you for the lunch conversations), Karin, Tove,

43 Young-A, Haipeng, Pikul (thanks for the very delicious date cakes and the dried mango), Wenlin (thank you for doing the band shift so many times), Xionghui, Chenglin, Apiruck, Chadanat, Suchao, Jinyao, Sirinit, Amornrat, Margaretha, and last but not least the “fish guys” Tobias, Daniel, PO and Svante.

Prof. Shun-ichiro Kawabata, Takahara, Ohwada, Shibata and the rest of the lab for giving me such a warm welcome during my wonderful stay in Fukuoka. I really enjoyed all the different food and the Sunday activities!

All my fellow lab-teachers: Paulo and Marie (your professional way inspired me and I still have to smile when I remember the times when we lubricated the separating funnels or sucked the KCl for the electrodes), Vendela (getting the genetics lab up and running again was great fun), Marie H (to know that you´ll take care of the genetics lab now feels good), Niklas, Carl, Oddny, Henrik, Sophia.

Marianne Svarvare for always being so friendly, well organized and for providing the solutions and equipment needed during lab-teaching. This made it much easier to focus on the students and the teaching and it was always a pleasure to organize lab-teaching with you.

Rose-Marie for being the “tough” but friendly administrator. You are always on top of things. Organizing the IFU x-mas party with you was a lot of fun as well!

This work was supported by grants of the Wenner-Gren foundations, the Carl Trygger's foundation (CTS08:226), Magn. Bergvall's foundation (2007, 2008) and Swedish Research Council (621-2008-3669) to Maria Lind Karl- berg and by a grant from Swedish Research Council (grant number 621- 2006-4658) to Kenneth Söderhäll.

Rektors resebidrag från Wallenbergstiftelsen and the ASCB travel scholarship allowed me to attend the ASCB meeting 2007 in Washington D.C., USA. The Anna Maria Lundins scholarship from Smålands Nation made it possible for me to attend the BioIron meeting 2007 in Kyoto, Japan, the ELSO meeting 2007 in Dresden, Germany, and the first IronFly meeting 2008 in London, UK. My visit at the Kawabata Lab, Fukuoka, Japan, in Oc- tober 2008 was funded by Stiftelsen för internationalisering av högre utbild- ning och forskning.

Mina “svenska” vänner: Erik, Maike och Laura: Jag kommer inte att glömma körmusiken innan jul! Hanna, Sine och Dennis Taylor: Kantareller, badtunnan, Loka,... det var alltid underbart att vistas i er sommerstuga!! Erik

44 och Greta. Det var härligt med havsbrusfest, långfärdsskridskoåkning, laga julgodis och ha fest hos er. Tim, Lei and Lois: Thank you for hot pot, home made sushi, your wedding and game evenings. Erik och Lisa: tack för en riktik svensk julfest, kommer aldrig att glömma den. Ina; Norbert, Steffi und Bela; Sebastian und Elke; Steffen und Ulrike: Danke für die Spieleabende, das Hochseilabenteuer (Norbert und Steffi – ihr habt euch wacker geschla- gen), Grillabende, Badminton, Eislaufen... .Ben: Alien vs. Predator :-).My climbing partners: Maike, Bupi, Per, Fredrik, Lina, Martin L, Uttama and Jo- han as well as all the friendly climbers at Stallet...errr campus1477 science park...who I met and climbed with. Special thanks to campus1477 and all in- structors/employees.

Martin, Volkmar, Doreen (66 km!!), Gabi, Kerstin, Wiebke, Mini, Sarah und Dirk: Auch wenn die Distanz grösser war und die Treffen selten, waren sie immer schön.

Meinen Eltern Marion und Jupp danke ich von ganzem Herzen, dass ich immer machen durfte was ich wollte...auch wenn es Biologie studieren war :-)

Meinen Brüdern Matze (ich sag nur Wasserrutschbahnfahren) und Alex (schön dass es mit Idre geklappt hat!!) und meinem Neffen Philipp.

Nicole danke ich von ganzem Herzen, für alles :-).

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