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Biochem. J. (2009) 421, 463–471 (Printed in Great Britain) 463 doi:10.1042/BJ20082231 Overexpression of Drosophila mitoferrin in l(2)mbn cells results in dysregulation of Fer1HCH expression Christoph METZENDORF, Wenlin WU and Maria I. LIND1 Department of Comparative Physiology, Uppsala University, Norbyv. 18A, Uppsala, Sweden Mrs3p and Mrs4p (Mrs3/4p) are yeast mitochondrial iron carrier proteins that play important roles in ISC (iron-sulphur cluster) and haem biosynthesis. At low iron conditions, mitochondrial and cytoplasmic ISC protein maturation is correlated with MRS3/4 expression. Zebrafish mitoferrin1 (mfrn1), one of two MRS3/4 orthologues, is essential for erythropoiesis, but little is known about the ubiquitously expressed paralogue mfrn2. In the present study we identified a single mitoferrin gene (dmfrn) in the genome of Drosophila melanogaster, which is probably an orthologue of mfrn2. Overexpression of dmfrn in the Drosophila l(2)mbn cell line (mbn-dmfrn) resulted in decreased binding between IRP1A (iron regulatory protein 1A) and stem-loop RNA structures referred to as IREs (iron responsive elements). mbn-dmfrn cell lines also had increased cytoplasmic aconitase activity and slightly decreased iron content. In contrast, iron loading results in decreased IRP-1A–IRE binding, but increased cellular iron content, in experimental mbn-dmfrn and control cell lines. Iron loading also increases cytoplasmic aconitase activity in all cell lines, but with slightly higher activity observed in mbndmfrn cells. From this we concluded that dmfrn overexpression stimulates cytoplasmic ISC protein maturation, as has been reported for MRS3/4 overexpression. Compared with control cell lines, mbn-dmfrn cells had higher Fer1HCH (ferritin 1 heavy chain homologue) transcript and protein levels. RNA interference of the putative Drosophila orthologue of human ABCB7, a mitochondrial transporter involved in cytoplasmic ISC protein maturation, restored Fer1HCH transcript levels of iron-treated mbn-dmfrn cells to those of control cells grown in normal medium. These results suggest that dmfrn overexpression in l(2)mbn cells causes an ‘overestimation’ of the cellular iron content, and that regulation of Fer1HCH transcript abundance probably depends on cytoplasmic ISC protein maturation. INTRODUCTION tions, MRS3/4 expression is correlated with haem synthesis as well as with mitochondrial and cytosolic ISC protein maturation [13]. The yeast Atm1 gene, coding for a mitochondrial ABC (ATPbinding-cassette) half-transporter, is the link between mitochondrial ISC synthesis and cellular regulation of iron homoeostasis [15] as it transports a substrate necessary for cytosolic ISC protein maturation [16]. Abcb7, the mammalian orthologue of Atm1p [17], has been shown to be essential for cytoplasmic ISC protein maturation [18] and for erythropoiesis [19]. Orthologues of MRS3/4 have been reported in Dictyostelium discoideum [20], Cryptococcus neoformans [21], zebrafish [22], mice [23] and humans [24] and are called mitoferrins in metazoa. Zebrafish mfrn1 (mitoferrin1) is involved in mitochondrial haem synthesis and is essential for erythropoiesis [22]. Less is known about the ubiquitously expressed mfrn1-paralogue mfrn2. Even though it cannot complement mfrn1 mutations in zebrafish, it rescues the MRS3/4 yeast mutant [22]. This underscores the specialized role that mfrn1 plays in erythropoiesis. In the present work we identified the Drosophila homologue of mfrn2 and studied the effects that its overexpression has on cellular iron homoeostasis. A complex system of iron acquisition, transport, storage, and sensing proteins has evolved to keep the delicate balance between iron overload and iron deficiency (reviewed in [1]). Iron deficiency interferes with many essential cellular processes, whereas excess free iron is implicated in the formation of free radicals via the Fenton reaction, causing oxidative stress and diseases related to oxidative stress (e.g. Friedreich ataxia [2] and Alzheimer’s disease [3]). Several of these diseases are caused by disorders in the mitochondrial iron metabolism in particular (reviewed in [4–6]) and therefore a better understanding of the regulatory cues of cellular iron homoeostasis exerted by mitochondrial iron metabolism is essential. Members of the mitochondrial carrier protein family are structurally related and their role is to translocate a broad spectrum of solutes between the mitochondrial intermembrane space and the matrix (reviewed in [7]). The yeast genes MRS3 and MRS4 (MRS3/4) code for functionally redundant mitochondrial iron carrier proteins [8,9] and have been shown to interact with frataxin in haem and ISC (iron-sulphur cluster) synthesis [10,11]. The deletion of MRS3/4 suppresses mitochondrial iron accumulation in frataxin mutants [12] and both deletion or overexpression of MRS3/4 causes activation of the iron regulon, a group of genes which are normally transcriptionally activated during low-iron conditions [13]. Overexpression of MRS3 or MRS4 results in a decrease in total cellular iron content [14] and at low-iron condi- Key words: Drosophila, iron homoeostasis, iron transport, mitochondrial iron metabolism. EXPERIMENTAL Bacteria and yeast cultures Escherichia coli TOP10 chemically competent bacteria (Invitrogen) were used for cloning, according to the supplier’s protocols. Abbreviations used: 䉭Mrs3䉭Mrs4, yeast strain with deletion of MRS3/4 , 2-ME, 2-mercaptoethanol; DFO, deferoxamine mesylate; dmfrm, Drosophila mitoferrin; dsRNA, double-stranded RNA; DTT, dithiothreitol; EMSA, electrophoretic mobility-shift assay; FAC, ferric ammonium citrate; Fer1HCH, ferritin 1 heavy chain homologue; Fer2LCH, ferritin 2 light chain homologue; GFP, green fluorescent protein; GFPi, RNAi against GFP protein; IRE, iron responsive element; IRP, iron regulatory protein; ISC, iron-sulphur cluster; mfrn, mitoferrin; ORF, open reading frame; RNAi, RNA interference; RT–PCR, reverse transcription PCR; UTR, untranslated region; WT, wild-type. 1 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2009 Biochemical Society 464 C. Metzendorf, W. Wu and M. I. Lind Yeast strains: W303-A (MATa, ade2-1, his3-11,15, leu2-3,112, trp1-1, ura3-1, can1-100) was termed WT and NR82 (W303-A mrs3::HIS3 mrs4::LEU2) was termed 䉭Mrs3䉭Mrs4 (provided by Dr R. Lill, Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Germany). Yeast cells were grown at 30 ◦C in rich YPD medium [1 % (w/v) yeast extract, 2 % (w/v) peptone and 2 % (w/v) glucose] and on minimal SC-URA medium agar plates [0.67 % yeast nitrogen base without amino acids and bases, 0.07 % drop-out mix (self-made and from BIO 101® ) and 2 % (w/v) glucose], containing 75 μM bathophenanthrolinedisulfonic acid (Sigma–Aldrich) as indicated in Figure 3, and were incubated at 30 ◦C under aerobic conditions for 2– 4 days. Construction of p426TDH3-CG4963 and transformation of yeast The ORF (open reading frame) of CG4963 was PCR amplified from cDNA from adult Drosophila w1118 flies (forward primer introducing a BamHI restriction site 5 -GTT CGG ATC CAC CAT GAA CAT CGA CGA C-3 ; reverse primer introducing an EcoRI restriction site 5 -TAA TGA ATT CCT ACG TGC TGA AGC CCC G-3 ) and cloned into p426TDH3 (provided by Dr R. Lill, Institut für Zytobiologie und Zytopathologie der PhilippsUniversität Marburg, Germany) [25], yielding p426TDH3CG4963. Yeast was transformed following the quick protocol from Gietz and Woods [26]. l(2)mbn cell culture The Drosophila l(2)mbn cell line was maintained in standard medium as described in [27]. Penicillin G and streptomycin (Sigma–Aldrich) were used at concentrations of 60 μg/ml and 50 μg/ml respectively. Cells co-transfected with pCoHygro (Invitrogen) were cultured in the presence of 300 μg/ml Hygromycin B (Invitrogen). When indicated, transfected cells were also cultured in the presence of DFO (desferrioxamine) and/or FAC (ferric ammonium citrate; 20 μg/ml contains ∼ 60 μM Fe3+ ) at various concentrations as indicated in Figure 4. For iron-loading conditions, 1 × 106 cells/ml were seeded and after 7–8 h, FAC (or sterile water for normal conditions) was added to a final concentration of 40 μg/ml. The cells were incubated for an additional 16 h before harvesting. Gateway® cloning All Drosophila Gateway® collection plasmids were obtained from the Drosophila Genomics Resource Center, Bloomington, IN, U.S.A. The complete ORF of CG4963 (forward primer 5 -CAC CAT GAA CAT CGA CGA CTA CGA ATC-3 and reverse primer 5 -CTA CGT GCT GAA GCC CCG-3 ) and the ORF lacking the stop codon [CG4963(−)stop, reverse primer 5 CGT GCT GAA GCC CCG CTC-3 ] were PCR amplified from cDNA from l(2)mbn cells and cloned into the Gateway® Entry vector pENTR\ D-TOPO. Full length CG4963 was transferred into the Drosophila Gateway® Destination vector pAW and CG4963(−)stop was transferred into Gateway® vector pHWV via the LR-reaction (Invitrogen). Resulting plasmids were named pACG4963 and pHCG4963venus respectively. Gateway® vector pAVW was used as a negative control. Transient transfection of l(2)mbn cells and confocal laser scanning microscopy Cells in 24-well-plate format were transfected with 400 ng of pHVW or pHCG4963venus at a ratio DNA/Effectene c The Authors Journal compilation c 2009 Biochemical Society Transfection Reagent (Qiagen) of 1:25 (w/v). After 24 h the medium was changed and protein expression was induced by heat shock. After staining of mitochondria with MitoTracker® Deep Red 633 (Invitrogen), cells were washed and fixed. Fixed cells were washed in acetone and mounted in Vectashield® (Vectorlabs). Cells were imaged using a Leica TCS-SP confocal laser-scanning microscope (Leica Microsystems). Stable transfection of l(2)mbn cells Stable transfection was carried out as transient transfection (above), with these modifications: six-well plate format, 950 ng of pAVW or pACG4963 co-transfected 1:20 (w/w) with 50 ng of pCoHygro (Invitrogen). Three days after transfection, the medium was replaced with new medium containing 300 μg/ml Hygromycin B for 6–8 weeks, to select for co-transfected cells. Co-transfection was verified by fluorescence (pAVW) and by realtime RT–PCR (reverse transcription–PCR) (pACG4963). RNAi (RNA interference) of l(2)mbn cells dsRNA (double-stranded RNA) synthesis and transfection was performed as in Liu et al. [28] with primers for GFPi [GFP (green fluorescent protein) interference] [28] and CG7955 (probe set HFA08603, GenomeRNAi Data Base [29]; T7-CG7955, forward 5 -taa tac gac tca cta tag ggA CGG TGA AGT ACT TCA ACA AC-3 and reverse 5 -taa tac gac tca cta tag ggC GAC GAT TTT CCT GAG CC-3 ). Cells were treated three times in seven days with dsRNA and cells were used for experiments three days after the last RNAi treatment. Real-time RT–PCR l(2)mbn cells were harvested by scraping. Total RNA was extracted using TRIzol® reagent (Invitrogen), treated with DNase I (Ambion) and re-extracted with an equal volume of watersaturated phenol/chloroform (1:1, v/v) (Q-Biogene) followed by precipitation with 50 % isopropanol. The RNA pellets were resuspended in nuclease-free water and then tested in RT–PCR reactions. Absence of genomic DNA was checked by PCR, using Fer1HCH (ferritin 1 heavy chain homologue) primers that anneal on either side of the first intron. Total RNA (2.5 μg), primed by oligo(dT)20 -primers, were reverse transcribed with ThermoScript (Invitrogen). Real-time RT–PCR was carried out using QuantiTect SYBR green (Qiagen) in a RotorGene 3000 (Corbett Research) thermocycler. Primer performance was assayed by performing real-time RT–PCR with a dilution series of cDNA. The method of ‘comparative quantification’ was used to quantify relative transcript levels. Relative transcript levels of the house-keeping gene Rp49 were used to normalize data obtained for genes of interest. The primers used were: Rp49 (CG7939), forward 5 CCG CTT CAA GGG ACA GTA TCT G-3 , reverse 5 -CAC GTT GTG CAC CAG GAA CTT-3 [30]; dmfrn (CG4963), forward 5 -TTT GCC GCC TAC GAG ATG-3 , reverse 5 -TAG AAA TGG CGT CGT GTA TG-3 ; Fer1HCH (CG2216), forward 5 CTG CTC CTG TTG GCC GTG GT-3 , reverse 5 -TCC TTC ATG TCC ACC CAG TCC T-3 ; Fer2LCH (ferritin 2 light chain homologue, CG1469), forward 5 -TAA TCA CCG CAT GCT CTA CG-3 , reverse 5 -TTG GCG TTG ATG TAG GAC TG-3 ; dABCB7 (CG7955), forward 5 -GCC TTC TCT TCC GCT TCT, TT-3 reverse 5 -GGC ACC ACT GCA ATA ACC TT-3 ; Act42A (CG12051), forward 5 -GCT TCG CTG TCT ACT TTC CA-3 reverse 5 -CAG CCC GAC TAC TGC TTA GA-3 ; Rbf (CG7413), forward 5 -GGA GGA CCC CAA GTT CAG TG-3 , reverse 5 CCT TTG GCT CCC CGT CCT CG-3 ; and CycE (CG3938), Drosophila mitoferrin 5 -GGA CGA GTA CCT GGG CGA-3 , reverse 5 -GTT GCT GCT GCT CTT GCC-3 . Western blot analysis l(2)mbn cells were dissolved in 1 × Laemmli buffer, and 20 μg total protein was separated by SDS/PAGE (12.5 % gel). Proteins were transferred to PVDF membranes (Amersham) and visualized by immunoblotting with anti-actin antibody C11 (1:2000, v/v) (Santa Cruz Biotechnology) and anti-Fer1HCH antiserum (1:2000, v/v) as described in [31]. Iron quantification of cell lysates The improved ferrozine-based assay for the quantification of total iron, including haem-iron, was performed according to Riemer et al. [32] with cell type specific adjustments as indicated in Figure 6. EMSA (electrophoretic mobility-shift assay) l(2)mbn cells were incubated as indicated, and homogenized in band shift buffer [40 mM KCl, 25 mM Tris/HCl, pH 7.5, 1 % (v/v) Triton X-100, 5 mM DTT (dithiothretiol), and protease inhibitor cocktail (Roche)]. Extracts were centrifuged twice (5 min, then 30 min) at 16000 g (4 ◦C). Total protein (20 μg) was added to a final volume of 12.5 μl of band shift buffer with, or without, 2 % (v/v) 2-ME (2-mercaptoethanol). Samples were incubated with 32 P-labelled IRE [33] in reaction mixture [20 % (w/v) glycerol, 2 μg/μl yeast tRNA, 200 μM DTT and RNase inhibitor] and separated on 4 % acrylamide (59:1)/TBE (Tris/borate/EDTA, 89 mM Tris, 89 mM boric acid and 10 mM EDTA, pH 8.0) gels. Fixed gels were exposed for autoradiography. QuantityOne software (BioRad) was used for densitometry and values were background corrected. Relative IRE-binding activity of IRP-1A (iron regulatory protein 1A) was calculated as density(no 2-ME) /density(2-ME) . Preparation of cytoplasmic extract l(2)mbn cells were incubated as indicated in Figure 5. Cells (40 × 106 per treatment) were harvested and washed three times in PBS. Cells were resuspended in a buffer containing 0.25 M sucrose, 50 mM Hepes, pH 7.4, and proteinase inhibitor cocktail (Roche). Cytoplasmic extract was prepared according to Drapier and Hibbs [34]. The protein concentration of cytosolic extract was 1–2 mg/ml. Samples were kept at − 80 ◦C before measurement of aconitase activity. Aconitase activity Cytosolic aconitase activity was determined spectrophotometrically at 240 nm by following the disappearance of cis-aconitate (Sigma–Aldrich). For the assay, 50 μg protein was used and the assay was performed according to Drapier and Hibbs [34]. One unit (U) corresponds to one μmol of substrate consumed per min. Software and internet resources DNA and amino acid sequences were retrieved and subjected to BLAST 2.2.17 searches at NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/) and Flybase (http://flybase.bio.indiana.edu/). Primers were designed using the Oligo 4 (Molecular Biology Insights) and Primer3 (http://frodo.wi.mit.edu/) programs. Sequence alignments were 465 created with BioEdit 7.0.5.1 and at the Biology Work Bench (http://workbench.sdsc.edu) using ClustalW with the BLOSUM matrix. Alignment shading was done with TexShade within Biology Work Bench. RESULTS AND DISCUSSION CG4963 is the only orthologue of Mrs3/4p in Drosophila We performed BLASTP searches against the Drosophila melanogaster protein database at FlyBase to search for orthologues of Mrs3/4p. CG4963 was the only probable candidate retrieved (bit scores 205 and 191 and E-values 3 × 10−53 and 3 × 10−49 respectively). The alignment of CG4963’s amino acid sequence with yeast Mrs3/4p and zebrafish mfrn1 and mfrn2 showed a ∼ 10 % higher degree of sequence conservation between the metazoan proteins (38 % and 32 % sequence identity, 50 % and 44 % sequence similarity to mfrn1 and mfrn2) than between CG4963 and the yeast proteins (28 % and 27 % sequence identity, 41 % sequence similarity to both Mrs3p and Mrs4p) (Figure 1). A putative substrate-binding site and three contact sites in mitochondrial carrier proteins have been proposed by Robinson and Kunji [35,36]. The putative substrate-binding site and contact sites I and II are identical in CG4963, Mrs3/4p and Mfrn1/2, while contact site III is identical within the group of eumetazoa and within the two yeast mitochondrial carriers (Figure 1). CG4963 expression rescues yeast ΔMrs3ΔMrs4 mutants Not all proteins with similarity to mitochondrial carrier proteins actually localize to mitochondria [7]. To investigate the subcellular localization of CG4963, we transiently transfected l(2)mbn cells with plasmids that allowed heat-inducible expression of venus protein or C-terminally venus-tagged CG4963 protein. Expression of venus protein resulted in a pancellular signal, whereas CG4963–venus protein colocalized with the mitochondrial stain MitoTracker® Deep Red (Figure 2), confirming CG4963 as a mitochondrial protein. Next, we tested whether CG4963 was able to complement the yeast ΔMrs3ΔMrs4 double mutant, which shows a growth defect on low-iron medium. The WT (wild-type) strain was able to grow on iron-replete and low-iron medium, while the double mutant transfected with empty vector grew only on iron-replete medium (Figure 3). This growth defect was rescued by ectopic expression of CG4963 (Figure 3). These results show that CG4963 codes for a functional orthologue of MRS3/4, and we therefore named it dmfrn (Drosophila mitoferrin) to avoid confusion with vertebrate mitoferrin, which is sometimes referred to as mitoferrin. Insect and invertebrate genomes code for only one mitoferrin gene Yeast [8,12,13], zebrafish, mouse and humans [22–24] have two mfrn genes, but only one gene was found in the genome of D. melanogaster. To investigate whether this was common in insects and invertebrates, the amino acid sequence of Mrs3p was used in BLASTP searches against six insect genomes as well as the databases of Caenorhabditis elegans, Strongylocentrotus purpuratus, human, mouse and chicken (Table 1). The E-values for previously identified mitoferrins ranged from 3 × 10−58 to 4 × 10−54 and the best off-target hit was 1 × 10−24 . Amino acid sequences of all hits with E-values lower than 1 × 10−40 and contained the putative c The Authors Journal compilation c 2009 Biochemical Society 466 Figure 1 C. Metzendorf, W. Wu and M. I. Lind CG4963 protein shares high sequence identity and similarity with mitoferrin1/2 and Mrs3/4p Alignment of the amino acid sequences of Drosophila melanogaster CG4963 (CG4963), zebrafish mitoferrin1 (Mfrn1) and mitoferrin2 (Mfrn2) as well as yeast Mrs3p (Mrs3) and Mrs4p (Mrs4). Identities in all four sequences (black boxes) and identities (dark grey boxes) and/or similarities (light grey boxes) between CG4963 and any other sequence are highlighted. Arrowheads indicate putative substrate-binding sites [35], asterisks indicate putative contact sites and bars above the sequences indicate transmembrane helices H1-H6 and matrix side helices h12, h34 and h56 [35]. GenBank accession numbers: CG4963, NP_651600; mitoferrin1, NP_001035060; mitoferrin2, NP_998284; Mrs3p, NP_012402; and Mrs4p, NP_012978. Figure 2 CG4963venus localizes to mitochondria l(2)mbn cells were transiently transfected with plasmids for expression of fluorescence protein venus (pHVW) or C-terminally venus-tagged CG4963 protein (pHCG4963venus). After heat shock induction of expression, cells were stained with MitoTracker® Deep Red 633, fixed, washed, and analysed by laser scanning microscopy. substrate binding site, whereas the first hits outside the threshold did not (see Supplementary Table S1 and Figure S1 at http://www.BiochemJ.org/bj/421/bj4210463add.htm). This analysis identified two mfrn genes in all vertebrates, whereas only one mitoferrin gene was identified in each of the six insect and the two other invertebrate (nematode and sea urchin) genomes (Table 1). The ubiquitously expressed mfrn2 cannot rescue mfrn1 mutants, even though mfrn2 rescues MRS3/4 double mutants, c The Authors Journal compilation c 2009 Biochemical Society which makes it a functional homologue of mfrn1 [22]. Therefore it was proposed that mfrn2 may have a specialized function in haem synthesis during erythropoiesis. Recently, the reason for the different functions of mfrn1 and mfrn2 was identified. Only mfrn1, but not mfrn2, accumulates in mitochondria of erythropoietic cells at sufficient quantities to support erythropoiesis [37]. Considering these reports, the most compelling reason for the presence of only one mfrn gene in invertebrate genomes may be the fact that they lack the Drosophila mitoferrin 467 Growth of the yeast mutant 䉭Mrs3䉭Mrs4 on low-iron minimal medium is restored by expression of CG4963 Figure 3 Yeast strains WT and 䉭Mrs3䉭Mrs4 were transformed with expression plasmids p426TDH3 or p426TDH3-CG4963 and grown in YPD medium overnight, washed with milliQ water and diluted to a D 600 ∼ 0.07. Serial dilutions (1:10) were spotted in parallel on SC-URA (control) and SC-URA 75 μM bathophenanthrolinedisulfonic acid (low-iron) agar plates and incubated at 30 ◦C under aerobic conditions for 2–4 days. Table 1 Mrs3p BLASTP against reference protein sequences (refseq) of completed genomes (NCBI) with their respective genes and chromosome Hits with E -values 10−49 . Organism Accession number Bit-score/E -value Gene Chromosome Anopheles gambiae Apis mellifera Drosophila melanogaster Drosophila pseudoobscura Nasonia vitripennis Tribolium castaneum Caenorhabditis elegans Strongylocentrotus purpuratus Danio rerio XP_316075 XP_625179 NP_651600 XP_001358253 XP_001604399 XP_973746 NP_496447 XP_001177451 NP_001035060 NP_998284 XP_417682 XP_421702 NP_080607 NP_660138 NP_057696 NP_112489.3 200/3 × 10−51 207/2 × 10−53 205/3 × 10−53 215/9 × 10−56 215/1 × 10−56 210/2 × 10−54 201/4 × 10−52 223/2 × 10−58 218/4 × 10−57 208/4 × 10−54 230/7 × 10−61 209/1 × 10−54 224/1 × 10−58 220/1 × 10−57 222/3 × 10−58 217/1 × 10−56 ENSANGP00000022876 LOC552801 CG4963-PA (dmfrn ) GA18557-PA LOC100120798 LOC662563 W02B12.9 LOC584279 Slc25a37 (mfrn1 [22]) Slc25a28 (mfrn2 [22]) SLC25A37 SLC25A28 Slc25a37 [22] Slc25a28 [22,23] SLC25A37 [22] SLC25A28 [22,24] 2L LGUn 3R 2 LG9 II 10 13 22 6 14 19 8 10 Gallus gallus Mus musculus Homo sapiens specialized haem synthesis system of vertebrate erythropoiesis. This would also suggest that invertebrate mitoferrins are functional orthologues of vertebrate mfrn2. Interestingly, the unicellular yeast does not seem to fit into this picture as it also has two mitoferrin genes despite its obvious lack of erythropoiesis. It is very likely that MRS3 and MRS4 are the product of a genome duplication after the divergence of Saccharomyces from Kluyveromyces [38,39]. Since only MRS4 is under the control of the transcriptional iron regulators Aft1p and Aft2p [40], the two mitoferrins in yeast may have adapted to different regulational control under specific conditions. dmfrn mRNA levels are increased after iron starvation with DFO MRS4 is under the control of the transcription factors Aft1p and Aft2p, and its transcript abundance is increased under low-iron conditions [40]. To test whether dmfrn transcript was also affected by low-iron concentrations, l(2)mbn cells were grown for 48 h in the presence of the iron chelator DFO. dmfrn mRNA abundance (as assessed by real-time RT–PCR) increased 4-fold in ironstarved cells compared with control cells. Iron-loading of the cells, using FAC, had no effect on dmfrn transcript levels (Figure 4). Accordingly, partially iron-loaded chelator still resulted in an increase in dmfrn transcript, whereas iron-saturated chelator did Figure 4 cells dmfrn transcript is increased at low-iron conditions in l(2)mbn l(2)mbn cells were grown for 48 h in standard medium or medium supplemented with either DFO or FAC (20 μg/ml contains ∼ 60 μM Fe3+ ) at concentrations as indicated. Real-time RT–PCR was used to record fold changes of treated cells and results are shown relative to cells grown in standard medium. Results are the means + − S.D. of four independent experiments. not (Figure 4). This shows that the change in dmfrn transcript level is probably due to decreased cellular iron levels and is not a result of chemical side effects from the chelator itself. Thus dmfrn is likely to play a role in the iron metabolism of l(2)mbn cells. c The Authors Journal compilation c 2009 Biochemical Society 468 C. Metzendorf, W. Wu and M. I. Lind dmfrn overexpression in normal conditions results in higher cytoplasmic ISC protein maturation IRP-1A is a cytoplasmic protein that, depending on the availability of ISCs, shifts between two conformations and functions. In the cytoplasm, IRP-1A has an ‘aconitase’ conformation if an ISC is bound, and the protein has aconitase activity. If the ISC is lost, IRP-1A can bind stem-loop RNA structures that are referred to as IREs (reviewed in [1,41]). In Drosophila, two IREs have been identified and shown to be functional. One is located in the 5 UTR (untranslated region) of Sdhb (succinate dehydrogenase B) mRNA [42] and the other is in the 5 -UTR of one of three splice variants of Fer1HCH mRNA [43]. Mrs3/4p co-operate with frataxin during ISC and haem synthesis [10,11] and Mrs3/4p overexpression at low-iron conditions results in increased cytoplasmic ISC protein maturation. dmfrn, which rescues the MRS3/4 double mutant and shows high similarity in amino acid sequence to other mitoferrins, is very likely to have the same function in Drosophila. If so, dmfrn overexpression should decrease IRP-1A–IRE binding activity and increase cytoplasmic aconitase activity. To test this hypothesis, we first made stable cell lines that overexpressed either venus (mbn-venus) or dmfrn (mbn-dmfrn). The mbn-venus cell line was included as an additional control, as it was not known whether a changed protein metabolism would effect iron homoeostasis. Next, the relative IRE-binding activity of IRP-1A and cytoplasmic aconitase activity was measured in cell extracts from the different cell cultures. IRP-1A–IRE binding activity and cytoplasmic aconitase activity of WT and mbn-venus cells were not different (Figure 5), indicating that protein overexpression does not interfere with this aspect of iron homoeostasis. During normal growth conditions, less IRP-1A was in the IREbinding conformation in mbn-dmfrn cells than in WT and mbnvenus cells (Figure 5A). In contrast, no difference in relative IRE-binding activity was observed between the different cell lines in iron-loading growth conditions induced by FAC treatment (Figure 5A). Nevertheless, FAC treatment resulted in an overall decrease in relative IRP-1A–IRE binding activity to a ratio lower than that of WT and mbn-venus cells, in normal growth conditions (Figure 5A). Conversely, we observed significantly increased cytoplasmic aconitase activity in mbn-dmfrn cells when compared with control cells at normal growth conditions (Figure 5B). FAC-induced iron loading resulted in a general increase in aconitase activity in all cell lines, but mbn-dmfrn cells still had marginally higher activity than the controls (Figure 5B). These results indicate that cytoplasmic ISC-protein maturation at normal growth conditions is increased in mbn-dmfrn cells compared with control cells. To test if the observed phenotype in mbn-dmfrn cells was simply caused by higher cellular iron content, we quantified total iron. Although WT and mbn-venus cells behaved similarly, a small but significant decrease in iron content of mbn-dmfrn cells at normal conditions was observed (Figure 6). At iron-loading conditions, as with relative IRE-binding and aconitase activity, no difference was evident between all three cell lines, but cellular iron content was increased to similar levels (Figure 6). The combination of a decreased cellular iron content and an increase in cytoplasmic ISC protein maturation in mbn-dmfrn cells under normal growth conditions supports our hypothesis that ISC synthesis is stimulated by the overexpression of dmfrn. Interestingly, iron-loaded mbn-dmfrn cells exhibited slightly increased cytoplasmic aconitase activity when compared with iron-loaded control cells (Figure 5B), whereas relative IRPbinding activity was similar in all three cell lines after ironloading (Figure 5A). It is probable that IRP-1A was saturated c The Authors Journal compilation c 2009 Biochemical Society Figure 5 In normal medium, relative IRE-binding activity of IRP-1A is decreased and cytoplasmic aconitase activity is increased in l(2)mbn cells overexpressing dmfrn l(2)mbn cells (WT) and stably transfected cell lines overexpressing venus (mbn-venus) or dmfrn (mbn-dmfrn) were grown in fresh standard medium or in iron-loading conditions of fresh standard medium supplemented with 40 μg/ml FAC for 16 h and either used for EMSA or a cytoplasmic aconitase activity assay. (A) Upper panel. Relative IRE-binding activities of IRP-1A. Intensities of EMSA IRP-1A–IRE-bands were measured and the relative IRE-binding activities of IRP-1A were calculated. At normal growth conditions, relative IRE-binding activity of IRP-1A is decreased in mbn-dmfrn cells when compared with control cells. Data represent the means + S.D. of three independent experiments. Lower panel. A representative EMSA. FP, free probe. − (B) Cytoplasmic aconitase activity is significantly increased in mbn-dmfrn cells when compared with control cells (*t test P 0.05). In iron-loading conditions cytoplasmic aconitase activity of mbn-dmfrn cells is slightly increased when compared with control cells. Data represent the means + − S.D. of three independent experiments. with ISC after iron-loading. However, as IRP-1A can lose one iron atom of the ISC and remain an inactive IRE-binding protein, its aconitase activity can be diminished [43a]. The slightly increased aconitase activity in iron-loaded mbn-dmfrn cells could indicate that the supply of functional IREs in these cells is increased when compared with that of control cells. In summary, these results are compatible with the model in the yeast system, where MRS3/4 overexpression at low-iron conditions results in increased ISC synthesis and ISC protein maturation, in mitochondria and the cytoplasm respectively [13]. Apparently, the iron content of l(2)mbn cells at normal conditions is not too high to interfere with the dmfrn overexpression phenotype. dmfrn overexpression in l(2)mbn cells alters Fer1HCH expression In whole flies, increased transcript levels of Fer1HCH can be used as an indicator for iron-loading [44]. As one Fer1HCH mRNA Drosophila mitoferrin 469 Figure 6 Iron levels in dmfrn -overexpressing cells are not increased compared with control l(2)mbn cells (WT) and stably transfected cell lines overexpressing venus (mbn-venus) or dmfrn (mbn-dmfrn) were grown in fresh medium or in iron-loading conditions of fresh medium supplemented with 40 μg/ml FAC for 16 h. Total iron was measured using the Ferrozine method [32] and data was normalized to the total protein concentration. Iron levels of mbn-venus cells are significantly decreased in normal medium (t test *P 0.05). Results shown are the means + − S.D. of four experiments. splice variant contains an IRE [43], Fer1HCH protein levels may give an additional indication of the cell-sensed iron status. Using real-time RT–PCR, we confirmed that dmfrn was actually overexpressed in mbn-dmfrn cells (samples 3a and 3b in insert of Figure 7A). Next, we measured relative transcript levels of Fer1HCH and performed Fer1HCH immunoblots of cells grown at normal conditions and iron-loading conditions. Both Fer1HCH mRNA and protein were increased in all three cell lines after treatment with FAC (Figures 7A and 7B). This increase in Fer1HCH expression (transcript and protein) occurs simultaneously with the elevated cellular iron levels described above (Figure 6), showing that cell-sensed iron levels are reflected in both Fer1HCH transcript and protein. In iron-loaded flies, Fer2LCH mRNA was also previously found to be elevated [45], but in our cell culture model this effect was not observed (Figure 7A). Interestingly, mbn-dmfrn cells had significantly increased levels of Fer1HCH transcript when compared with WT and mbnvenus cells grown under the same conditions (normal medium and high-iron medium) (Figure 7A). Fer1HCH protein levels were also higher in mbn-dmfrn cells compared with control cells (Figure 7B). These results indicate that mbn-dmfrn cells ‘overestimate’ the cellular iron content. The higher amount of cytoplasmic ISCs, as determined by reduced IRP-1A–IRE binding activity and increased cytoplasmic aconitase activity, in mbn-dmfrn cells at normal conditions and in all cell lines at high-iron conditions is probably responsible, to some degree, for the increase in Fer1HCH protein, i.e. the reduction of relative IRP-1A–IRE binding activity would be expected to result in a higher rate of translation of the Fer1HCH splice variant containing the IRE. RNAi of the putative Drosophila ABCB7 transporter restores basic Fer1HCH transcript levels Although the decrease in IRP-1A–IRE binding activity is probably responsible for some of the increase in Fer1HCH protein expression observed in mbn-dmfrn cells at normal conditions, and in all cell lines at iron-loading conditions, the reason for a higher level of Fer1HCH transcript in mbn-dmfrn cells remains unclear. As relative Fer1HCH transcript levels correlate with the activity of cytoplasmic aconitase in mbn-dmfrn cells, we suspected that mitochondrial ISC synthesis might play a role in Fer1HCH Figure 7 dmfrn Fer1HCH expression is increased in l(2)mbn cells overexpressing l(2)mbn cells (1) and stably transfected cell lines overexpressing venus (2) or dmfrn (3) were grown in normal medium or in iron-loading conditions of medium supplemented with 40 μg/ml FAC for 16 h. (A) Relative transcript levels of Fer1HCH and Fer2LCH were obtained by real-time RT–PCR. Fer1HCH is significantly (t test **P 0.01) upregulated in cells overexpressing dmfrn , compared with WT and cells overexpressing venus . After FAC treatment, Fer1HCH is significantly (t test *P 0.05) increased in dmfrn -overexpressing cells compared with dmfrn -overexpressing cells grown in normal medium and FAC treated WT and cells overexpressing venus . Fold changes are relative to WT cells at normal growth conditions. Results shown are the means + − S.D. of four independent experiments. Insert, representative experiment showing relative expression of dmfrn in WT cells (1) and in stably transfected cell lines overexpressing venus (2) or dmfrn (3a and 3b, two independent cell lines). (B) Western blot of Fer1HCH protein expression of cells treated as in (A). Representative data of three experiments is shown. transcriptional regulation. So far, a regulation mechanism that acts on the transcript level and is influenced by mitochondrial ISC synthesis has only been described in yeast. Chen et al. [46] found that mitochondrial ISC synthesis inactivates Aft1p and Aft2p under iron-replete conditions. The signal which mediates this inactivation comes from mitochondrial ISC synthesis itself, and depends on Atm1p [15]. Therefore we investigated whether such a signal could also be responsible for the increase in Fer1HCH transcript of mbn-dmfrn cells. Using BLASTP search, we identified CG7955 (referred to as dABCB7) as a putative ABCB7 orthologue. Its amino acid sequence shares very high sequence similarity and identity with Abcb7 and Atm1p (see Supplementary Table S2 and Figure S2 at http://www.BiochemJ.org/bj/421/bj4210463add.htm). A dsRNA probe targeting all three possible transcripts of dABCB7 was chosen from the Heidelberg RNAi database. Knockdown of dABCB7 resulted in a depletion of dABCB7 transcript to 20–30 % of the levels seen in mock-treated cells (Figure 8A) but did not effect dmfrn expression (see Supplementary Figure S3 at http://www.BiochemJ.org/bj/421/bj4210463add). Fer1HCH transcript levels in mock-treated mbn-dmfrn cells grown for 16 h under iron-loading conditions were increased when compared with mock-treated mbn-venus cells, which were grown in similar conditions (Figure 8A). Treatment of mbn-dmfrn cells with dABCB7 dsRNA decreased Fer1HCH transcript levels significantly and to the level observed in mock-treated control cells, not grown under iron-loading conditions (Figure 8A). Fer1HCH expression levels in mock-treated mbn-venus cells c The Authors Journal compilation c 2009 Biochemical Society 470 C. Metzendorf, W. Wu and M. I. Lind Figure 8 RNAi of dABCB7 suppresses increased Fer1HCH expression in dmfrn overexpressing cells l(2)mbn cells overexpressing venus (mbn-venus) or overexpressing dmfrn (mbn-dmfrn) were treated with dsRNA probes for either GFP (GFPi, mock treatment) or dABCB7 (dABCB7i). Cells were then grown in iron supplemented medium for 16 h and used for real-time RT–PCR. Fold changes are relative to the control. (A) normal medium, mbn-venus cells, GFPi treated. (B) The respective GFPi control of each cell line. Results shown are the means + − S.D. of three (A) and two (B) independent experiments (*t test P 0.05). that fly ferritin expression is most likely regulated on both the translational and transcriptional level to avoid overproduction of ferritin protein. In mosquitoes, a dipteran insect closely related to flies, it was shown in cell culture that ferritin is under transcriptional regulation [49], and in Drosophila, metalresponsive elements have been identified in the ferritin promoter region [50]. Furthermore, two alternative polyadenylation sites in Drosophila Fer1HCH mRNA have also been reported [43,44]. We assume that the change in Fer1HCH transcript in our experiments is probably due to a combination of both elevated transcription and transcript stability. It should also be mentioned that holo-ferritin in Drosophila is not located in the cytoplasm, but in the secretory pathway, which allows export of iron-loaded ferritin (for a review on insect iron metabolism see [51]). Total iron content of cells, as measured in the present study, is the sum of iron influx and efflux mediated by this system and possibly by other, yet unidentified, mechanisms. A special region of the fly midgut, which is very ferritin-rich in ironloaded flies, is purported to be responsible for iron storage. A yet unidentified post-transcriptional regulatory mechanism balances the ratio of Fer1HCH and Fer2LCH subunits in holo-Ferritin of the midgut [52]. We were not able to detect Fer2LCH protein in the l(2)mbn cell line, and one possible reason for this could be that it is exported as holo-ferritin from l(2)mbn cells, which being blood cells [53] are unlikely to be involved in iron storage. In summary, our results establish dmfrn as the Drosophila orthologue of mitoferrin2 and show that its overexpression causes changes in several components of the cellular iron homoeostasis system in l(2)mbn cells. Furthermore, we show that knockdown of putative dABCB7 blocks the effects of dmfrn overexpression on Fer1HCH expression, indicating that both dABCB7 and dmfrn act in a similar pathway. AUTHOR CONTRIBUTION treated with FAC to give iron-loading conditions were only slightly increased, but also returned to levels comparable with that of control cells after dABCB7 RNAi treatment (Figure 8A). It has been reported that RNAi of ABCB7 in HeLa cells resulted in decreased cell viability [47]. As further controls, we therefore assayed Actin5C, cyclinE and Rbf transcript levels and found no difference in their expression (Figure 8B) when comparing dABCB7-dsRNA treated cells with mock-treated cells. From this, and our observation that cells treated with dABCB7dsRNA did not noticeably stop proliferating (results not shown), we concluded that cell viability was not affected during the experimental period. Since RNAi of dABCB7 restored Fer1HCH transcript levels of FAC treated mbn-dmfrn cells to levels of control cells, we suggest that regulation of Fer1HCH transcript levels is connected with mitochondrial ISC synthesis. Conclusions In yeast it has been established that cytoplasmic ISC protein maturation is dependent on substrates provided by mitochondrial ISC synthesis, whereas in mammals this issue is controversial (reviewed in [48]). Our results indicate, in a Drosophila cell line, stimulation of mitochondrial ISC synthesis, through the overexpression of dmfrn, results in more ISC protein maturation in the cytoplasm. This is in favour of a model in insects where mitochondrial ISC synthesis stimulates cytoplasmic ISC protein maturation. The fact that none of the Fer2LCH transcripts and only one of three Fer1HCH transcripts contains an IRE makes it clear c The Authors Journal compilation c 2009 Biochemical Society Christoph Metzendorf designed the experiments, wrote the manuscript and performed all experiments, except for the aconitase and EMSA assays. Wenlin Wu performed the EMSA assay. Maria Lind designed the experiments, discussed the manuscript and performed the aconitase assay. ACKNOWLEDGEMENTS We thank Dr R. Lill (Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Germany) for the yeast strains W303-A and NR82 as well as for the plasmid p426TDH3. We also thank Dr F. Missirlis (Queen Mary, University of London, U.K.) for providing ferritin antibodies. We received The Drosophila GateWay® plasmid collection through the Drosophila Genomics Resource Center. FUNDING This work was supported by grants from the Wenner–Gren foundations; the Lennander’s foundation [grant number CTS08:226]; the Magnus Bergvall’s foundation; and by the Swedish Research Council [grnat number 621-2008-3669] to M.I.L. W.W. is a recipient of a postdoctoral fellowship from Wenner–Gren foundations. The work was also supported by a grant from the Swedish Research Council [grant number 621-2006-4658] to Kenneth Söderhäll (Department of Comparative Physiology, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden). REFERENCES 1 Dunn, L. L., Rahmanto, Y. 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Science 200, 1448–1459 Received 17 November 2008/8 May 2009; accepted 19 May 2009 Published as BJ Immediate Publication 19 May 2009, doi:10.1042/BJ20082231 c The Authors Journal compilation c 2009 Biochemical Society Biochem. J. (2009) 421, 463–471 (Printed in Great Britain) doi:10.1042/BJ20082231 SUPPLEMENTARY ONLINE DATA Overexpression of Drosophila mitoferrin in l(2)mbn cells results in dysregulation of Fer1HCH expression Christoph METZENDORF, Wenlin WU and Maria I. LIND1 Department of Comparative Physiology, Uppsala University, Norbyv. 18A, Uppsala, Sweden Table S1 Bit scores and E -values of Mrs3p blast hits (BLASTP 2.2.17) Organism Abbreviation Protein accession number Gene Bit score/E -value Drosophila melanogaster Dmel1 Dmel2 Dsp1 Dsp2 Agam1 Agam2 Amel1 Amel2 Nvit1 Nvit2 Spur1 Spur2 Tcas1 Tcas2 Mfrn1 Mfrn2 Drer3 Mrs3 Mrs4 Ggal1 Ggal2 Ggal3 Mmus1 Mmus2 Mmus3 Hsap1 Hsap2 Hsap3 Cele1 Cele2 NP_651600 NP_733366 XP_001358253 XP_001357671 XP_316075 XP_316535 XP_625179 XP_395257 XP_001604399 XP_001606469 XP_001177451 XP_796953 XP_973746 XP_970499 NP_001035060 NP_998284 NP_001070070 NP_012402 NP_012978 XP_417682 XP_421702 XP_418818 NP_080607 NP_660138 NP_659042 NP_057696.2 NP_112489.3 NP_060345.1 NP_496447 NP_501552 CG4963-PA aralar1 GA18557-PA GA15263 ENSANGP00000022876 ENSANGP00000009995 LOC552801 PREDICTED: similar to CG4963-PA LOC100120798 LOC100122862 LOC584279 LOC592330 LOC662563 LOC662563 Slc25a37 (mfrn1) Slc25a28 (mfrn2) zgc:153036 MRS3 MRS4 SLC25A37 SLC25A28 PREDICTED: similar to Solute carrier family 25, member 38 Slc25a37 Slc25a28 SLC25A38 SLC25A37 SLC25A28 SLC25A38 W02B12.9 D1046.3 205/3×10−53 102/4×10−22 215/9×10−56 104/1×10−23 200/3×10−51 103/7×10−23 207/2×10−54 111/2×10−25 215/1×1056 109/1×10−24 223/2×10−58 110/2×10−24 210/2×10−54 105/1×10−23 218/4×10−57 208/4×10−54 107/9×10−24 – – 230/7×10−61 209/1×10−54 111/5×10−25 224/1×10−58 220/1×10−57 110/1×10−24 222/3×10−58 217/1×10−56 109/3×10−24 201/4×10−52 108/3×10−24 Drosophila pseudoobscura Anopheles gambiae Apis mellifera Nasonia vitripennis Strongylocentrotus purpuratus Tribolium castaneum Danio rerio Saccharomyces cerevisiae Gallus gallus Mus musculus Homo sapiens Caenorhabditis elegans 1 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2009 Biochemical Society C. Metzendorf, W. Wu and M. I. Lind Table S2 Result of BLAST 2 sequence alignments (BLASTP 2.2.18) for comparison of human, yeast and Arabidopsis ABCB7 amino acid sequences with that of the putative orthologues in Drosophila Gi number, NCBI sequence identifier; Bit., Bit score; Exp., E -value; Id., identities; and Pos., positives. Human Yeast Arabidopsis Drosophila Gi number gi|42490749 gi|6323959 gi|15237155 gi|45551501 Human – Bit. 582 (1501) Exp. 5e-164 Id. 295/598 (49%) Pos. 412/593 (69%) Gaps 11/593 (1%) Bit. 664 (1713) Exp. 0.0 Id. 339/604 (56%) Pos. 447/604 (74%) Gaps 9/604 (1%) Bit. 723 (1867) Exp. 0.0 Id. 371/645 (57%) Pos. 486/645 (75%) Gaps 18/645 (2%) – Bit. 587 (1512) Exp. 3e-165 Id. 332/714 (46%) Pos. 462/714 (64%) Gaps 44/714 (6%) Bit. 542 (1397) Exp. 6e-152 Id. 294/609 (48%) Pos. 408/609 (66%) Gaps 9/609 (1%) – Bit. 640 (1652) Exp. 0.0 Id. 341/599 (56%) Pos. 434/599 Gaps 13/599 (2%) Yeast Arabidopsis Figure S1 Alignment of the first transmembrane helix region of putative and known mitoferrins The Mrs3p amino acid sequence was used in BLASTP against whole genome databases of invertebrates and vertebrates. Amino acid sequences of hits with E -values lower than 1 × 10−20 were aligned. The putative substrate-binding site of mitochondrial carrier proteins as proposed by Kunji and Robinson [1,2] is conserved within all known mitoferrins and all BLAST hits with E -values lower than 1 × 10−40 (underlined). Table S1 lists the sequence abbreviations, BLAST scores and accession numbers of the proteins and the genes. c The Authors Journal compilation c 2009 Biochemical Society Drosophila mitoferrin Figure S2 Alignment of human ABCB7 with yeast Atm1, Arabidopsis ABCB7 orthologues and the three putative Drosophila orthologues of ABCB7 One putative ABCB7 gene (CG7955), with three hypothetical transcripts, was identified in Drosophila by BLASTP analysis. The alignment with known ABCB7 transporters from human (ABCB7), yeast (ATM1) and plant (AtAtm) shows very high sequence identity and similarity. c The Authors Journal compilation c 2009 Biochemical Society C. Metzendorf, W. Wu and M. I. Lind Figure S3 dmfrn expression is not affected by CG7955 (putative ABCB7 orthologue) RNAi treatment in cells overexpressing venus (mbn-venus) or dmfrn (mbn-dmfrn) Results from two sets of independently transfected cell lines are shown. REFERENCES 1 Robinson, A. J. and Kunji, E. R. (2006) Mitochondrial carriers in the cytoplasmic state have a common substrate binding site. Proc. Natl. Acad. Sci. U.S.A. 103, 2617–2622 2 Kunji, E. R. and Robinson, A. J. (2006) The conserved substrate binding site of mitochondrial carriers. Biochim. Biophys. Acta 1757, 1237–1248 Received 17 November 2008/8 May 2009; accepted 19 May 2009 Published as BJ Immediate Publication 19 May 2009, doi:10.1042/BJ20082231 c The Authors Journal compilation c 2009 Biochemical Society