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In memory of Maria Knapp

“Und Lust und Liebe sind dieFittige zu großen Thaten”Iphigenie auf Tauris, J.W. vonGoethe, 1787.

List of Papers

This thesis is based on the following papers, which will be referred to in thetext 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 Fer1HCHexpression. Biochem. J. 2009. 421:463-471.

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

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

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

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

Contents

Introduction ................................................................................................... 9

Iron Homeostasis ......................................................................................... 11Vertebrate iron homeostasis .................................................................... 11

Systemic iron metabolism and homeostasis ....................................... 11Regulation of cellular iron homeostasis – IRE/IRP ........................... 12

Invertebrate iron homeostasis ................................................................. 14Regulation of cellular iron homeostasis ............................................. 14Evolution of the IRE/IRP system ....................................................... 16Iron storage and transport ................................................................... 18Invertebrate iron homeostasis: Summarizing remarks ....................... 24

Mitochondrial iron metabolism ............................................................... 25Iron-sulfur cluster biosynthesis .......................................................... 26Heme biosynthesis .............................................................................. 27Mrs3/4 and mitoferrins ....................................................................... 27

Spermatogenesis in Drosophila melanogaster ............................................ 30

Objectives .................................................................................................... 32Results and discussion ............................................................................ 32

Overexpression of Drosophila mitoferrin in l(2)mbn cells results indysregulation of Fer1HCH expression. (Paper I) ............................... 32Drosophila mitoferrin is essential for male fertility: evidence for arole of the mitochondrial iron metabolism during spermatogenesis.(Paper II) ............................................................................................ 35Characterization of Drosophila mitoferrin and CG18317 mutants. (Paper III) ........................................................................................... 37The 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

Tf transferrin

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 fewbacilli and lactobacilli. As it can easily be reduced and oxidized, it is an idealcofactor for oxidases, reductases as well as dehydrogenases and plays essen-tial roles in almost all cellular functions; e.g. cellular respiration (iron ispresent in all respiratory chain complexes) and DNA synthesis (iron is acofactor of ribonucleotide reductase which converts ribonucleotides to de-oxyribonucleotides). The two most common iron-containing prostheticgroups are heme and iron-sulfur clusters (ISC). Synthesis of both is eithercompletely or in part carried out in mitochondria, which therefore play acentral role in the cellular iron metabolism and influence cellular ironhomeostasis.

The high reactivity of free iron with hydrogen peroxide also poses a threatto living organisms. Free iron generates reactive oxygen species via theFenton reaction (Fig. 1). These radicals can damage proteins, lipids andDNA. In mammals, chronic iron overload results in tissue damage of nervetissue, the liver, heart and adrenal gland (iron related diseases are reviewedin (Andrews, N. C. 2008)). In Drosophila, limiting the iron load of the dietresults in a longer life span (Massie, H. R. et al. 1993) and overexpression ofthe metal-responsive transcription factor increases life span of flies on foodwith 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 inthe 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 thefollowing 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 con-served aspects of the iron metabolism can help to understand and treat hu-man iron-related diseases. In invertebrates several different and uniquestrategies exist to achieve iron homeostasis. A better overview of the di-versity and function of invertebrate and vertebrate iron homeostasis will un-ravel the evolutionary background of iron homeostasis and provide know-ledge that is needed to choose the appropriate model organism and to inter-pret findings from model organisms (e.g. C. elegans, D. melanogaster). Inthe cases of blood-borne pathogens and their blood sucking invertebrate vec-tors, such knowledge is anticipated to provide new tools and possibilities tofight malaria, yellow fever and tick-borne diseases.

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Iron Homeostasis

Vertebrate iron homeostasisSystemic iron metabolism and homeostasisThe high interest in mammalian iron homeostasis, due to several severe hu-man diseases (e.g. Friedreich´s ataxia and hemochromatosis) caused bymutations 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 synthesisduring erythropoiesis. Most of this iron is recycled from senescent erythro-cytes, 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 involvesduodenal cytochrome b or antioxidants in the food that reduce ferric iron-which is insoluble at physiological pH - to soluble, ferrous iron. Thedivalent metal transporter 1 (DMT1, also known as Nramp2) can then trans-port iron across the cytoplasmic membrane. The other system utilizes heme,which is transported into cells by the heme carrier protein-1 and oxidized byheme oxygenase to release the iron. Iron within duodenal enterocytes is re-leased at the basal membrane by the transmembrane protein ferroportin.

After oxidation of exported iron by the ferroxidase hephaestin, ferric ironis bound by transferrin (Tf). Tf, the major iron transport molecule in verteb-rates, is composed of two similar lobes, which bind one iron atom each.Erythroblasts and most other cells present the transferrin receptor (TfR) ontheir surface when they are in need of iron. Iron loaded Tf binds the TfR andTf/TfR complexes are endocytosed. Acidification of the endosome causesconformational changes in Tf that result in the release of iron. Both apo-Tfand TfR are recycled to the cell surface, while endosomal iron is reduced bySTREAP3 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 macro-phages. Iron, which is released from hemoglobulin is exported via ferro-portin and bound by Tf after being oxidized by ceruloplasmin. This way ironis continuously recycled in the system.

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

Regulation of cellular iron homeostasis – IRE/IRPSeveral genes involved in cellular iron homeostasis are regulated on thetranslational level through the interaction of iron-regulatory proteins (IRP)and iron-responsive elements (IREs). IREs are conserved stem-loop RNAstructures (Fig.2) in target mRNAs.

In vertebrates two different IRPs exist. Both bind the same IREs but theirbinding 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 havebeen identified and functionally characterized for several genes (Table I). Ageneral pattern is, that IRP binding to IREs in the 5´ untranslated region(UTR) of a mRNA results in inhibition of translation. Binding of IRP toIREs in the 3´UTR of a mRNA on the other hand results in the stabilizationof 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 theIRP/IRE system is essential for development. Deletion of either IRP genealone results only in rather mild phenotypes, which indicates that the func-tions of both IRPs are overlapping enough to complement the function of themissing IRP.

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

A classic example for the regulation by the IRE/IRP system is the expressionof ferritin, a cellular iron storage protein, and the TfR. At low iron condi-tions, IRP binding to several IREs in the 3ÚTR of the TfR mRNA result inits stabilization, while translation of ferritin is blocked by IRP binding to theIRE in the 5ÚTR of ferritin mRNA. Increased TfR expression results in anincreased uptake of iron via the Tf/TfR pathway, until iron levels are highenough to stabilize ISCs, shifting the IRP function from IRE-binding to cyto-plasmic aconitase. As IRPs are lost from the IREs of TfR and ferritinmRNAs, TfR mRNA is degraded and ferritin translation is increased. Thisresults in a decrease in cellular iron uptake via Tf/TfR and increase in ironstorage within ferritin (vertebrate iron homeostasis is excessively reviewedin (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 thistable 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 bindingH-Ferritin iron storage 5ÚTR Inhibition of translationL-Ferritin -“- -“- -“-Ferroportin iron export -“- -“-eALAS erythroid heme synthesis -“- -“-HIF2alpha erythropoiesis -“- -“-MAconitase TCA Cycle -“- -“-

TfR1 iron uptake 3ÚTR RNA stabilizationDMT1 -“- -“- unknownCDC14A cell cycle -“- RNA stabilizationMRCKα cytoskeletal reorganization -“- RNA stabilization

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Invertebrate iron homeostasisEven 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 organ-isms within the group of invertebrates. Often, findings from one group of in-vertebrates do not apply to invertebrates in general. Some general featuresare conserved, but their specific function or regulation may differ. For in-stance, ferritins are conserved from invertebrates to vertebrates. While manyferritins 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 theIRE/IRP system shows a great deal of variability as well. It is absent in nem-atodes, restricted to heavy chain homologues in Diptera, present in both sub-units in Lepidoptera (Table II) and not studied at all in most invertebrates. Inthe following sections, some of the special features of invertebrate ironhomeostasis will be highlighted, to give an impression of the diversity, butalso to identify common patterns.

Regulation of cellular iron homeostasisIn an early IRE binding activity assay with lysates from twelve different spe-cies, yeast and plant lysates failed to produce a mobility shift of human H-ferritin IRE, whereas vertebrate (human, mouse, chicken, frog and fish), in-sect 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 activityhave 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 func-tional 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 insponges (Piccinelli, P. and Samuelsson, T. 2007), Cnidaria (Piccinelli, P. andSamuelsson, 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) comefrom IREs identified in the 5´UTRs of ferritin mRNAs of these organisms.

The nematode, Caenorhabditis elegans, on the other hand has a cytoplas-mic protein with more similarity to human IRP-1 than to mitochondrialaconitase (Muckenthaler, M. et al. 1998), but despite its iron dependent cyto-plasmic aconitase activity it does not bind IREs (Gourley, B. L. et al. 2003).Neither a bioinformatic search for conserved IRE sequences, nor binding as-

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says with recombinant human or nematode IRP to C. elegans ferritin (FTN-1and 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 regulationof C. elegans ferritins is solely regulated by transcription and that it is unlikelythat C. elegans has a functional IRE/IRP system (Gourley, B. L. et al. 2003).

Transcriptional regulation of ferritin expression also plays a role in organ-isms that have the IRE/IRP system. Cloned ferritin heavy subunit from thegiant fresh water prawn, Macrobrachium rosenbergerii, contains a well con-served IRE, indicating that it is likely to be regulated on the post-transcrip-tional level. Still, injection of iron into the hemolymph increased the tran-script abundance of this ferritin in muscle, ovary and heart (Qiu, G. F. et al.2008). In dipterans, which express ferritin heavy chain homologue mRNAsthat contain an IRE, a similar transcriptional response of ferritin expressionwas observed in mosquito cell culture (Pham, D. Q. et al. 1999) and in fruitflies (Georgieva, T. et al. 1999). Mapping of the promoter region of the fer-ritin heavy chain homologue in the mosquito identified the presence of en-hancer and silencer elements of which some are iron responsive (Pham, D.Q. et al. 2005). Expression of the light chain homologue subunit which hasno IRE in its mRNA has also been shown to be regulated transcriptionally inthe 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 viathe IRE/IRP system in vertebrates, it should be noted, that both H and L fer-ritins are transcriptionally regulated during development (leading to tissuespecific 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 theIRE/IRP-system have been identified in vertebrates (Table I), while in inver-tebrates mRNAs with IREs have been identified in only three different genes:ferritin heavy and light chain homologue subunits and succinate dehydro-genase (Table II). In invertebrates, most IREs have been found in ferritinheavy chain homologue genes, while only a small number of ferritin lightchain homologues with an IRE are known. Drosophila is the only invertebratefor which a functional IRE has been reported in the mRNA for a citric acidcycle enzyme (Table II). Furthermore, alternative splicing in the 5´UTR ofDrosophila melanogaster Fer1HCH pre-mRNA results in different transcripts.Some of these transcripts do not have an IRE (Lind, M. I. et al. 1998) and theirabundance is increased under iron loading conditions (Georgieva, T. et al.1999).

Searching the Drosophila melanogaster 5´ and 3´UTR sequence librariesfor IREs using PatSearch (Grillo, G. et al. 2003) followed by folding of can-didate mRNA sequences did not identify any new putative IREs; while bothpreviously identified Drosophila IREs (Fer1HCH and SdhB) were dis-

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covered and folded correctly (unpublished data). In a screen to discover newmammalian 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 systemIn D. melanogaster two IRPs (IRP-1A and IRP-1B) have been identified andthey share more similarity with human IRP-1 and with each other than withhuman IRP-2 (Muckenthaler, M. et al. 1998). While both have aconitaseactivity, only IRP-1A is able to bind IREs (Lind, M. I. et al. 2006). Thesefindings and the fact that C. elegans lacks the IRE/IRP system, were taken asevidence that during evolution an ancient gene for cytoplasmic aconitasewas duplicated in the common ancestor of vertebrates and insects and onevariant had acquired IRE-binding activity (Lind, M. I. et al. 2006). One func-tional 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 anIRE-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 Plasmodiumfalciparum, a cytoplasmic aconitase with IRP-binding activity as well asthree 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 aboutthe IRE/IRP-system, the absence of the IRE/IRP-system in plants and thepresence of an IRE-binding aconitase and IREs in operons for cytochromeoxidase and iron uptake genes in Bacillus subtilis have led to the formulationof two alternative hypothesis (Leipuviene, R. and Theil, E. C. 2007): Eitherthe IRE/IRP system is ancient and was lost in plants and other organismsthat lack this system, or it has arisen independently in several groups of or-ganisms. A thorough bioinformatic search by Piccinelli and Samuelssonidentified putative IREs in ferritin mRNAs in many metazoan taxa, all theway 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 ofthe IRE/IRP-system in the regulation of ferritin within Metazoa is in favor of aconvergent evolution of IRE/IRP systems in bacteria, protozoa and the Meta-zoan stem group. From the bioinformatic study, it can be concluded, that in-vertebrates possess IREs only in ferritin mRNAs (with the exception of Dro-sophila, where an IRE is present in succinate hydrogenase b) and that moregenes came under the control of the IRE/IRP system during the evolution ofChordata, Vertebrata and Mammalia (Piccinelli, P. and Samuelsson, T. 2007).

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Table II: Presence and absence of IREs in invertebrate mRNAs

Organism Ferritin SdhB References

IRE ferroxidasecenter

Annelida Periserrula leuco-phryna

yes yes (Jeong, B. R. et al. 2006)

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)

CnidariaHydra 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. andLocke, M. 1999; Zhang, D. et al. 2001a)

yes no (Kim, B.S. et al. 2002; Nichol, H. andLocke, 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)

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Table II continued.

Organism Ferritin SdhB References

IRE Ferroxidasecenter

Nematoda

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

PlatyhelminthesSchistosoma mansoni soma and yolk ferritin

no yes(Schüssler, P. et al. 1996)

Clonorchis sinensis yolk ferritin no (Tang, Y. et al. 2006)

PoriferaSuberites domunculaFTN1 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/cyto-plasmic by WolfPSORT (http://wolfpsort.org/). Two very similar cytoplasmic fer-ritins (probably gene duplication or database errors) contain an IRE-like structure.** IREs were identified bioinformatically by Piccinelli and Samuelsson in ferritingenes of unspecified subunit type.

Iron storage and transportFerritinFerritin 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 spherethat 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 aheteropolymer of heavy and light chain homologue subunits. In several otherinvertebrates, such as nematodes (Romney, S. J. et al. 2008), snails (vonDarl, 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 fer-ritin subunits have been identified, even though it is not known for all ofthese organisms whether these subunits form hetero- or homopolymers.

Heavy chain subunits and their homologues are characterized by the pres-ence of seven conserved amino acid residues that form a ferroxidase center,needed for oxidation of ferrous iron to ferric iron. Light chain subunits lackthese conserved residues, but expose basic side chains to the cavity, whichpromote crystallization and stabilization of the iron core. Ferritins with more

18

heavy chain subunits show increased protection from iron induced oxidativestress, while ferritins with more light chain subunits improve long term stor-age of iron (Arosio, P. et al. 2008).

While mammalian ferritin has a tissue specific ratio of light and heavychain subunits, the crystal structure of lepidopteran secreted ferritin revealeda fixed ratio of 12 light and 12 heavy chain subunits (Hamburger, A. E. et al.2005). Further inspection of the structure identified another difference tovertebrate ferritins. Inter-subunit disulfide bonds, which do not occur in ver-tebrate ferritins, between pairs of H and L subunits are formed and it wasproposed that hetero-dimers of H and L subunits assemble in a first step, be-fore 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 thatthe ratio between both subunits is fixed in the fruit fly as well (Missirlis, F. etal. 2007). Furthermore, it was shown that overexpression of either subunitdoes not increase the protein level of that subunit, unless the other subunit isoverexpressed as well (Missirlis, F. et al. 2007). It was argued, that due tothis unknown post-translational regulation mechanism, regulation of Fer-2LCH by the IRE/IRP-system was not needed, as the regulation of Fer1HCHby 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 theabsence of an IRE in the message of Fer2LCH. Nonetheless, both H- and L-ferritin messages of Lepidoptera, where the ratio between the ferritin sub-units in holo-ferritin is fixed as well, contain an IRE. Therefore, the lack ofan IRE in the light chain ferritin mRNA of dipterans may have another reas-on.

Unlike as in vertebrates, were ferritin is mainly a cytoplasmic iron storageprotein, 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. etal. 1994) secreted ferritins, contain an N-terminal leader sequence that tar-gets them to the endoplasmatic reticulum. Vertebrate ferritins lack such sig-nal peptides, but are present in blood plasma, cerebrospinal fluid and synovi-al fluids. Plasma levels of vertebrate ferritin are low compared to total bodyferritin, but plasma ferritin serves as a clinical parameter, since its concentra-tion is increased during the acute phase of infection, oxidative stress and ironloading, while iron deficiency results in lower levels. The mechanism of fer-ritin secretion in vertebrates is still unknown, but most likely dissimilar tothat 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 alsobeen 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 orferritin mRNA in hemocytes were observed after induction of the immuneresponse. In Drosophila melanogaster 2D-gel electrophoresis indicates thatferritin is post-translationally modified after infection (Vierstraete, E. et al.2004b). It has also been shown that iron-loading has a negative impact onfruit fly survival after fungal infection, whereas iron-starvation has a positiveimpact (Chamilos, G. et al. 2008).

In ticks and snails, two types of ferritin subunits, that each contain con-served amino acids of the ferroxidase center and that are expressed in a tis-sue 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 somaticand yolk ferritin. Somatic ferritin mRNA contains an IRE and the deducedprotein sequence lacks a secretion signal, which is in line with its cytoplas-mic 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 tran-scriptional regulation during development (von Darl, M. et al. 1994). Yolkand soma specific heavy chain homologue ferritins have also been reportedfor 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 ubiquit-ously 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, isindependent of the IRE/IRE-system and strongest in the gut and present atlow levels in ovary and salivary glands (Hajdusek, O. et al. 2009). The studyof Hajdusek et al. also demonstrated that Fer2 is very likely transporting ironfrom the gut to peripheral tissues, as RNAi of Fer2 resulted in decreasedFer1 protein levels in peripheric tissues of blood fed ticks compared to mocktreated 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 intissues with high metabolic activities (e.g. testis, heart, nervous tissue) andabsent from tissues that have iron storage functions (e.g. liver), which wastaken as an indication that mitochondrial ferritin may not perform iron stor-age functions but that it instead serves primarily to protect mitochondria ofthese cell types from oxidative stress (Santambrogio, P. et al. 2007). In thefruit fly, mitochondrial ferritin message is also highly abundant in testis(Missirlis, F. et al. 2006). Overexpression of vertebrate mitochondrial ferritinin HeLa cells resulted in an increased mitochondrial iron retention, downregulation 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 ofsecreted ferritin (Missirlis, F. et al. 2006). Instead, Fer3HCH overexpressionreduced 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).

TransferrinTransferrins are high-affinity, ferric iron binding proteins that can be orderedin 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 ofpacifastin) proteins. Transferrin is the major iron transport molecule in ver-tebrates (Andrews, N. C. 2008; Dunn, L. L. et al. 2007), the function oflactoferrin 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 inegg 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 – anarthropod proteinase inhibitor - is a three-lobed protein with similarity totransferrins (Liang, Z. et al. 1997). Iron binding of pacifastin was observedand from analysis of the conservation of iron binding residues it was specu-lated 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-terminaland 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 anancient monolobal precursor in the vertebrate stem group. It was argued, thatsmall proteins with masses less than 70 kDa are easily lost during ultrafiltra-tion in the glomuleri of vertebrates, and that bilobal transferrins with mo-lecular masses around 80 kDa solved that problem. Experimental support tothis theory was provided by the finding that oTf half-molecules from heneggs, each with a molecular mass of ~40 kDa, were readily lost with the ur-ine after injection into mice (Williams, J. et al. 1982). The identification of a40 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 bi-lobal 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 overmonolobal transferrins (Tinoco, A. D. et al. 2008) and is currently the besttheory for explaining bilobal transferrins. From a large scale phylogeneticanalysis of transferrin evolution, including amino acid sequences of 71 trans-ferrins from 51 species, it was concluded that the duplication of the transfer-rin lobes occurred before the split of the deuterosomes and protostomes(Lambert, L. A. et al. 2005).

Another striking difference between insect and mammalian transferrin be-came 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 transfer-rins from holometabole insects (e.g. Drosophila melanogaster (Yoshiga, T. etal. 1999), Manduca sexta (Bartfeld, N. S. and Law, J. H. 1990), as well asApis melifera, Bombyx mori and mosquitoes (Dunkov, B. and Georgieva, T.2006)) contain deletions and lack the conserved iron binding residues, whilehemimetabole insects (Blaberus discoidalis (Jamroz, R. C. et al. 1993), Mas-totermes darwiniensis (Thompson, G. J. et al. 2003)) have retained the con-served 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 hemo-lymph and upon infection the level of its mRNA is increased (Yoshiga, T. etal. 1997). As some pathogenic bacteria can express Tf-receptors that mainlyinteract 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 transfer-rin plays a role in insect immune response comes from a multitude of re-ports, where transferrin transcript abundance or protein was increased afterimmune 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. etal. 1997; Yoshiga, T. et al. 1999). Therefore, it was suggested that it may ful-fill a dual role in insects, that of iron transport (like serum transferrin) and asan 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 couldbe identified (Nichol, H. et al. 2002). Delivery of transferrin iron to fat bodycells has been demonstrated in vitro in Manduca sexta and radioactive ironfrom food was bound by hemolymph ferritin and transferrin (Huebers, H. A.et al. 1988). Transferrin expression was downregulated in iron fed Aedes ae-gypti and Drosophila melanogaster, and it was reasoned that iron transport isnot needed in an already iron loaded organism (Harizanova, N. et al. 2005;Yoshiga, T. et al. 1999). The injection of iron into hemolymph of Protaetiabrevitarsis increased transferrin mRNA levels above those after PBS injec-tion (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. Avitellogenic function, that is somewhat related to iron transport, has been at-tributed to transferrin from Sarcophaga peregrina. It was shown to transferiron from the hemolymph into oocytes (Kurama, T. et al. 1995).These find-ings show that transferrin is likely to act as an iron transporter in insects, butto which extend is unclear, as insects also use ferritin for iron transport.

It should also be mentioned, that insect genomes revealed the presence oftwo (in mosquitoes three) more transferrin-like proteins (Dunkov, B. andGeorgieva, 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 latershown 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 trans-port 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 in-sects may also turn out to transport other metals than iron.

Divalent metal transporters in invertebratesGenes coding for proteins with high similarity to divalent metal transporters(DMTs) have been identified in insects and the nematode Caenorhabditis el-egans. Other than that, knowledge about iron uptake mechanisms in inver-tebrates is scarce.

Mutations in malvolio, a Drosophila melanogaster gene coding for a pro-tein with high similarity to human Natural resistance-associated macrophageproteins (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 expressionof human Nramp1 (D'Souza, J. et al. 1999). Besides its ability to transportMn2+ and Fe2+, malvolio - in analogy to its human orthologue- has also beenshown to transport copper (Southon, A. et al. 2008). It is expressed in thenervous 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 plas-matocytes suggests, that malvolio might carry out a function in both im-munity (as Nramp1 does in vertebrates) and metal absorption in the gut.

After a blood meal, the malvolio homologue in mosquitoes, was downreg-ulated in the midgut and upregulated in malpighian tubules (Martinez-Barnetche, J. et al. 2007). In honey bees, it was shown that Mn2+ levels andmalvolio expression in the head correlated with division of worker bees inpollen foragers, nectar foragers and nurses, indicating that feeding-relatedgenes 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 sequencesimilarity to DMT have been identified in Caenorhabditis elegans. SMF-1

23

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

Invertebrate iron homeostasis: Summarizing remarksCellular iron homeostasis in many invertebrate taxa relies at least partiallyon the IRE/IRP system, but transcriptional regulation is very likely to playthe major role. Furthermore, apart from the presence of two heavy chain likeferritins 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 homeo-stasis in basal groups of invertebrates. Nevertheless, there seems to be a trendin the usage of transcriptional regulation to post-transcriptional regulation ofiron homeostasis from invertebrates to vertebrates. Most likely this trend is aproduct of the functional adaptation of the regulation of iron homeostasis dur-ing evolution. One cause for this shift to more post-transcriptional regulationin vertebrates may be the need for a much closer and faster regulation or amore complex integration of multiple signals. Identifying the driving force inthis shift of regulation would be extremely exciting, as it would also teach us alot about the evolution of vertebrates and the dynamics of iron homeostasis.

While the function of ferritin, despite certain adaptations (e.g. iron trans-port and immunity), is to store iron, it is much more difficult to ascribe aclear function to transferrins in invertebrates. It binds iron, but whether fortransport or as an iron scavenger to suppress microbial growth, or both, isnot clear. Furthermore, insect Tf2 and Tf3 (and Tf4 in mosquitoes), whichhave only been annotated in insect genomes, have not been characterized inthe slightest. The polylobal transferrin-like subunit of pacifastin from Paci-fastacus leniusculus is an indication of the divers roles that transferrin mayplay in invertebrates, whereas the lack of reports about transferrins in inver-tebrate groups other than crustaceans, insects and tunicates, may suggestseveral things. Either it was a trait developed rather close to the deutero-stome/protostome split, and lower animals lack transferrins or the putativelymonolobal ancestral transferrins in lower invertebrates became unrecogniz-able during the millennia. The third explanation, and simplest, would be, thatno one has looked for transferrins in lower invertebrates. Clearly, the field ofinvertebrate 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 theimmune response. To identify this role may prove to be a difficult task, asmanipulations of either gene will certainly impact the iron household andlead 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 metabolismCurrently the mitochondrial iron metabolism is mainly studied in yeast andvertebrate systems (zebrafish, mouse and human cell lines), leaving a biggap. As the mitochondrial iron metabolism is highly conserved this may notbe a big problem in itself, but yeast lacks the IRP/IRE system, ferritins andtransferrins, which are all present in Drosophila. Therefore, a better under-standing of Drosophila mitochondrial iron metabolism and its integrationinto the cellular iron metabolisms may provide better models than yeast. Mitochondria are the hot-spot of the eukaryotic iron metabolism. Here, ISCsare synthesized and iron is inserted into protoporphyrin IX – the final step inheme biosynthesis (Fig. 3). As ISCs and heme are prosthetic groups of manyproteins with essential functions (Fig. 4) the disruption of their synthesisleads 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 biosynthesisThe ISC biosynthesis pathway is well conserved from bacteria to higher euk-aryotes and about 20 different proteins are involved in the eukaryotic ISC as-sembly machinery. ISC biosynthesis requires four key steps: (1) abstractionof sulfur from cystein by a cystein desulfurase (NFS1p in yeast, ISCS in hu-man), (2) iron delivery in bioavailably form (mitoferrin and probably byfrataxin), (3) assembly of an ISC on a scaffold protein (ISU1p yeast, ISCUhuman) and (4) transfer of the ISC to an apo-ISC protein with help of theISC-protein assembly machinery. The transfer of assembled ISCs from thescaffold proteins to mitochondrial recipient proteins is probably carried outby ATP dependent conformational changes that are facilitated by co-chaper-one HSCB and heat shock 70-kDa protein 9 (HSCPA9).

In yeast, mitochondrial ISC synthesis is necessary for extramitochondrialISC-protein maturation, but it seems very unlikely that complete clusters areexported. Atm1p (ABCB7 in humans (Csere, P. et al. 1998)) has been shownto transport an eagerly sought but still unidentified compound that is bothnecessary for cytoplasmic ISC maturation (Kispal, G. et al. 1999; Pondarre,C. et al. 2006) and modulation of cellular iron homeostasis (Cavadini, P. etal. 2007; Pondarre, C. et al. 2007; Rutherford, J. C. et al. 2005). Both Atm1pand ABCB7 depleted cells accumulate mitochondrial iron, indicating thatmitochondrial ISC synthesis is emitting a signal that controls cellular ironhomeostasis. In vertebrates a direct integration of ISC synthesis into the regu-

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lation of cellular iron homeostasis is conveyed by the IRP-1/IRE system. Inyeast – which lacks the IRP/IRE system – mitochondrial ISC synthesis regu-lates cellular iron homeostasis via a signalling cascade containingFra1/Fra2/Grx3/Grx4. Recently it has been shown in vitro that Grx3/Grx4 andFra2 form heterodimeric complexes that contain an ISC (Li, H. et al. 2009)giving first mechanistic insights how ISC synthesis may be integrated into theregulation 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).

Fig. 3: Iron-sulfur cluster and heme biosynthesis in the eukaryotic cell. Unknown orhypothetical functions are marked by “?”. “X” is an unidentified compound essentialfor cytoplasmic ISC synthesis and involved in the regulation of cellular iron homeo-stasis.

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mfrn - mitoferrin/Mrs3p/Mrs4p

iron

mfrn

Cys Ala

-SSHdesulph

desulph cystein desulphurase

?frataxin?

frataxin

scaffold protein

apo

holo

X apo

holo

ABCB7

ABCB7/Atm1p

scaffold

CIA

CIA (cytoplasmic ISC assembly machinery)

mitochondrial aconitase and succinate-DH

ALA delta-aminolevulinic acid

ALA

X

?

ALAD

HMBS UROS

UROD

COPP III

CPOX

PP IX

ferrochelatase

heme

ALAS

ALAD

HMBS

UROS

UROD

CPOX

PPOX

PPOX

ALA synthase

ALA dehydrogenase

Hydroxymethyl bilane synthase

Uroporphyrinogen III synthase

Uroporphyrinogen III decaboxylase

Coproporphyrinogen III oxidase

Protoporphyrin III oxidase

?

glycine ALA

succinyl-CoACAC

iron regulatory protein (IRP)cytoplasmic aconitase

RNA binding form

iron uptake &

conservation

iron utilization &

storage

ferrochelatase

Citric acid cycleCAC

unidentified mitochondrial transporters

apo / holo ISC protein

ISC protein assembly machinery

?

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

Heme biosynthesisThe biosynthesis of heme comprises eight steps, of which the very first andthe last three are carried out in mitochondria, whereas the other four stepstake place in the cytoplasm (Fig. 3). Amino-levulinic acid synthase convertssuccinly-CoA – a metabolite from the citric acid cycle – and glycine, into d-Amino-levulinic acid (dALA), which is exported to the cytoplasm where it isconverted in four steps to coproporphyrinogen III (CPP III). Two oxidationsteps inside mitochondria turn CPP III into protoporphyrin IX. Finally theISC-protein Ferrochelatase inserts an iron ion into protoporhyrin IX to pro-duce heme (Fig. 3).

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Respiratory chain:- complex I & II (ISC)- complex III (ISC & heme)-cytochrome c (heme)-complex IV (heme)

ISC Biosynthesis-ferredoxins (ISC)

Heme Biosynthesis-ferrochelatase (ISC)

Krebs Cycle-Aconitase (ISC)-Succinate DH (ISC)

mitochondrialiron homeostasis

Energy metabolism

cellular ironhomeostasis

IRP1/IRE system- ferritin - mammals: TfR, ferroportin...

cytosolic/nuclearISC protein maturation

DNA repairFancJ, XPD (ISC)DNA helicaseRad3 (ISC)DNA replicationPri2 (ISC)

Apoptosiscyt c (heme)

Amino AcidSynthesis

-Glt1, Lys4 (ISC)

Ribosome Assembly-RLi1/ABCE1 (ISC)Amino acid synthesis-Leu1, Ecm17 (ISC)Nucleotide metabolism-GPAT, XOR (ISC)t-RNA modifications -Elp3, Tyw1 (ISC)Receptor signaling-SPROUTY (ISC)

Mrs3/4 and mitoferrinsMitochondria as the central sub-cellular site of the iron metabolism are sur-rounded by two lipid bilayers. The inner membrane is an efficient diffusionbarrier 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 mitochondrialcarrier proteins is their tripartite structure, composed of three about 100amino acid residues long repeats. Each repeat contains two transmembranehelices, 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 acrossthe inner mitochondrial membrane (Froschauer, E. M. et al. 2009). Mutantsof 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 overexpres-sion of MRS3/4 cause the activation of the iron regulon (Mühlenhoff, U. etal. 2003), a group of genes transcriptionally activated during low-iron condi-tions. MRS4 itself is a target of the iron regulon and is preferentially regu-lated by Aft2p (Rutherford, J. C. et al. 2003). On isolated mitochondria fromiron starved MRS3/4 double mutants and MRS3/4 overexpressing yeast cells,it was shown that mitochondrial iron accumulation correlated with the levelof Mrs3/4p expression (Mühlenhoff, U. et al. 2003). A similar correlationwas observed for the formation of heme (Foury, F. and Roganti, T. 2002) andthe maturation of ISC-proteins, and together these findings were interpretedas indirect evidence that Mrs3/4p are involved in mitochondrial iron trans-port (Foury, F. and Roganti, T. 2002; Mühlenhoff, U. et al. 2003).

Mutations of MRS3/4 suppress mitochondrial iron overload in a yeastfrataxin-deficiency strain (Foury, F. and Roganti, T. 2002) and MRS3/4 to-gether with yeast frataxin homologue (YFH1) have been shown to be in-volved 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. etal. 2006) and humans (Li, F. Y. et al. 2001; Shaw, P. J. et al. 2002) and arealso referred to as mitoferrins. The frascati mutations in the zebrafish mito-ferrin1 gene, result in decreased heme synthesis, which in turn causes asevere defect in erythropoiesis leading to embryonic death (Shaw, G. C. etal. 2006). Mitoferrin1 is expressed mainly in erythropoietic tissues, while itsparalogue mitoferrin2 is ubiquitously expressed. Interestingly, expression ofmitoferrin2 cannot rescue frascati mutants, although both mitoferrins do res-cue the yeast Mrs3/4 double mutant. A study in cell cultures showed, that the

28

half-life of mitoferrin1 was specifically increased in mitochondria oferythroid cells, while the half-life of mitoferrin2 was unchanged (Paradkar,P. N. et al. 2008). More recently it was shown that mitoferrin1 physically in-teracts with the uncharacterized ABC transporter ABCB10, which is ex-pressed at increased levels during erythroid maturation and causes mitofer-rin1 stabilization (Chen, W. et al. 2009). The difference in turnover rates ofmitoferrin1 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.

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Spermatogenesis in Drosophila melanogaster

Spermatogenesis in Drosophila begins in the wandering third instar larvaeand progresses throughout metamorphosis, allowing male flies to mateshortly after eclosion. Each male has two testes that each contain a seminalvesicle and open into the ejaculatory duct. Drosophila testes are terminallyblind tubes that contain all stages of spermatogenesis (Fig. 5 A). At the tip ofa testis, the hub contains the germ line stem cells and the cyst progenitorcells. After asymmetric stem cell division, the parental stem cells remain at-tached to the hub, while the daughter cells detach. A single spermatogoniumis encapsuled by two cyst cells and undergoes development to 64 maturesperm that are finally released from the cyst.

The spermatogonium first undergoes four rounds of mitosis, producing 16syncytial (interconnected by cytoplasmic bridges called ring canals) earlyspermatocytes which grow in size and accumulate transcripts needed duringlater steps of spermatogenesis. Late primary spermatocytes undergo meiosis,resulting in 64 round spermatids. The mitochondria of spermatids grow inmass 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 alength of 1.8 millimeters, with one axoneme and two mitochondrial derivat-ives extending along the complete length. In elongated spermatids, nuclei areneedle shaped and form a parallelly organized pack at the base of the testiswith the spermatid tails extending apically. An individualization complexforms at the position of the nuclei and moves as a parallelly organized com-plex along all 64 spermatids, removing all organelles and most of the cyto-plasm, except for the major and the minor mitochondrial derivative as wellas the axoneme. Through this process each spermatid is wrapped in its ownmembrane (Fig. 5 B1-4). At the site of individualization, a cytoplasmic bulgeforms and cones of filamentous actin (actin cones) can be visualized withrhodamine stains. When the individualization complex reaches the end of thespermatids, the removed material is cast off as a structure referred to aswaste bags, the mature sperm curl and are transferred from the cyst into theseminal vesicle. The major mitochondrial derivative of post-individualiza-tion spermatids is characterized by the accumulation of an uranophilic parac-rystalline structure of unknown origin and function. (the above summaryabout Drosophila spermatogenesis is based on these references: (Castrillon,

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

Fig. 5: Spermatogenesis in Drosophila melanogaster. (A) Drosophila testes are blindtubes with the stemcells at the apical end. Spermatogenesis proceeds toward the bas-al end and mature sperm are stored in the seminal vesicle. Only few germcells percyst are depicted and only one cyst of each stage is shown. The cystcells are notshown for elongated and individualized spermatids/sperm (adapted from (Castrillon,D. H. et al. 1993)). (B) Spermatid individualization is a coordinated process thattakes occurs in all 64 elongated spermatids at the same time. In the image, only threespermatids are shown for sake of simplicity (adapted from (Fabrizio, J. J. et al.1998))

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Germline and somatic stem cells

Gonial cell surrounded by two cyst cells

16 primary spermatocytes

4 x mitosis

16 spermatocytes before meiosisgrowth

asymetric stem cell divisions

meiosis64 onion stage spermatids

MATURESPERM

transfer into seminal vesicle

elongated spermatidsindividualized spermatids

B. Spermatid individualization

A. Spermatogenesis in Drosophila melanogaster

cyst cell

nucleus

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pre-individualized spermatid

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Objectives

Drosophila melanogaster offers a large pool of mutants and with theUAS/Gal4 system it also offers the possibility of tissue specific overexpres-sion and RNAi of a great many genes, readily available from stock collections.

The mitochondrial iron metabolism is highly conserved from yeast to ver-tebrates and a homologue of frataxin, the gene that causes Friedreich ataxia,was identified in Drosophila (Canizares, J. et al. 2000). It was shown thatfrataxin-depletion by RNAi (Anderson, P. R. et al. 2005) causes oxidativestress (Llorens, J. V. et al. 2007) and can be partially suppressed by hydrogenperoxide scavenging through the overexpression of catalase (Anderson, P. R.et al. 2008). These reports show that Drosophila can be used to elucidate thecauses underlying human iron metabolism related diseases, even thoughDrosophila lacks erythropoiesis.

At the beginning of this work, zebrafish mitoferrins were still unknownand all work on mitochondrial iron import via Mrs3/4 had been done inyeast. Work on the two mitoferrins in zebrafish mainly focused on the func-tion of mitoferrin1, while the role of the ubiquitously expressed mitoferrin2is still unclear. We were interested in the function of mitoferrin in metazoanorganisms and wanted to identify Mrs3/4 homologues in the Drosophila gen-ome, characterize their role in cellular iron homeostasis and study their rolein whole flies.

Results and discussionOverexpression of Drosophila mitoferrin in l(2)mbn cells resultsin dysregulation of Fer1HCH expression. (Paper I)In this article we describe the identification of a Drosophila mitoferrin andits involvement in cellular iron homeostasis in a Drosophila cell line system.

BLAST search of the Drosophila genome for mitoferrin candidates gaveonly one likely hit, the previously uncharacterized gene CG4963. We foundthat CG4963 protein localizes to mitochondria and rescues the growth defectof ΔMRS3/4 double mutants on low iron medium, confirming CG4963 as amitoferrin. To avoid confusion of CG4963 with the erythropoiesis specificvertebrate mitoferrin1, which is sometimes referred to as mitoferrin, wechose to name it Drosophila mitoferrin (dmfrn).

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Yeast has two mitoferrin genes, MRS3 and MRS4. It was therefore ratherunexpected to find only one mitoferrin gene in Drosophila. Further BLASTanalysis of genome databases of several invertebrates and vertebrates re-vealed, that the presence of one mitoferrin gene is common in invertebrates,while vertebrates possess two mitoferrin orthologues. As mitoferrin1 has aspecific role in vertebrate erythropoiesis (Shaw, G. C. et al. 2006), the mostcompelling reason for one mitoferrin gene in invertebrates is that they lackerythropoiesis. The reason for two mitoferrin genes in yeast (MRS3 andMRS4) is not clear, but could have to do with fine tuning of regulation. OnlyMRS4, but not MRS3, is regulated by the iron dependent transcriptional regu-lator Aft2p (Rutherford, J. C. et al. 2001) and only MRS3/4 double deletioncauses a phenotype.

Overexpression of dmfrn in the l(2)mbn cell line (mbn-dmfrn cells) duringnormal growth conditions resulted in decreased IRP/IRE binding, increasedcytoplasmic aconitase activity and an increase in Fer1HCH mRNA and pro-tein. This is indicative for iron loaded cells, but to our surprise, iron levels inmbn-dmfrn were not increased, but slightly decreased, indicating that thesecells may overestimate iron levels. The difference in cellular iron contentand IRE/IRP binding disappeared in iron loaded cells, where iron levels wereincreased and IRE/IRP binding decreased in control and mbn-dmfrn cellsalike. Interestingly, the increase in Fer1HCH mRNA and protein was furtherenhanced 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 ISCsynthesis 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 cel-lular 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 transcriptwas increased in mbn-dmfrn cells, we wanted to find out if this was connec-ted to the unknown compound emitted by mitochondria. We identified a pu-tative ABCB7 gene in Drosophila, and found that decreasing its expressionby RNAi resulted in Fer1HCH expression in mbn-dmfrn cells that was sim-ilar to control cells.

From these findings we deduce the model depicted in Fig.6. In mbn-dmfrncells, iron uptake by mitochondria is increased compared to control cells,resulting in increased ISC synthesis. Consequently more of the unknowncompound is emitted by mitochondria, which positively effects cytoplasmicISC generation, reflected by the decrease in IRE-binding activity of IRP, in-creased cytoplasmic aconitase activity and increased Fer1HCH mRNAlevels. RNAi of putative dABCB7 counters these effects by limiting theamount of the unknown compound released by mitochondria. This in turnwould decrease cytoplasmic ISC synthesis and Fer1HCH mRNA levels.

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These results show that a mechanism exists in Drosophila that regulatesthe expression of Fer1HCH on the transcript level in an ISC-synthesis de-pendent way. The integration of ISC synthesis and transcriptional regulationin 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 homeo-stasis (e.g. Fer1HCH expression) on the transcriptional level in Drosophila.

Fig. 6: Model for the dmfrn overexpression phenotype in Drosophila l(2)mbn cellsand 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 isnot clear if mitoferrins transport iron or modulate the function of an iron transporter.

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mfrn - mitoferrin/Mrs3p/Mrs4p

iron

dmfrn

X

dABCB7

ABCB7/Atm1p

CIA

CIA (cytoplasmic ISC assembly machinery)

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iron regulatory protein (IRP)cytoplasmic aconitase form

RNA binding form

IRP binding

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apo / holo ISC protein

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control mbn-2 cell mbn-2 cell overexpressing dmfrn

mbn-2 cell overexpressing dmfrn andRNAi of dABCB7

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aconitaseactivity

apo

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Fer1HCH transcript Fer1HCH transcript

Fer1HCH transcript

Drosophila mitoferrin is essential for male fertility: evidence fora role of the mitochondrial iron metabolism duringspermatogenesis. (Paper II)After studying the effects of dmfrn overexpression on iron homeostasis inl(2)mbn cells we started to study the role of this mitoferrin2 homologue inthe whole organism and found that dmfrn plays a role in spermatogenesisand 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 tothe putative transcriptional start site of dmfrn (25 bp and 40 bp downstreamrespectively) whereas dmfrnSH115 is located furthest downstream of the tran-scriptional start (254 bp) and closest to the translational start (446 bp) of dm-frn. A fourth allele of dmfrn is the deletion (deficiency, Df) Df(3R)ED6277which deletes ~10 kbp of the genome at cytogenic map position 98B6, in-cluding the genes dmfrn and CG5514, as well as parts of the 5´UTRs ofMes-4 and Gp93 and can be used to uncover alleles in dmfrn. The insertionsclosest to the transcriptional start site did not cause any annotated phenotypeby themselves, but dmfrnEY01302 can be used to drive the expression of dmfrnusing the Gal4/UAS system. Gal4 is a DNA binding protein that binds UASsites and thereby activates transcription of downstream regions. In a geneticscreen, 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 es-sential genes (Oh, S. W. et al. 2003) and annotated at FlyBase (http://fly-base.org) to cause recessive lethality.

P-elements mobilization, which is used in many screens to generate newmutants, can result in mutations during the excision process. These muta-tions are no longer associated with the P-element and can result in pheno-types that are not related to the gene of interest. To remove such unmarkedbackground mutations, female flies carrying the marked P-element (redeyes) can be outcrossed for several generations to male flies of a suitablestrain (e.g. w1118 white eyed flies with a genetic background widely used) toallow homologous recombination during female gametogenesis (there is nohomologous recombination in male flies). After only three generations ofoutcrossing, we found that dmfrnSH115 flies were no longer recessive lethalbut 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 removeby outcrossing (the probability of homologous recombination to occurbetween proximate genomic regions is lower than in distal regions), we re-

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mobilized the P-element and selected for flies that had lost the P-elementmarker (red eyes) and most likely the P-element as well. All flies that hadlost the P-element were not sterile, showing that indeed, the P-element ofdmfrnSH115 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 theinsertion site of the P-element P{lacW}dmfrnSH115, the translational start siteand a small part of the first intron. These flies showed a certain degree of re-cessive lethality and male escapers were recessive sterile.

To finally show that the sterility phenotype caused by dmfrnSH115 was asso-ciated with the dmfrn gene, and not caused by genetic interactions betweenthe P-element and another gene in its proximity, we made transgenic flieswith a genomic construct of dmfrn. We also included a fluorescense tag inthe form of the coding sequence for venus to the 3´ end of dmfrn to allow thevisualization of dmfrn protein expression and localization. The genomic con-struct (dmfrnvenus) rescued male fertility of homozygous dmfrnSH115 flies andvarious combinations of alleles dmfrnSH115, dmfrnDf13 and Df(3R)ED6277, butnot homozygous Df(3R)ED6277. The gene CG5514, which is deleted togeth-er with dmfrn, is expressed more abundantly in testes than in other tissues. Itis most likely that CG5514 is essential for male fertility as well. Neverthe-less, dmfrnvenus rescued viability of dmfrnDf13 and Df(3R)ED6277 flies, show-ing that dmfrn-venus protein is biologically active and the rescue of malesterility of dmfrnSH115 flies shows that dmfrn is essential for male fertility inDrosophila.

Male fertility can result from behavioral, morphological or spermatogen-esis 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 typetestis and had very few elongated spermatids or could contain many elong-ated spermatids and look almost as WT testes. But all testes we analyzedlacked mature sperm. Spermatids form mutant testes showed abnormalitiesin the morphology of their mitochondrial derivatives, had signs of delayedelongation and often accumulated unidentifiable masses of spherical struc-ture. At the ultramicroscopic level we observed defects in mitochondrial de-rivatives as well as signs for spermatid individualization defects. Confocallaser scanning microscopy revealed that the normal organization of the nuc-lei of elongated spermatids in parallel bundles was disturbed in dmfrnSH115

testes. As the individualization complex forms at the nuclei, it is possiblethat individualization defects observed in dmfrnSH115 testes resulted from de-fects during elongation. On the other hand, some nuclei bundles did lookmore or less intact, and therefore it can not be ruled out that dmfrn plays arole in spermatid individualization. Furthermore, spermatid individualizationin Drosophila depends on an apoptosis-like process that requires the testisspecific cytochrome c cyt-c-d gene. Cytochromes are heme proteins, and

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with the involvement of dmfrn in the mitochondrial iron metabolism, a roleof dmfrn during spermatid individualization could be possible.

The P-element P{lacW} of allele dmfrnSH115 contains the coding sequenceof the β-galactosidase gene in the same orientation as dmfrn and thereby al-lows 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 dm-frn-venus showed that dmfrn is expressed in spermatids. During spermato-genesis, dmfrn-venus accumulated in the mitochondrial derivatives and alarge fraction of it is removed during individualization and ends up in wastebags.

Since mitoferrins are involved in the mitochondrial iron metabolism, andyeast MRS3/4 mutants develop a strong phenotype only on low iron medi-um, we wanted to know if sterility of dmfrn mutants was also effected byiron loading. Using the Df(3R)ED6277 genetic background, we quantifiedmale fertility (fertile male flies in %) of flies with either of the three allelesdmfrnBG00456, dmfrnEY01302 and dmfrnSH115 cultured on low iron food, normalfood and high iron food. While the insertions most upstream of the transla-tional start caused sterility phenotypes that were stronger on low iron food,dmfrnSH115 males were completely sterile on all food types. Sterility of thehypomorphs on low iron food was accompanied by small testes with few andincompletely elongated spermatids. The weaker mutant alleles may sustainspermatogenesis through low level expression of dmfrn, but in flies with thestrong 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 islikely to consume large amounts of energy. Many enzymes of the energymetabolism need prosthetic iron groups to function (Fig. 4). Defects in theexpression of dmfrn are likely to cause defects in the mitochondrial ironmetabolism and thereby hamper the energy metabolism.

This work shows for the first time, that spermatogenesis depends on themitochondrial iron metabolism and that dmfrn plays an essential role withinthis process. Mammalian and Drosophila spermatogenesis share several sim-ilarities, one of which is that testes are the tissue with highest expressionlevels of mitochondrial ferritin, indicating that our findings might be applic-able in mammals as well.

Characterization of Drosophila mitoferrin and CG18317mutants. (Paper III)During the study of P-element insertions in the Drosophila mitoferrin (dm-frn) 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 se-quence and a small part of the first intron are deleted. In the Df(3R)ED6277

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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 thegenes Mes-4 and Gp93 are truncated in their 5´UTR region (Metzendorf, Cand 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 onnormal food (Metzendorf, C and Lind, MI, paper II). In the current study weanalyzed 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 deletionmutants might show a similar dependence. We found that iron supplementa-tion of the food increased the number of adult escapers, whereas lethalitywas absolute if food iron availability during development was reduced by theiron chelator bathophenanthrolinedisulfonic acid (BPS). As the deletions ofthe parental strains that we used for the crosses were heterozygous to a bal-ancer chromosome with the dominant marker mutation Tubby, which resultsin short and tubby looking larvae and pupae, we were able to quantify thenumber of pupae with or without the balancer. On low iron food we detectedno pupae with unbalanced dmfrn deletions, indicating that low iron condi-tions enhance the lethality phenotype. The presence of third instar larvaewithout the balancer indicates, that dmfrn deletion might not cause larvallethality, but may either slow down development or cause developmental ar-rest. Introduction of the genomic construct dmfrnvenusB32 (Metzendorf, C andLind, MI, paper II) rescued dmfrn deletion flies to pupal stage on low ironfood, and on normal and high iron food it increased the fraction of adultswith 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 ofthe dmfrn deletion strains. However, RNAi of dmfrn caused a weaker pheno-type: only low iron food caused notable lethality. Interestingly strong over-expression of dmfrn, using a transgenic UAS-dmfrn construct, caused lethal-ity 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 irondeficiency and it was suggested that this was due do the depletion of thecytosol of iron (Mühlenhoff, U. et al. 2002). This could also be the case indmfrn overexpressing flies. On low iron food, cytoplasmic iron levels maynot be repleted by the uptake of more food iron, whereas this would be pos-sible on iron rich food.

The presence of ISC-proteins and heme-proteins in the electron transportchain make the mitochondrial iron metabolism essential. As MRS3/4 dele-tions do not result in lethality, it was proposed that they either are not theonly iron transporters or that they modulate iron transport of another mito-chondrial transport protein (Mühlenhoff, U. et al. 2003). The survival of ho-mozygous Df(3R)ED6277 flies to adulthood indicates that the function of

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dmfrn is either similarly substituted by another low affinity iron carrier, orthat 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 transportinto mitochondria and to be a multi-copy suppressor of the MRS3/4 deletionphenotype (G. Wiesenberger, unpublished data).

As a putative homologue of Rim2p/Mrs12p in Drosophila we identifiedthe protein encoded by the gene CG18317 on the second chromosome. Wefound that CG18317 protein localizes to mitochondria and that its transcriptis ubiquitously present in all adult male fly tissues tested. We also confirmedthe presence of two alternative splice variants, one of which has a 21 bplonger second exon.

In yeast, RIM2/MRS12 deletion causes loss of mitochondrial DNA andresults in the slow growth phenotype on glucose medium (Van Dyck, E. etal. 1995). If CG18317 has a similar function as yeast Rim2/Mrs12p its dys-function should cause lethality, as mitochondrial DNA is essential for respir-ation, and therefore for life. To analyze CG18317 we obtained two PiggyBactransposon mutants and two large deletions, which delete several genes inthe CG18317 gene region. Both deletions cause recessive lethality, and thecombination of the two different deletions also causes lethality. This is to beexpected, as a the genomic region covered by both deletions contains geneswith potential essential function (genes for a rRNA processing protein and aribosomal protein). One of the PiggyBac insertions (allele CG18317e01575) isin the first intron of CG18317, while the other (allele CG18317e00411) is loc-ated at the end of the 3´UTR of the gene. While allele CG18317e00411 did notcause any detectable lethality in combination with either of the two dele-tions, CG18317e01575 resulted in partial lethality in combination with either ofthe deletions when flies were grown on low iron food. On normal food, al-lele CG18317e01575 in combination with one of the deletions caused partiallethality, while it did not with the other. This indicates, that there might besome genetic interaction between the allele CG18317e01575 and one of thegenes that is deleted by only one of deletions. To find out if CG18317 is es-sential, 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 usethese in screens for genes that are involved in iron homeostasis or interact inother ways with dmfrn. Our initial study of CG18317 provides some evid-ence that this gene might be involved in the mitochondrial iron transport inthe fruit fly as well and using these mutants together with our dmfrn mutantswe can now investigate their genetic interaction.

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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 thel(2)mbn cell line, we also explored iron chelation to reduce iron levels of thecell culture and mimic low iron conditions. In mammalian systems it hasbeen shown, that iron chelation of tumorous cell lines blocks cell cycle pro-gression (Richardson, D. R. et al. 1995). To test whether insect cells wouldbehave similarly, we monitored cell viability at different concentrations ofthe 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 de-creased at concentrations above 10 μM DFO. To determine the cause of thedecreased cell viability, we manually counted live and dead cells at controlconditions and conditions that decrease cell viability (25-100 μM DFO).While the number of control cells doubled every 2-3 days, the number ofcells that were treated with 25-100 μM DFO decreased slightly. The fractionof dead cells in the control group did not increase (~5%), while it was a bitincreased under growth inhibited conditions (10-15%), which may be attrib-uted to the accumulation of dead cells while no new cells are added by celldivisions. Furthermore, restoration of the iron levels of the growth mediumof DFO treated cells, was sufficient to restore cell viability in cells that weretreated 48 h with the iron chelator.

Fluorescence assisted cell sorting (FACS) of cells stained with a nucle-otide specific dye, allows to determine the fractions of cells that have not du-plicated 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 with25 μM DFO showed that the cell cycle was arrested in G1, while there wereonly 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 of25 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 con-served from insects to mammals. Mammalian genetic systems are oftenhighly redundant, with several isoforms that substitute each others function,which makes it more difficult to study a process or the function of a gene. InDrosophila often only one isoform exists, reducing redundancy and the over-all complexity. Therefore, this study may lay the foundation for further re-search 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 copewith low iron levels. In retrospect, we have to add the possibility that dmfrnexpression may be dependent on the cell cycle, directly or indirectly.

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Svensk sammanfattning(Summary in Swedish)

Järn är ett spårämne som är nödvändigt för nybildande och överlevnad avceller och därmed hela organismens välbefinnande. Dess oundgänglighetberor på att det deltar i många processer, till exempel syretransport och bild-ning av arvsmassa. Trots dess oundgänglighet kan järn i större mängd varagiftigt, eftersom det kan bilda s.k. fria syreradikaler som ger upphov till far-liga metaboliter. Därför kan överskott av järn orsaka allvariga cell- ochvävnadsskador.

Varje organism, till och med varje enskild cell i organismen, måste nog-grant reglera upptag, lagring och utnyttjande av järn för att hålla mängden påen 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 settganska konserverade. Av detta följer att järnmetabolismen hos lägre djur somt ex insekter sker, i princip, på samma sätt som hos människan. Därför finnsstora möjligheter att i dessa djur studera järnmetabolismens basala mekanis-mer.

I denna avhandling har modelldjuret bananfluga (Drosophilamelanogaster) används. Bananflugan har många fördelar. Exempelvis är debilligare och etiskt mindre problematiska att arbeta med än t ex däggdjur.Det är enkelt att skapa mutantflugor, d v s flugor med kända defekter, vilketär en stor fördel vid forskningsstudier. Dessutom kan mutanter användas föratt påverka mängden av utvalda proteiner i hela flugan eller i ett visst organvid en speciell tidpunkt i flugans utveckling. Till fördel i avhandlingensstudier är att bananflugan inte har några röda blodkroppar. Djur som behöverröda blodkroppar för sin syretransport är mycket känsliga för förändringar ijärnbalansen eftersom det lätt resulterar i blodbrist. Därför kan det varaproblematiskt att studera reglering av vissa delar av järnmetabolismen,speciellt mitokondriell järnmetabolism eftersom det är där som detsyrebildande 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 därförviktigt för cellens överlevnad.

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Första studien visar att mitoferrin är evolutionärt mycket konserverateftersom mitoferrin från bananflugan kan ersätta mitoferrins motsvarighet ijästsvampar. Vidare visar studien att om mängden mitoferrin ökas artificielltså tror cellen att mängden järn har ökats och cellen börjar producera järnlag-ringsproteinet, ferritin, för att ta hand om järnet. Detta bevisar att den mi-tokondriella järnmetabolismen även är viktig för reglering av den cellulärajärnmetabolismen. 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, kanstoppas genom att tillsätta en kelator som binder upp järn och därmed redu-cera mängden tillgängligt järn för cellerna. Denna blockering var reversibeleftersom tillsättning av nytt järn återskapade tumöregenskapen.

Med hjälp av olika mitoferrinmutantflugor och manipulering av mängdenjärn i flugornas föda kunde vi visa att mitoferrin har betydelse för flugansutveckling till vuxna flugor. Mitoferrin är framför allt nödvändigt då flugor-na äter järnfattig kost: Vid nedreglering av funktionellt mitoferrin så utveck-lades inte flugorna till vuxna flugor utan stannar på larvstadiet om deodlades på föda med låg järntillgänglighet. Våra resultat visar också att mito-ferrin behövs för spermatogenesen, d v s bildningen av spermier. I mutant-flugor hos vilka produktionen av funktionellt mitoferrin var nedreglerat såvar hanarna sterila. Dessa resultat visar för första gången en direkt kopplingmellan spermatogenes och mitokondriell järnmetabolism och att mitoferrinhar en avgörande roll i denna process. Bildningen av spermier hos högre djursom människa och andra däggdjur delar många likheter med spermatogen-esen 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 kundeöverleva till larvstadiet eller längre då de fick järnrik kost indikerar att detfinns ytterligare proteiner som är involverade i transport av järn in i mi-tokondrier. Vi föreslår att bananflugemotsvarigheten till det mitokondriellaproteinet Rim2p/Mrs12 hos jäst kan fungera som en järntransportör.

Resultaten i denna avhandling kommer att öka förståelse för hur mitokon-driell järnmetabolism fungerar och påverkar olika utvecklingsprocesser ochkan komma att användas vid utveckling av metoder för att kunna behandlasterilitet hos män.

42

Acknowledgments

I would like to thank:

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

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

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

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

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

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

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

43

Young-A, Haipeng, Pikul (thanks for the very delicious date cakes and thedried 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 andSvante.

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

All my fellow lab-teachers: Paulo and Marie (your professional way inspiredme and I still have to smile when I remember the times when we lubricatedthe separating funnels or sucked the KCl for the electrodes), Vendela (gettingthe genetics lab up and running again was great fun), Marie H (to know thatyou´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 forproviding the solutions and equipment needed during lab-teaching. Thismade it much easier to focus on the students and the teaching and it was al-ways a pleasure to organize lab-teaching with you.

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

This work was supported by grants of the Wenner-Gren foundations, the CarlTrygger'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 scholar-ship allowed me to attend the ASCB meeting 2007 in Washington D.C.,USA. The Anna Maria Lundins scholarship from Smålands Nation made itpossible for me to attend the BioIron meeting 2007 in Kyoto, Japan, theELSO meeting 2007 in Dresden, Germany, and the first IronFly meeting2008 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 attglömma 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, lagajulgodis och ha fest hos er. Tim, Lei and Lois: Thank you for hot pot, homemade sushi, your wedding and game evenings. Erik och Lisa: tack för enriktik svensk julfest, kommer aldrig att glömma den. Ina; Norbert, Steffi undBela; 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 :-).Myclimbing partners: Maike, Bupi, Per, Fredrik, Lina, Martin L, Uttama and Jo-han as well as all the friendly climbers at Stallet...errr campus1477 sciencepark...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 undDirk: Auch wenn die Distanz grösser war und die Treffen selten, waren sieimmer schön.

Meinen Eltern Marion und Jupp danke ich von ganzem Herzen, dass ich im-mer 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 :-).

45

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