From the Eccles Program in Human Molecular Biology and Genetics and Department of Medicine, Divisions of Hematology and Oncology, University of Utah, Salt Lake City, Utah 84112
Received for publication, October 9, 2002, and in revised form, November 12, 2002
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ABSTRACT |
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Iron regulatory protein-1 (IRP-1) is a cytosolic
RNA-binding protein that is a regulator of iron homeostasis in
mammalian cells. IRP-1 binds to RNA structures, known as
iron-responsive elements, located in the untranslated regions of
specific mRNAs, and it regulates the translation or stability of
these mRNAs. Iron regulates IRP-1 activity by converting it from an
RNA-binding apoprotein into a [4Fe-4S] cluster protein exhibiting
aconitase activity. IRP-1 is widely found in prokaryotes and
eukaryotes. Here, we report the biochemical characterization and
regulation of an IRP-1 homolog in Caenorhabditis elegans
(GEI-22/ACO-1). GEI-22/ACO-1 is expressed in the cytosol of cells of
the hypodermis and the intestine. Like mammalian IRP-1/aconitases,
GEI-22/ACO-1 exhibits aconitase activity and is post-translationally
regulated by iron. Although GEI-22/ACO-1 shares striking resemblance to mammalian IRP-1, it fails to bind RNA. This is consistent with the lack
of iron-responsive elements in the C. elegans ferritin genes, ftn-1 and ftn-2. While mammalian
ferritin H and L mRNAs are translationally regulated by iron, the
amounts of C. elegans ftn-1 and ftn-2 mRNAs
are increased by iron and decreased by iron chelation. Excess iron did
not significantly alter worm development but did shorten their life
span. These studies indicated that iron homeostasis in C. elegans shares some similarities with those of vertebrates.
Iron is an essential element required for growth and survival of
most organisms. The importance of iron is implicit in the role it plays
in oxygen transport and heme synthesis as well as its ability to serve
as a cofactor for enzymes involved in a variety of biological processes
including DNA synthesis, energy production, and neurotransmitter
synthesis. Abnormally high concentration of cellular iron is toxic due
to its ability to catalyze the generation of free radicals that damage
DNA, lipids, and proteins. In humans, the accumulation of excess
cellular iron can result in cirrhosis, arthritis, cardiomyopathy,
diabetes mellitus, and increased risk of cancer and heart disease. To
provide adequate iron for cellular needs yet prevent the accumulation
of excess iron, the concentration of iron within cells is tightly controlled.
In vertebrates, the iron regulatory proteins 1 and 2 (IRP-1 and
IRP-2)1 regulate iron
homeostasis. IRPs are cytosolic RNA-binding proteins that regulate the
translation or the stability of mRNAs encoding proteins involved in
iron and energy homeostasis (1-4). IRPs bind to RNA stem-loop
structures, known as iron-responsive elements (IREs), that are located
in either the 5'- or 3'-untranslated regions (UTRs) of specific
mRNAs. These mRNAs encode proteins involved in iron storage
(ferritin), iron utilization (erythroid aminolevilunate synthase and
mitochondrial aconitase), and iron transport (transferrin receptor and
divalent metal transporter-1). When iron is scarce, IRP binding to the
5' IRE in ferritin mRNA represses translation, whereas IRP binding
to the 3' IREs in the transferrin receptor mRNA stabilizes this
mRNA. When iron is abundant, IRPs lose affinity for the IREs,
leading to enhanced ferritin synthesis and to the rapid degradation of
transferrin receptor mRNA. By regulating the amount of iron taken
up by transferrin receptor and the amount of iron sequestered by
ferritin, cellular iron concentration is maintained, and iron toxicity
is avoided.
Iron regulates the RNA binding activity of IRP-1 and IRP-2, but the
mechanism of regulation differs. In the presence of iron, a [4Fe-4S]
cluster assembles in IRP-1, converting it from an RNA-binding protein
into a cytosolic aconitase. Aconitases are [4Fe-4S] cluster proteins
that are found in the cytosol, mitochondria, and glyoxysomes and
catalyze the reversible isomerization of citrate and isocitrate via
cis-aconitate (5). Despite information regarding the role of
aconitase in mitochondria and in the glyoxylate cycle in microorganisms and plants, the function of cytosolic aconitase in higher eukaryotes is
not clear. Unlike IRP-1, IRP-2 lacks a [4Fe-4S] cluster and consequently lacks aconitase activity (6). Rather, iron regulation of
IRP-2 involves iron-induced IRP2 degradation by the proteasome (7-9).
IRP-1s have been identified in a wide variety of organisms,
including bacteria (10-12), plants (13-15), and animals (16, 17). IRP-1s share a high degree of amino acid identity among different species. For example, mammalian IRP-1s are >90% identical and are
highly homologous to IRP-1s from other organisms, including Caenorhabditis elegans (64% identity) (16),
Arabidopsis thaliana (59% identity) plants (13),
Trypanosome brucei (64% identity) (17), and
Escherichia coli (52% identity) (10). In contrast, IRP-1s
share only ~20% amino acid identity to mitochondrial aconitases. Although these IRP-1s show striking similarity to mammalian IRP-1 and
in most cases exhibit aconitase activity, only vertebrate and insect
IRP-1s (18-21) bind RNA. Exceptions are Bacillus subtilis and Plasmodium falciparum IRP-1s, where studies show that
these proteins are capable of binding to a mammalian consensus IRE (11, 22).
Here, we report on the biochemical properties and regulation of
C. elegans cytosolic aconitase (GEI-22/ACO-1). C. elegans is a multicellular organism that shares many basic
cellular mechanisms with vertebrates, and consequently, it has been
used to study development, neurobiology, stress, and aging. Many of the
same genes that are involved in iron and energy homeostasis in
vertebrates are conserved in C. elegans, including
aconitases, ferritin, divalent metal transporter-1, frataxin, and iron
sulfur cluster assembly proteins, suggesting that iron and energy
homeostasis are also conserved. These features prompted us to
characterize the biochemical properties and the regulation of
GEI-22/ACO-1 and mechanisms regulating iron homeostasis in C. elegans.
C. elegans Culture--
Wild-type C. elegans (variety
Bristol, strain N2) were cultivated on nematode growth medium (NGM)
agar plates or in large scale liquid cultures seeded with E. coli strain OP50 at 22 °C (23). For large scale cultures, mixed
stage worms were placed in complete S-basal medium (500 ml) containing
OP50 bacteria (15 g) and grown until the bacteria were gone. S-basal
medium was supplemented with 0.003-6.6 mg/ml ferric ammonium citrate
(FAC) or 100 µM deferoxamine (DF), an iron chelator.
Because FAC lowered the pH of S-basal medium, it was adjusted to pH
7.0. Worms were grown for 4 days with gentle shaking at 180 rpm and
were collected and washed free of bacteria by sucrose flotation. The
toxic concentration of FAC and DF was determined by iron toxicity
assays (see below) and by growth of worms in different DF
concentrations followed by assaying worm survival.
RNA for Northern blots was made from L4 worms prepared by alkaline
hypochlorite of gravid adults (24). Embryos were grown on NGM agar
plates (12 × 60-mm plates) supplemented with FAC (0.003-6.6 mg/ml) and adjusted to pH 7.0 or DF (100 µM) for 4 days
at 22 °C. DF-supplemented plates were prepared by seeding NGM plates with 0.1 ml of OP50 bacteria (380 mg/ml) to which 100 µM
DF was added.
For iron toxicity life span analysis, 10 L4 larval stage worms were
placed on NGM agar plates supplemented with FAC (0.03-6.6 mg/ml) at
22 °C. Each day, the worms were moved to a new plate and were scored
as dead if they failed to move when provoked or lacked pharyngeal
pumping. Three distinct experiments were carried out, with 10 worms in
each experiment. Statistical comparisons were made using a Cox
regression model testing for trend in survival explained by the dose of iron.
Construction of a cDNA Encoding GEI-22/ACO-1 and
of a gei-22/aco-1::GFP Reporter Gene--
C. elegans
gei-22/aco-1 cDNA was synthesized from RNA isolated from mixed
stage worms using TRIzol reagent (Invitrogen) and Superscript II
reverse transcriptase (Invitrogen). PCR was performed using
Pfu polymerase and an upstream primer containing a
KpnI site followed by the first 18 nt of the
gei-22/aco-1 coding sequence (5'-GCGCGGTACCGCCATGCGTTTCAACAACCTT-3') and a downstream primer containing the last 18 nt of gei-22/aco-1 coding
region in frame with a FLAG epitope, a stop codon, and an
XbaI site
(5'-GCGCTCTAGATTACTTGTCATCGTCGTCCTTGTAGTCTTGGATCAACTTTCTGATCAT-3'). These primers were chosen based on the alignment of the GEI-22/ACO-1 amino acid sequence predicted from the ZK455.1 with those of other IRP-1s using the ClustalW multiple sequence alignment program (Fig. 1).
The 2.9-kb fragment was cloned into the KpnI-XbaI
sites of pcDNA3 (Invitrogen), yielding the mammalian expression
construct pACO-1FLAG.
A translational green fluorescent protein (GFP) construct
GEI-22/ACO-1::GFP was constructed by digesting ZK455.1 with
Eco47III-NruI and inserting the 3,895-bp fragment
into the SmaI site of the promoterless GFP reporter vector
pPD95.77 (Dr. Andrew Fire). This construct contained 939 nt of 5'
regulatory sequences and 2.9 kb of gei-22/aco-1
coding and intronic sequences (7 introns) fused in frame to GFP. The
construct lacks sequences encoding the last 110 amino acids of
GEI-1/ACO-1 and the 3'-UTR, resulting in a nonfunctional protein.
pGFP-ACO-1 (30 ng/µl) was co-injected with pRF4 DNA (30 ng/µl)
containing a dominant mutant gene for rol-6 allele, and
transformed worms were selected by their rolling behavior. Four
transgenic worms were generated, and all showed the same GFP expression pattern.
Expression of GEI-22/ACO-1 in Yeast and Generation of
Antibodies--
A His8 tag was cloned onto the N terminus
of pACO-1FLAG, and the insert was subcloned into the
KpnI-XbaI sites of pYes2 (Invitrogen), yielding
the yeast expression construct pYhis-ACO-1FLAG. pYhis-ACO-1FLAG was
transformed into yeast strain JEL1 (25). Yeast were grown overnight in
synthetic complete medium minus uracil (SC-Ura Expression of GEI-22/ACO-1 in Human Embryonic Kidney
293 Cells--
A 10-cm plate of HEK 293 cells was cotransfected with 8 µg of pACO-1FLAG and 2 µg of pEGFP (Clontech)
DNAs. The cells were equally split after 5 h to six 35-mm plates,
and duplicate plates received either FAC (50 µg/ml) or DF (50 µM) or no addition. The cells were incubated for 16 h and then harvested in 0.125 ml of lysis buffer (0.02 M
HEPES, pH 7.6, 0.025 M KCl, 1 mM
dithiothreitol, 0.25% Nonidet P-40) containing a protease inhibitor
tablet (Roche Molecular Biochemicals). The lysate was centrifuged at
15,000 × g for 20 min at 4 °C, and the supernatant
was assayed for protein using the Coomassie Blue Plus Protein Reagent (Pierce).
Isolation of C. elegans Protein Extracts--
Worms were
harvested from 500-ml cultures containing 0.33 mg/ml FAC or 100 µM DF. The worms were washed free of bacteria by sucrose
flotation and suspended in a 2× volume of homogenization buffer (0.05 M Tris-HCl, pH 7.9, 25% glycerol, 0.1 mM EDTA,
0.32 M NH4SO4) containing a
protease inhibitor tablet (Roche Molecular Biochemicals). The lysates
were homogenized on ice using a Brinkmann Instruments Polytron
homogenizer at full power for 15 s, repeated seven times. The
homogenates were centrifuged at 160,000 × g for 1 h, and the supernatants were assayed for protein using the Coomassie Blue Plus protein reagent (Pierce).
RNA-binding Gel Shift and Aconitase Assays--
RNA-binding
assays were performed as described (26), using protein (12 µg) from
yeast, HEK 293 cells, or C. elegans lysates and a
32P-labeled rat ferritin L-IRE (R. norvegicus
fer-l) (25) or a C. elegans ferritin-2
(ftn-2) RNA. The ftn-2 RNA was synthesized from
DNA that corresponded to 530 nt 5' to the start codon of ftn-2 gene (C. elegans cosmid D1037.3). The
forward primer contained a T7 promoter sequence (19 nt) followed by 20 nt of ftn-2 sequence (5'-TAATACGACTCACTATAGGGGTTGAAGCATAATACTATTACG-3'), and the
reverse primer contained the first 20 nt 5' to the start site in
ftn-2 (5'-GGTAGTTTGTGGCTGGTAAG-3'). After incubation
of the RNA-protein complexes for 20 min, RNase T1 (1 unit) was added to
the reaction for 5 min followed by the addition of 1.5 µg of heparin
(50 mg/ml) for 5 min. A 5% native polyacrylamide gel was used to
resolve the RNA-protein complexes.
Aconitase assays were performed using lysates obtained from worms and
from HEK 293 cells transfected with pcACO-1FLAG. Aconitase activity was
assayed by the addition of cis-aconitate (0.2 mM final concentration) to 50 µg of protein in 0.5 ml of aconitase buffer (0.05 M Tris-HCl, pH 7.5, 0.1 M NaCl)
(27). The disappearance of cis-aconitate was measured at 240 nm over time. For aconitase assays in worms, four separate experiments
were carried out. The differences between FAC- and DF-treated worms
were determined by paired Student's t test, and
p < 0.05 was considered significant.
Immunoblot Analysis--
Protein from HEK 293 cells (50 µg),
yeast (20 µg), and worms (50 µg) was separated by 8% SDS-PAGE, and
the protein was transferred onto nitrocellulose membranes. Membranes
were incubated with the following primary antibodies: rabbit anti-FLAG
(Sigma) (1:5,000), rabbit anti-GEI-22/ACO-1 (1:5,000), mouse anti-GFP
(1:2,000), chicken anti-Rattus norvegicus IRP-1
(1:6,000) (6), and rabbit anti-R. norvegicus IRP-2 (1:8,000)
(6). The appropriate goat horseradish peroxidase-conjugated secondary
antibodies (Pierce) were used at 1:10,000. Antibodies were visualized
using the Renaissance detection system (PerkinElmer Life Sciences).
Northern Blot Analysis--
Total RNA was isolated from
age-synchronized L4 worms grown on FAC (0.003-6.6 mg/ml)-supplemented
or DF (100 µM)-supplemented NGM plates for 4 days. Worms
(~100 mg) were homogenized in TRIzol (1 ml) using a Dounce
homogenizer. Total RNA (25 µg) was resolved using a 1.2%
formaldehyde agarose gel and transferred to a nylon membrane. The
membrane was hybridized with 32P-labeled C. elegans
ftn-1, ftn-2, gei-22/aco-1, and
act-1 DNA probes prepared by the amplification of worm
genomic DNA. DNA templates for amplification were prepared by washing
worms in PCR lysis buffer (50 mM KCl, 10 mM
Tris-HCl, pH 8.2, 2.5 mM MgCl2, 0.45% Nonidet
P-40, 0.45% Tween 20, 0.01% gelatin). The worms were placed in 25 µl of lysis buffer containing proteinase K (1 mg/ml) for 1 h at
65 °C followed by a 15-min incubation at 95 °C. The lysate (1 µl) was added to a PCR (50 µl) containing 20 µM of
the appropriate forward and reverse primers. Forward and reverse
primers, respectively, for PCR were obtained from sequences of the
following C. elegans cosmid clones: ftn-1
(C54F6.14), 5'-ACGTAGAACTCTACGCCTCC-3' and
5'-CTCCGAGTCCTGGGCCGG-3'; ftn-2 (D1037.3),
5'-TCCGAGGTTGAAGCTGCC-3' and 5'-CGGAAAAGTGTTCCTTATCG-3'; and
act-1 (TO4C12.6), 5'-GACAATCCATCCGGAATGTGCAAGGCC-3' and
5'-GAAGCACTTGCGGTGAATGGAT-3'. A probe for
gei-22/aco-1 was obtained by excising the DNA
insert from pACO-1FLAG. PCRs were resolved on 1% agarose gels, and the
DNA bands were purified and 32P-labeled using the RadPrime
DNA Labeling System (Invitrogen). 32P-Labeled DNA was
hybridized with membranes for 18 h at 42 °C and washed with 2×
SSC, 0.5% SDS at 50 °C. Band intensity was normalized to
act-1 and quantified by PhosphorImager analysis.
Cloning and Expression of a GEI-22/ACO-1 Homolog in
C. elegans--
The C. elegans genome possesses two
aconitase genes, which are designated
gei-22/aco-1 and aco-2.
gei-22/aco-1 encodes a protein that shares ~63%
identity with mammalian IRP-1 (16), D. melanogaster IRP-1A
and 1B (16), and A. thaliana (13) and T. brucei
(17) IRP-1s while sharing only ~24% identity to porcine (28) and C. elegans mitochondrial aconitases (Fig.
1). aco-2 encodes a protein
that shares ~74% identity to human and porcine mitochondrial aconitases.
To study the iron homeostasis in C. elegans, a
gei-22/aco-1 cDNA was generated by reverse
transcription-PCR using sequences obtained from the C. elegans cosmid clone ZK455.1. GEI-22/ACO-1 contains all 24 active-site residues required for aconitase activity, including the 3 cysteines that serve as ligands for the [4Fe-4S] cluster (29-31).
GEI-22/ACO-1 also contains residues that are similar to those
identified in mammalian IRP-1 implicated in RNA binding, including
amino acids 121-130, 685-689, and 732-737 (32, 33).
The expression pattern of GEI-22/ACO-1 in living worms was determined
using GFP-reporter fusions constructs. A
gei-22/aco-1::GFP translational fusion gene was made that contained ~1 kb of 5' regulatory sequences and 2.9 kb of coding and intronic sequences of
gei-22/aco-1 fused in-frame to gfp
(Fig. 2A). This construct lacks 3'-UTR sequences and those encoding the last 110 amino acids GEI-22/ACO-1. Transgenic embryos, L2 larval stage, and adult animals carrying the reporter construct showed high levels of cytosolic GFP
expression in the hypodermal seam cells and in the intestine (Fig. 2,
B-D). No significant GFP expression was observed in muscle cells or in neurons.
GEI-22/ACO-1 Does Not Bind RNA--
Mammalian IRP-1
exhibits the mutually exclusive activities of RNA binding and
aconitase. Iron causes IRP-1 to switch from an RNA-binding apoprotein
form to a non-RNA binding aconitase form containing a [4Fe-4S]
cluster. The switch between these forms occurs without changes in IRP-1
protein levels. Since GEI-22/ACO-1 shows significant amino acid
identity with mammalian IRP-1, we questioned whether GEI-22/ACO-1
binds RNA. The 5' sequences flanking the C. elegans genes encoding ferritin-1 (C. elegans FTN-1)
and ferritin-2 (C. elegans FTN-2), succinate dehydrogenase,
and ACO-2 were inspected for IREs, since 5' IREs are found in
homologous genes in other organisms (19, 34-36). No consensus IREs was
identified in these genes. It was possible, however, that nonconsensus
IREs might be present in the 5'-UTRs of these genes. To test for this, IRE binding activity was measured in extracts of worms grown in FAC or
the iron chelator DF using 32P-labeled RNAs
corresponding to sequences located ~500 nt upstream of the start site
of the ftn-1, ftn-2, aco-2, and
succinate dehydrogenase genes. Since C. elegans exons
and introns are generally shorter than in vertebrates (37), we reasoned
that if these genes harbor 5'-IREs, then they should be present within
these sequences. Worms were grown in FAC- or DF-supplemental
medium, because previous studies showed that FAC and DF can decrease
and increase, respectively, IRP-1 RNA binding activity (1, 2, 4). The
concentration of FAC and DF was chosen based on testing the growth of
worms in different concentrations of FAC and DF (see "Experimental
Procedures"). Fig. 3B shows
that no specific RNA binding activity was detected in worm extracts by
RNA-band shift gels with the 32P-labeled ftn-2
RNA or with other 32P-labeled C. elegans RNAs
(data not shown), although GEI-22/ACO-1 was detected in these extracts
by immunoblotting using an anti-GEI-22/ACO-1 antibody (Fig.
3C). As controls, HEK 293 cells were treated with FAC or DF,
and IRP RNA binding activity was measured using the 32P-labeled R. norvegicus fer-l IRE (Fig.
3A). DF increased IRP RNA binding activity as expected,
whereas FAC had little effect on IRP RNA binding activity due to the
high iron concentration in these cells. No RNA-protein complexes were
formed with a 32P-labeled R. norvegicus
fer-l IRE in worm extracts (Fig. 3A). C. elegans extracts spiked with R. norvegicus IRP-1 showed
that 32P-labeled R. norvegicus fer-l IRE bound
to R. norvegicus IRP-1, indicating that an inhibitor of RNA
binding activity was not present in these extracts (data not
shown).
To confirm that GEI-22/ACO-1 lacks RNA binding activity, we expressed
His/FLAG-tagged GEI-22/ACO-1 in yeast using a galactose-inducible promoter. The advantage of yeast is that they do not express endogenous IRPs, which might interfere with the detection of small amounts of RNA
binding activity. As controls, yeast strains expressing His-tagged
R. norvegicus IRP-1 and IRP-2 were also assayed for RNA
binding activity. Galactose induced the expression of GEI-22/ACO-1, R. norvegicus IRP-1, and R. norvegicus IRP-2, but
only R. norvegicus IRP-1 and R. norvegicus IRP-2
bound to the R. norvegicus fer-l IRE (Fig.
4, A and B). Taken
together, these data indicate that GEI-22/ACO-1 does not bind to a
mammalian consensus IRE or to C. elegans RNAs that might be
expected to harbor functional IREs.
GEI-22/ACO-1 Is an Aconitase--
The data indicate
that GEI-22/ACO-1 lacks RNA binding activity. The 24 active sites in
mitochondrial aconitases (29-31) are present in GEI-22/ACO-1,
suggesting that GEI-22/ACO-1 is an aconitase. To determine whether
GEI-22/ACO-1 exhibits aconitase activity and whether it is regulated by
iron, total aconitase activity was measured in HEK 293 cells
transfected with FLAG-tagged GEI-22/ACO-1 or pcDNA3 control. Some
cells were treated with either FAC or DF for 16 h before assaying
aconitase activity. Cells transfected with FLAG-tagged GEI-22/ACO-1
showed a ~2-fold increase in total aconitase activity compared with
pcDNA3-transfected cells (Fig. 5A). When cells were treated
with FAC, total aconitase activity increased ~4-fold in
GEI-22/ACO-1-transfected cells compared with pcDNA3- transfected
cells. Endogenous aconitase activity did not significantly increase in
pcDNA3-transfected cells treated with FAC, indicating that these
cells are iron-sufficient, and that the majority of IRP-1 is in the
aconitase form. In contrast, DF dramatically reduced aconitase activity
in GEI-22/ACO-1- and pcDNA3-transfected cells. The decreased
aconitase activity observed in pcDNA3-transfected cells is due
to decreased endogenous cytosolic and mitochondrial aconitase
activities. GEI-22/ACO-1 was expressed at similar levels in all
extracts, suggesting that the change in aconitase activity was due to
the post-translational conversion of the apoprotein form to the
[4Fe-4S] form (Fig. 5B). The fact that iron caused a
~3-fold increase in aconitase activity in GEI-22/ACO-1-transfected cells compared with GEI-22/ACO-1-untreated cells suggested that iron is
limiting under conditions of overexpression. Taken together, these data
showed that GEI-22/ACO-1 is an aconitase and that this activity can
be regulated by iron.
Iron Regulates Aconitase Activity in C. elegans--
To determine
whether aconitases are regulated by iron in worms, aconitase activity
was measured in lysates from mixed stage worms grown for 4 days in
liquid cultures supplemented with either 0.33 mg/ml FAC or 100 µM DF. We compared FAC and DF treatments because these
conditions represent the extremes of iron concentrations. In these
experiments, total aconitase activity (GEI-22/ACO-1 and ACO-2) was
measured, because the cytosol was routinely contaminated with
mitochondrial proteins. Total aconitase activity from four separate
experiments showed base-line variability; however, in all experiments a
significant reduction (12-16%) was observed in DF-treated worms
compared with FAC-treated worms (p < 0.003, paired
Student's t test) (Fig.
6A). A representative
immunoblot showed that the amount of GEI-22/ACO-1 did not change in DF-
or FAC-treated worms. Although we cannot attribute the change in aconitase activity specifically to GEI-22/ACO-1 because of ACO-2 contamination, these data indicated that aconitase activity can be
regulated by iron in worms. The data also showed that the decrease in
aconitase activity in worms grown in DF is not as great as in HEK 293 cells transfected with GEI-22/ACO-1 (compare Figs. 5A and
6A). This might be due to inefficient DF uptake into worms grown in liquid culture compared with DF uptake into cultured cells.
Iron Regulates C. elegans ftn-1 and ftn-2 mRNAs--
The lack
of discernible IREs in the C. elegans genome and the absence
of GEI-22/ACO-1 RNA binding activity suggested that C. elegans ferritin genes might be iron-regulated by mechanisms other
than translation. C. elegans expresses two ferritin-like proteins, which we designated C. elegans FTN-1 and FTN-2.
C. elegans FTN-1 and FTN-2 are more homologous to R. norvegicus FER-H (54 and 61%) than to R. norvegicus
FER-L (45 and 50%), and both proteins contain ferroxidase active-site
residues that are characteristic of R. norvegicus FER-H
subunits (38).
To determine whether ftn-1 and ftn-2 mRNAs
are regulated by iron, worms were grown on agar plates supplemented
with FAC (0.003-6.6 mg/ml) or with DF (100 µM) for 4 days, and ftn-1 and ftn-2 mRNAs were measured
by Northern blots. Fig. 7 shows that
ftn-1 mRNA steadily increases ~2-fold as FAC
concentration increases from 0.33 to 6.6 mg/ml, whereas a smaller, but
consistent, ~0.5-fold increase is observed for ftn-2
mRNA. In worms grown in the presence of DF, ftn-1 and
ftn-2 mRNAs decrease ~10- and ~2-fold, respectively. To quantify changes in ftn-1 and ftn-2 mRNAs,
band intensity was normalized to act-1 mRNA (Fig.
7B). These data indicated that ftn-1 and
ftn-2 mRNAs are regulated by iron but that
ftn-1 is more responsive to changes in iron concentration
than ftn-2. gei-22/aco-1 mRNA does
not significantly change with FAC or DF treatment (Fig. 7, A
and B), which is consistent with data indicating the amounts of GEI-22/ACO-1 protein are not affected by iron (Figs. 3C
and 6B).
Effect of Iron on the Development and Life Span of C. elegans--
Since little is known about iron homeostasis in
worms, we determined the concentration of iron that causes toxicity in
worms. First, we tested whether the development of worms was affected by iron. Embryos were grown on FAC-supplemented (0.033-6.6 mg/ml) NGM
plates. All worms grew into adults within 4 days, indicating that iron
did not affect worm development (data not shown). We then tested
whether the life span of worms was altered when grown on iron plates.
L4 larval stage worms were placed on agar plates supplemented with FAC
(0.033-6.6 mg/ml), and worms were counted each day until all were
dead. Fig. 8 shows that the mean life spans ± S.E. of worms grown on 3.3 and 6.6 mg/ml FAC plates were 12.9 ± 2.0 and 11.1 ± 1.2 days, respectively, compared with
15.8 ± 0.70 days for worms grown on control plates. These data
indicated that excess iron does not affect the development of worms but is toxic when worms are exposed to iron during their lifetime.
Here, we investigate the iron-dependent regulation of
GEI-22/ACO-1 and the regulation of iron homeostasis in C. elegans. We show that GEI-22/ACO-1 is similar to other vertebrate
IRP-1 proteins in that it exhibits aconitase activity and is
post-translationally regulated by iron. Unlike vertebrate IRP-1
proteins, GEI-22/ACO-1 does not bind RNA. Our data show that
GEI-22/ACO-1 is expressed in intestine and hypodermis, which are organs
that absorb and store nutrients, and is consistent with its role in
energy homeostasis. We show that high concentrations of iron reduce the
life span of worms. Finally, we demonstrate that iron regulates the
amounts of ftn-1 and ftn-2 mRNAs, unlike in
vertebrates, where ferritin H and L mRNAs are primarily regulated
by translation by the IRP-IRE system.
Aconitases are conserved [4Fe-4S] cluster proteins found in
prokaryotes and eukaryotes that catalyze the reversible isomerization of citrate and isocitrate in the Krebs and glyoxylate cycles. Aconitases can be subdivided into several groups (5). One group is
represented by IRP-1, which shares >47% identity with mammalian IRP-1, whereas the other group is represented by mitochondrial aconitase, which shares only ~24% identity with IRP-1. Since
GEI-22/ACO-1 and other IRP members are so similar to vertebrate IRP-1s,
which are RNA-binding proteins, it was expected that GEI-22/ACO-1 also binds RNA. Our data show that GEI-22/ACO-1 lacks RNA binding activity, which is consistent with the lack of discernible IREs in the C. elegans genome. Other IRP-1s, such as T. brucei (17),
A. thaliana (13), and Nicotiana tabacum
(15), were shown to be aconitases, but RNA binding activity was not
reported, and it is probable that these proteins also do not bind RNA.
However, some nonvertebrate IRP-1s bind RNA. For example, P. falciparum IRP-1 was reported to bind a mammalian consensus IRE
(22), and B. subtilis aconitase binds not only to a
mammalian consensus IRE but also to IRE-like stem-loop structures
present in bacterial genes (11). Whether these interactions are
functional in vivo is not clear. Overall, these studies show
that RNA binding is not a requisite feature of the IRP-1 family.
Iron post-translationally regulates GEI-22/ACO-1 activity. In
GEI-22/ACO-1-transfected HEK 293 cells, FAC and DF increased and
decreased aconitase activity, respectively. In worms, total aconitase
activity was 14% lower in worms grown in DF compared with FAC. Because
FAC and DF have no effect on the amounts of GEI-22/ACO-1 protein or
mRNA, this suggested that iron regulates GEI-22/ACO-1 activity by
the assembly and disassembly of the [4Fe-4S] cluster. These studies
also show that the [4Fe-4S] cluster machinery in human cells is
efficient in assembling a cluster in GEI-22/ACO-1. Although we cannot
attribute the changes in aconitase activity solely to GEI-22/ACO-1
because of contamination with mitochondrial ACO-2, the data indicated
that altering the iron concentration in worms could regulate aconitase
activity. Our data show that in worms grown in the presence of DF, the
decrease in aconitase activity was much less than in HEK 293 cells
transfected with GEI-22/ACO-1. The reasons for these differences are
not clear, but we suspect that DF uptake in worms grown in liquid
cultures might not be as efficient as DF uptake into cultured cells.
Despite information regarding the role of aconitase in mitochondria and
in the glyoxylate cycle, the function of cytosolic aconitase in higher
eukaryotes is not clear. The glyoxylate pathway takes place in plants
and microorganisms, allowing these organisms to convert lipids to
gluconeogenic precursors for the synthesis of carbohydrates. In plants,
IRP-1 participates in the glyoxylate pathway (14, 39), where its
expression increases with the growth of seedlings and coincides with
the increase in activity of other glyoxylate enzymes (14). A recent
study shows that aconitase along with other glyoxylate and Krebs cycle
enzymes are increased in A. thaliana during infection with
Pseudomonas syringae, suggesting a role for aconitase in
plant defense against pathogens (40). In T. brucei, IRP-1
expression is developmentally regulated, increasing in procyclic stage
when Krebs cycle enzymes also increase (17). Whether IRP-1 functions in
a glyoxylate pathway in T. brucei is unclear. In mammals,
the role of IRP-1 was hypothesized to be important in maintaining redox
control during periods of iron excess (41). Finally, some studies have hypothesized that the glyoxylate pathway may be operative in mammals during periods of stress, such as during starvation (42) and alcohol
consumption (43); however, this has yet to be tested.
Because the glyoxylate cycle is operative in C. elegans (44,
45), it is plausible that GEI-22/ACO-1 is involved in this pathway. In
C. elegans, the activities and expression of the glyoxylate cycle protein increase when carbohydrates are low and fatty acids are
elevated, which would occur in embryos and in larva during fasting
(46-48). We examined the expression of GEI-22/ACO-1 in L1 to L4 larval
stages and in adults and found no differences in amounts of
GEI-22/ACO-1.2 It is
possible, however, that GEI-22/ACO-1 activity might be altered during
these conditions. Other functions for GEI-22/ACO-1 are suggested by our
data showing that the expression patterns of GEI-22/ACO-1 are not
identical with glyoxylate cycle protein. For example, glyoxylate cycle
protein is expressed in body wall muscle and intestine (47, 48),
whereas GEI-22/ACO-1 is expressed in hypodermal seam cells and
intestine but not in body wall muscle. Furthermore, a recent study
showed that GEI-22/ACO-1 interacted with GEX-3 in a yeast two-hybrid
screen (49). GEX-3 is thought to be a Rac-interacting protein, which is
essential for proper cell migration and cell morphology, and in its
absence, hypodermal cells fail to become organized (50). The
localization of GEI-22/ACO-1 to hypodermal cells and its interaction
with a GEX-3 suggest that GEI-22/ACO-1 might have a distinct function
in development in hypodermis. However, when
gei-22/aco-1 expression was inhibited by RNA
interference, mutant worms showed no developmental abnormalities (49).
These data suggested that gei-22/aco-1 may have
other roles in hypodermal cells or that aco-2 can substitute
for gei-22/aco-1 function.
To understand how C. elegans controls iron homeostasis, the
regulation of C. elegans ftn-1 and ftn-2
mRNAs was studied. Ferritins are conserved iron storage proteins
found in bacteria, plants, and animals that can store up to 4,500 iron
atoms and, by so doing, prevent iron toxicity (38, 51). Vertebrate
ferritins are generally composed of H and L subunits (FER-H and FER-L),
whereas those of plants and bacteria contain mainly H subunits. FER-H
contains amino acids that are required for ferroxidase activity,
whereas the FER-L functions in enhancing iron nucleation and long term iron storage. In mammalian cells, FER-H and FER-L are regulated at the
translational level by the IRP-IRE system. Iron-dependent transcriptional regulation of ferritin has been described in mammalian cells (52), but this plays a minor role in iron regulation.
Since GEI-22/ACO-1 does not bind RNA, and C. elegans lacks
obvious IREs, we hypothesized that ftn-1 and
ftn-2 mRNAs might be regulated by iron through processes
other than translation. Our data showed that ftn-1 mRNA
increased ~2-fold in worms grown in the presence of iron, whereas
ftn-2 mRNA showed a small but consistent ~0.5-fold
increase. In contrast, DF treatment decreased ftn-1 and
ftn-2 mRNAs ~10-fold and ~2-fold, respectively. One explanation for the ~2-fold increase in ftn-1 mRNA by
iron is that worms are grown in iron-replete medium and fed E. coli and are iron-sufficient. The data also show that
ftn-1 mRNA is more responsive to iron concentration than
ftn-2 mRNA. A similar situation occurs in A. thaliana, where the fer-1 and fer-3
mRNAs are more responsive to iron than fer-2 or
fer-4 (53), and in maize, where fer-1, but not
fer-2, is iron-regulated (54-56). Although our data do not
conclusively show that ftn-1 and ftn-2 genes are
transcriptionally regulated by iron in worms, this mechanism would be
consistent with the observed transcriptional regulation of ferritin in
plants (53, 56, 57) and in insects (58).
The response of C. elegans to heavy metals, such as copper,
arsenate, and cadmium (59-64), has been investigated, but less is
known about iron regulation and toxicity in worms. Our first experiment
was to determine the effect of iron on the development of worms. No
significant change was observed in the timing of development of worms
grown on FAC-supplemented plates. High concentrations of iron, however,
did decrease the life span of worms. The mean life span of worms grown
on 3.3 and 6.6 mg/ml FAC plates was 12.9 ± 2.04 and 11.1 ± 1.2 days, respectively, compared with worms grown on control plates
(15.8 ± 0.70 days). Although the mechanism responsible for
iron-mediated decrease in life span is not known, it is possible that
worms maintained on high iron would accumulate iron as they age,
leading to increased iron-catalyzed free radical formation. An
elevation in the concentrations of free radicals would overwhelm the
antioxidant defense machinery, leading to cellular damage and eventual
cell death. Studies show that when reactive free radicals are decreased
(65, 66) or antioxidant enzymes, such as catalase and superoxide
dismutase, are increased (67-69), the life span of the organism can be
increased. The toxicity of iron in worms may be akin to iron overload
diseases in humans, where the accumulation of iron over a lifetime can
result in cirrhosis, cardiomyopathy, diabetes mellitus,
neurodegeneration, and increased risk of cancer (70). These studies
show that C. elegans may serve as a model system for the
study of iron homeostasis in mammals.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)
containing 2% dextrose. Yeast were then diluted 1:100 into
SC-Ura
containing 3% glycerol, 2.0% lactic acid and
grown to an A600 of 0.8. 2% galactose
was added to the culture for 10-18 h to induce GEI-22/ACO-1
expression. Yeasts were harvested, and the pellet was resuspended in 3 ml/g, wet weight, lysis extraction buffer (0.3 M NaCl, 0.05 M NaH2PO4, pH 8.0, 10 mM
-mercaptoethanol, 0.025% Nonidet P-40, 0.1 mM EDTA) containing a protease inhibitor tablet (Roche
Molecular Biochemicals). Cells were disrupted by vortexing with glass
beads, and the lysate was centrifuged at 27,000 × g
for 30 min. The lysate was mixed with 2.0 ml of equilibrated Ni2+-nitrilotriacetic acid resin (Qiagen) for 1 h at
4 °C. The slurry was poured into a column and was washed with column
buffer (0.3 M NaCl, 0.05 M
NaH2PO4, pH 8.0) containing 0.07 M
imidazole. GEI-22/ACO-1 was eluted with column buffer containing 0.2 M imidazole, and fractions containing GEI-22/ACO-1 were
dialyzed against assay buffer (0.05 M Tris, pH 7.3, 0.1 M NaCl, 1 mM dithiothreitol). For antibody
preparation, GEI-22/ACO-1 was further purified by preparative 8%
SDS-PAGE. The protein was visualized using a cold solution of 2.5 M KCl, and the band containing GEI-22/ACO-1 was excised and
used to inject rabbits. The polyclonal antibodies detected a band on
SDS-PAGE with a molecular mass of ~100 kDa, which corresponds to
the size of the predicted gene product of gei-22/aco-1 mRNA. Preimmune serum did not
detect this band.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (121K):
[in a new window]
Fig. 1.
Sequence conservation among IRP-1
proteins. Shown is an alignment of the predicted sequence of
GEI-22/ACO-1 (GenBankTM accession number Z66567) with
R. norvegicus (Rn) IRP-1 (71)
(GenBankTM accession number NM_017321), D. melanogaster (Dm) IRP-1A and IRP-1B (16)
(GenBankTM accession numbers AJ223247 and AJ223248),
A. thaliana (At) IRP-1 (13)
(GenBankTM accession number X82839), and pig mitochondrial
aconitase (28) (GenBankTM accession number J05224) using
the ClustalW multiple sequence alignment program. Aconitase active site
residues are indicated with white lettering in
black boxes. Ce, C. elegans.
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Fig. 2.
Expression patterns of
gei-22/aco-1::GFP reporter construct in C. elegans. A, structure of the
gei-22/aco-1::gfp gene. White boxes,
exons; black box, gfp fused to
sequences in exon 8. The gei-22/aco-1 regulatory region is
indicated. B, GFP expression in embryos. C, L2
larval stage worms. D, adults. Hypodermis (Hyp),
seam cells (seam), and intestine (Int) are
indicated.
View larger version (51K):
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Fig. 3.
C. elegans extracts lack IRE
binding activity. HEK 293 cells were grown in medium containing
FAC (100 µg/ml), DF (100 µM), or no treatment
(NT) for 16 h. L4 larval stage worms were grown in
liquid cultures containing FAC (1 mM), DF (100 µM), or no treatment for 4 days. Cytosolic extracts were
prepared from HEK 293 cells and C. elegans cultures, and
protein (12 µg) was incubated with either a 32P-labeled
rat ferritin L-IRE (R. norvegicus fer-l; Rnfer-l)
(A) or a 32P-labeled C. elegans ftn-2
RNA (Ceftn-2 RNA) (B). After 20 min,
RNase T1 (1 unit) was added to the RNA band shift reactions to degrade
nonspecific RNA (A and B, lanes 2-7).
The 32P-labeled RNA-protein complexes were analyzed by 5%
native PAGE. IRP-1·IRP-2 RNA complexes comigrate in HEK 293 cells; free RNAs are indicated. C, protein (25 µg) from
extracts in A and B were analyzed by 8% SDS-PAGE
using anti-rat IRP-1 (R. norvegicus IRP-1 Ab) or
anti-GEI-22/ACO-1 (GEI-22/ACO-1 Ab) antibodies.
View larger version (49K):
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Fig. 4.
Recombinant GEI-22/ACO-1 expressed in yeast
does not bind RNA. A, extracts (20 µg) from yeast
expressing His/FLAG-tagged GEI-22/ACO-1 and His-tagged R. norvegicus IRP-1 and R. norvegicus IRP-2 were assayed
by immunoblots using anti-FLAG, anti-rat IRP-1, or anti-rat IRP-2
antibodies (Ab). Galactose (+) was added to some cultures to
induce IRP expression. Note that there is some IRP expression in yeast
grown in the absence of galactose due to leaky expression.
B, the same extracts (12 µg) from A were
incubated with a 32P-labeled R. norvegicus fer-l
IRE followed by separation of the RNA-protein complexes by 5% native
PAGE.
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Fig. 5.
GEI-22/ACO-1 is an aconitase.
A, HEK 293 cells were transfected with FLAG-tagged
pGEI-22/ACO-1 or pcDNA3 (empty vector). Cells were treated with
either FAC (100 µg/ml), DF (50 µM), or no treatment
(NT) for the last 12 h of the transfection. After
36 h, cell lysates were prepared, and total aconitase activity was
assayed by using a spectroscopic assay that measures the disappearance
of cis-aconitate at 240 nm (27). Aconitase activity is
plotted as arbitrary activity units relative to pcDNA3-NT control.
Data are presented as means ± S.E. (n = 4);
asterisks denote significant differences using a paired
Student's t test (p < 0.01). B,
samples from two experiments in A were analyzed by 8%
SDS-PAGE, and immunoblots were carried out using anti-GEI-22/ACO-1
antibody.
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Fig. 6.
Aconitase activity is regulated by iron in
worms. A, mixed stage worms were grown in liquid
cultures containing FAC (0.33 mg/ml) or DF (100 µM) for 4 days. Worm lysates were prepared, and total aconitase was measured as
in Fig. 5A. Aconitase activity is plotted as arbitrary
activity units. Four independent experiments (represented by different
symbols) are shown with each line representing
worms grown in FAC or DF. The differences in aconitase activity between
FAC and DF groups were compared using a paired Student's t
test (p < 0.003). B, protein (20 µg) in
one of the experiments in A was analyzed by 8% SDS-PAGE,
and immunoblots were carried out using anti-GEI-22/ACO-1 antibody
(Ab).
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Fig. 7.
C. elegans ftn-1 and
ftn-2 mRNAs are regulated by iron.
A, embryos were grown on NGM plates containing FAC
(0.003-6.6 mg/ml), DF (100 µM), or no treatment
(NT) for 4 days until they reached the L4 larval stage.
Total RNA (25 µg) was analyzed from each condition by a 1.2%
formaldehyde-agarose gel. The RNA was transferred to a membrane that
was sequentially probed with 32P-labeled DNAs for
ftn-1, act-1 (actin), ftn-2, and
gei-22/aco-1. B, the data in
A were quantified by PhosphorImager analysis and normalized
to act-1, and band intensity was plotted relative to the
untreated control. Two independent DF experiments were carried out.
Data for FAC groups are presented as means ± S.E.
(n = 3). The asterisks denote significant
differences between untreated control and FAC groups using a paired
Student's t test (p < 0.05).
Ceftn-1 and 2, C. elegans
ftn-1 and -2; Ceact-1, C. elegans act-1.
View larger version (15K):
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Fig. 8.
Kaplan-Meier survival curve of C. elegans grown in the presence of iron. The
graph shows the percentage of worms alive on NGM agar plates
supplemented with or without FAC (0.03-6.6 mg/ml) at 22 °C. L4
larval stage worms were placed on FAC-supplemented NGM agar plates and
were counted and moved to fresh plates each day. Worms were scored as
dead if they failed to move when provoked. Three distinct experiments
were carried out, each containing 10 worms. Mean life spans for worms
were as follows: control (15.8 ± 0.70 days), 0.03 mg/ml FAC
(15.9 ± 0.70 days), 0.33 mg/ml FAC (16.7 ± 1.7 days), 3.3 mg/ml FAC (12.9 ± 2.04 days), and 6.6 mg/ml FAC (11.1 ± 1.2 days). A Cox regression model testing for trend in survival
explained by the dose of iron shows a highly significant effect
(p = 3 × 10 8).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Susan Mango and Adam Blaszczak and Mike Portereiko for advice on worm culture and for scientific discussion. We thank Drs. Eric Hanson and Bruce Bamber for critical reading of the manuscript and Diana Lim for assistance with figures. We acknowledge the C. elegans Genetics Center for cosmids and strains and the University of Utah DNA Core Facility for oligonucleotides. We thank Dr. Chris Rodesch at the University of Utah Cell Imaging Core Facility for assistance in worm imaging and Dr. Aniko Szabo at the Huntsman Cancer Institute Biostatistics Shared Resource Center for statistical analysis.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM45201 (to E. A. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University of Utah,
HMBG, 15 N. 2030 E. Rm. 3240, Salt Lake City, UT 84112. Tel.: 801-585-5002; Fax: 801-585-3501; E-mail:
betty.leibold@hmbg.utah.edu.
Published, JBC Papers in Press, November 15, 2002, DOI 10.1074/jbc.M210333200
2 B. L. Gourley and E. A. Leibold, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: IRP, iron regulatory protein; IRE, iron-responsive element; UTR, untranslated region; NGM, nematode growth medium; FAC, ferric ammonium citrate; DF, deferoxamine; GFP, green fluorescent protein.
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