Download mbn cells results in dysregulation of Fer1HCH expression

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).
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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