From the Unité de Biochimie Physiologique,
Place Croix du Sud, 2-20, 1348 Louvain-la-Neuve, Belgium and the
¶ Eurogentech, Parc Scientifique du Sart Tilman,
4102 Seraing, Belgium
Received for publication, July 3, 2000, and in revised form, November 17, 2000
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ABSTRACT |
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Deletion of YFH1, the yeast frataxin
homologue gene, elicits mitochondrial iron accumulation and alters
cellular iron homeostasis. Here, we report a genome wide analysis of
gene expression in a yfh1( Friedreich's ataxia is a neurodegenerative disease, associated
with cardiomyopathy and, in some cases, with diabetes (for a review,
see Ref. 1). It is caused by mutations in a nuclear gene encoding
frataxin, a small protein with no homology to proteins of known
function (2). Frataxin has been conserved during evolution from
Gram-negative bacteria to humans, and in eukaryotes it is located in
mitochondria (3-6). Deletion of the yeast frataxin gene
(YFH1) elicits a 10-15-fold increased mitochondrial iron accumulation in cells cultivated in glucose-rich iron-replete medium
(7), and moreover, the accumulated iron cannot be extruded from
mitochondria (8). Loss of mitochondrial DNA has been observed at high
iron concentrations (7-8). In parallel, expression of the
FET3 and FTR1 genes which encode the two subunits
of high affinity iron transporter is increased (4, 8). Analysis of the
solution and crystal structures of human frataxin (9-10) and its
bacterial Cyay homologue (11) has revealed a novel protein fold
consisting of a five-stranded antiparallel Iron is an essential cofactor for many reactions in the cell; in
particular, it is present in cytochromes, iron-sulfur proteins, and
ribonucleotide reductase. Moreover, mitochondria contain the protein
machinery responsible for iron-sulfur cluster biosynthesis and export
into the cytosol (for reviews, see Refs. 13 and 14). However, excess
iron can generate via the Fenton reaction highly toxic-free radicals
generating oxidative damage to the cell. Thus, cellular iron
concentration must be tightly controlled. We have found that
mitochondrial iron accumulation in a yfh1 deletion strain is
strongly dependent on culture medium composition (7, 15). In contrast
with cells grown in glucose-rich medium, only a 2-3-fold increase in
mitochondrial iron is observed in raffinose minimum medium, and no iron
accumulation is detected in mitochondria from cells grown in
glycerol-rich medium (see below). Mitochondrial iron overload in a
Thus, we thought that loss of frataxin could alter the expression of a
number of genes, involved either in iron metabolism or oxidative
stress, or in any other unexpected metabolic pathway. To address this
question, we have carried out a genome wide analysis of gene expression
in wild-type and YFH1-deleted strains using DNA microarrays
including all available Saccharomyces cerevisiae open
reading frames.
Strains and Growth Media--
S. cerevisiae strains
were W303-1B (MAT
The glucose-rich medium (YD) contained 2% glucose and 2% yeast
extract (KAT). The glycerol-rich medium (YG) contained 3% glycerol and
2% yeast extract (KAT). Raffinose synthetic medium contained 2%
raffinose, 0.67% yeast nitrogen base (Difco), 0.5% ammonium sulfate,
a mixture of amino acids and the required auxotrophic supplements. Mitochondria were prepared from strains grown in glucose minimum medium supplemented with 100 µM
bathophenantroline disulfonate
(BPS)1 and 100 µM desferroxiamine B (Desferal, Sigma) and iron
concentration was measured as previously described (7).
RNA Preparation and Northern Blotting Analysis--
Cells were
grown to an OD of 0.6-0.7, harvested in ice-cold water as quickly as
possible and frozen in liquid nitrogen before use. Total RNA prepared
by hot acidic phenol extraction was used for Northern blot analysis and
was further purified for DNA microarray hybridizations by the RNeasy
purification kit from Qiagen. For each sample, equal amounts of RNA (20 to 30 µg) were loaded on 1.5% agarose-formaldehyde gels, blotted
onto a nylon membrane, and hybridized with appropriate
32P-labeled probes. Hybridizations were carried out using
standard procedures.
cDNA Synthesis for Microarray Hybridizations--
The
reverse transcription reaction was performed in a final volume of 40 µl. Up to 50 µg of purified total RNA was combined to 8 µl of 5X
First Strand reaction buffer (Superscript II, Life Technologies), 3 µl of AncT mRNA primer (T20VN, 100 pmol), 3 µl of 20 mM dNTP minus dCTP (6.67 mM of each dATP, dGTP,
dTTP), 1 µl of 2 mM dCTP, 1 µl of 1 mM
Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech), and 4 µl of 0.1 M dithiothreitol. The mixture was incubated at 65 °C for
5 min, then at 42 °C for 5 min. 2 µl of reverse transcriptase Superscript II (400 units) (Life Technologies) and 1 µl of RNasin (Promega) were added to the reaction and incubated at 42 °C for 2 h. The reaction was stopped by adding 7 µl of a 5:2 mixture of
50 mM EDTA (pH 8), and 1 N NaOH and incubated
for 20 min at 65 °C. The unincorporated fluorescent dNTP, AncT
primers, and salt were removed by precipitating the cDNA on ice for
at least 30 min in the presence of 4 µl of 0.5 M acetic
acid, 4 µl of 3 M sodium acetate, and 50 µl of
isopropyl alcohol. The fluorescent cDNA was dissolved in 2.5 µl
of water.
Microarray Hybridizations and Data Analysis--
The yeast DNA
chips manufactured by the Microarray Center, Ontario Cancer Institute,
Toronto, Canada, are a generous gift from Dr. Bryan Mcneil in this
Center. The microarray was placed in a sealed humidified chamber
and prehybridized using 57 µl of warmed DIG Easy Hybridization buffer
(Rochem Molecular Biochemicals) and 3 µl of 10 µg/ml fragmented
denatured salmon sperm DNA. A coverslip of 24 × 50 mm was placed
on the top and the microarray was incubated in a water-bath at 37 °C
for at least 2 h. The slides were then washed twice with 0.1 × SSC at room temperature, briefly rinsed with bi-distilled water and
dried by centrifugation at 500 rpm. For hybridization, 2.5 µl of
Cy3-cDNA (wild-type) and 2.5 µl of Cy5-cDNA ( Cells were cultivated in glucose-rich medium and harvested in the
early exponential phase of growth. The proportion of cytoplasmic petites in the culture medium was below 5% and could not influence the
results. Microarray hybridization experiments were repeated three times
using independent cultures. Only those genes whose expression was
stimulated at least 2.5 times on average in the YFH1) deleted strain. Frataxin
deficiency results in enhanced expression of some 70 genes including a
set of genes, called the iron regulon, that are under the control of
the iron-sensing transcription factor AFT1. Five new
AFT1-dependent genes, YOR382w,
YOR383c, YDR534c, YLR136c, and
YLR205c were found. The first three genes presumably encode
cell-wall glycosylphosphatidylinositol anchor proteins and
exhibit a 30-100-fold increased expression. The triple deletion of
these genes decreases efficiency in utilization of the iron of
ferrioxamine B by the yeast cell. YLR136c bears homology to
tristetraproline proteins, which are post-transcriptional regulators in
mammalian cells. Deletion of YLR136c increases the mRNA levels of
iron regulon members. YLR205c bears homology to heme oxygenases. Our
data show that frataxin deficiency elicits iron mobilization from all
iron sources in an AFT1-dependent manner.
Wild-type and
YFH1 glycerol-grown cells exhibit similar high
respiration rates, no mitochondrial iron accumulation, and high
expression of the iron regulon, suggesting that under these conditions
little iron is extruded from mitochondria. These data suggest that the
activity of Yfh1p is not essential in cells grown on glycerol. This
study has also revealed unexpected links between mitochondria and
remote metabolic pathways since frataxin deficiency also enhances the expression of genes such as HSP30, that escape to
AFT1 control. Finally, no oxidative stress gene is induced.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sheet packing against a
pair of parallel
helices. A patch of anionic residues at the
surface could be involved in iron binding. It has recently been
reported (12) that recombinant purified yeast frataxin is a soluble
monomer that contains no iron but self-aggregates upon addition of
ferrous iron as a high molecular weight product that sequesters more
than 3000 iron atoms in an available form. Thus, frataxin might be an
iron storage protein regulating iron homeostasis (12).
YFH1 strain can be enhanced by increasing external iron
concentration or can be avoided by preventing iron import into
mitochondria (15, 16).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ura3-52, leu2-3, 112, trp1-1,
his3-11, 15, ade2-1) and its isogenic derivative W303-1B
YFH1 (MAT
ura3-52, leu2-3, 112, trp1-1, his3-11,15,
ade2-1 yfh1
::KanR) (7). All
deletion mutants were constructed in strain W303-1B and/or
W303-1B
YFH1. Briefly, the deletion mutants were obtained as follows.
AFT1 and FET3 deletion cassettes were constructed in a pSK vector by insertion of the URA3 gene between two
polymerase chain reaction-amplified DNA fragments homologous to
the 3'- and 5'-flanking regions of the AFT1 gene. YLR136c
deletion cassette was obtained from strain BY4741 (17) by polymerase
chain reaction amplification of the kanamycin resistance marker and
yeast DNA-flanking regions. YOR382w and YOR383c were disrupted in a
single step using a loxP-kanMX-loxP gene disruption cassette
(18). The kanMX module was excised by the Cre recombinase
expressed in the presence of galactose and the same procedure was used
to delete YDR534c in
YOR382
YOR383 strain. All deletion strains
were verified by polymerase chain reaction amplification of the 3' and
5' regions flanking the target gene using specific oligonucleotide
primers. Deletion cassettes were used to transform W303 diploid and
W303-1B haploid strains. Introduction of the
YFH1 deletion in the
YOR382
- YOR383
YDR534 strain was obtained by mating, zygote
isolation, and tetrad dissection. The absence of the wild-type gene and
presence of the deletion markers were verified by appropriate
polymerase chain reaction amplifications using specific oligonucleotide primers.
YFH1) were
mixed, added to 55 µl of DIG Easy Hybridization solution, and applied
to the yeast DNA chips. The dual color hybridization was carried out at
37 °C for 16 h. The glass slides were washed twice, 15 min
each, with 0.1 × SSC, 0.1% SDS washing solution prewarmed
at 50 °C, then transferred to 0.1 × SSC at room temperature
for 5 min and dried by centrifugation at 500 rpm. The hybridization
signal was detected by scanning the microarrays using the GenePix 4000 laser scanner (Axon Instruments). The signal quantification was
performed using the integrated GenePix software version 3.01. The Yeast
Proteome (YPD), Saccharomyces cerevisiae Genome (SGD) and
MIPS data bases have been used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
YFH1 strain are
reported in Table I. This is certainly an
underestimate of the number of genes up-regulated by frataxin loss as
the three experiments gave appreciable fluctuations in the expression
of several genes, probably reflecting tight dependence on subtle physiological conditions. Reliable identification of down-regulated genes was often limited by low expression levels in the wild-type strain. However, the expression of several genes was significantly reduced in the
YFH1 strain, in particular GLT1 and
LEU1 which possess iron-sulfur clusters (Table I).
Gene expression in a YFH1 deleted mutant
Up-regulated Genes--
The most widely represented class of
up-regulated genes in a frataxin null mutant encodes proteins that
localize, or are predicted to localize to the cell surface (Table I).
More specifically, the expression of YOR382w, YOR383c, and YDR534c was
increased 30 to 100 times as compared with wild-type strain (Table I). These data were confirmed by Northern blot analysis (Fig.
1, lanes 1 and 2).
The encoded proteins have no homology to other proteins even though
they exhibit the typical features of cell-wall GPI-anchor glycoproteins
(19). They are acidic, extremely rich in serine/threonine residues, and
their amino acid sequence is characterized by reiterated motifs.
YOR383c and YDR534c are very similar except that their common motif is
repeated four times in YDR534c and only twice in YOR383c.
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The expression of all known members of the "iron regulon" was
increased in a YFH1 strain (Table I and Fig. 1). The iron regulon includes the genes of the ferric reductases
(FRE1, FRE2, and FRE3) (20, 21), the
high affinity iron uptake system (FET3/FTR1) (22,
23), siderophore transporters (ARN1-4) (24-27), vacuolar iron transporter (FET5/FTH1) (28), and copper
delivery to the Fet3p oxidase (ATX1 and CCC2)
(29-31). All are under the control of the iron-sensing transcription
factor, Aft1p (32). This protein recognizes a specific sequence in the
promoter of the iron regulon genes and activates their transcription
(32-34). Iron prevents binding of Aft1p to the target genes, and
therefore, represses their expression. Alteration of iron homeostasis
in the
YFH1 strain is also associated with increased expression of
several genes involved in heavy metal uptake or homeostasis
(CTR2, ATX2, COT1, and SMF3) (35-37)
that are not under AFT1 control (Table I). This is in
agreement with the observation that
YFH1 strains are more sensitive
to copper, cobalt, and manganese than the wild-type strain.2
The high increase in the expression of several genes of unknown function might be particularly interesting (Table I). HSP30 (Fig. 1) encodes a seven-transmembrane helix protein related to the opsin family (38-39). YOR285w bears homology to sulfuryltransferases of the mitochondrial rhodanese protein family. YLR205c shares high amino acid sequence identity with heme oxygenases. These are enzymes associated with the endoplasmic reticulum that open the heme ring and release iron. YLR136c (Fig. 1) bears homology to the mammalian tristetraproline (TIS) proteins which are characterized by CX8CX5CX3H repeats and play a role in mRNA stability (40-42).
By comparison, the effect of YFH1 deletion on mitochondria was
modest. However, the expression of the IDH1 and
IDH2 genes which encode isocitrate dehydrogenases was 3 to 4 times increased (Table I), in agreement with the observation that
isocitrate dehydrogenase activity was substantially increased in
YFH1 mitochondrial extracts (data not shown). The expression of the
ISU1 and ISU2 genes which are involved in
mitochondrial iron-sulfur cluster biosynthesis (43, 44) was also
enhanced by a factor of 2.5-3.5 in a
YFH1 strain (Table I).
Surprisingly, the expression of the genes involved in the response to oxidative stress was not significantly increased. We have verified by Northern blotting (Fig. 1) that neither SOD1, which encodes the zinc-copper superoxide dismutase in the cytosol (45), nor SOD2, which encodes the mitochondrial manganese superoxide dismutase (46, 47), were up-regulated.
The Iron Regulon Is Up-regulated in a Frataxin-deficient
Strain--
We addressed the question whether the most representative
genes of unknown function identified by our microarray analysis, YOR382w, YOR383c, YDR534c, YLR136c, YLR205c and HSP30 belong
to the iron regulon. A typical feature of the members of the iron regulon is that they are induced upon iron deprivation and are not
expressed in a AFT1 strain (32-34).
We performed two types of Northern blot experiments. First, we used RNA
from iron-starved wild-type cells grown in the presence of 100 µM BPS, a nonpermeant iron chelator. YOR382w, YOR383c, YDR534c, and YLR136c were strongly induced in the presence of BPS while
HSP30 was not (Fig. 1, lanes 5 and 6).
Second, AFT1 was deleted in YFH1/YFH1
and YFH1/YFH1 diploid strains and tetrads were
dissected. It has previously been reported that
AFT1-deleted strains
cannot grow on a respiratory carbon source such as glycerol (34). We
found that strains W303-1B and W303-1B
YFH1 with an AFT1
gene deletion could grow slowly on glycerol medium. Optimal growth was
restored by addition of iron in the culture medium (Fig.
2a). While in a glucose-rich
medium containing 250 µM iron 70% petite mutants
accumulated in the
YFH1 strain, less than 10% petite colonies were
observed in the
YFH1
AFT1 strain (Fig. 2b). However,
the double mutant was still sensitive to high iron concentrations, so
that optimal growth on glycerol medium was obtained for a narrow range
of iron concentrations (Fig. 2a). Therefore, AFT1
deletion did not prevent mitochondrial iron accumulation in a
YFH1
background provided enough iron was imported into the cell. Tetrad
analysis showed that AFT1 deletion considerably decreased the expression of YOR382w and YLR136c both in wild-type and
YFH1 strains (Fig. 3). Only a very slight
decrease in HSP30 expression was observed (Fig. 3). These
data show that YOR382w, YOR383c, YDR534c, and YLR136c belong to the
iron regulon, in agreement with the presence of several copies of the
consensus sequence recognized by Aft1p in their promoter. YLR205c is
probably also under AFT1 control since the consensus
sequence recognized by Aft1p is present twice in the promoter region at
positions 582 and 292 upstream of the start codon. In contrast,
HSP30 promoter does not contain this sequence and,
therefore, is not directly regulated by AFT1.
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The New Members of the Iron Regulon Play a Role in Iron
Metabolism--
All known iron regulon members play a role in iron
mobilization, or copper delivery to Fet3p. Induction of YOR382w,
YOR383c, and YDR534c upon iron deprivation is so great that it is
difficult to imagine that these proteins have no role in iron
mobilization. In addition to ferrous iron which is used by the high
affinity iron uptake system (Fet3p/Ftr1p), S. cerevisiae can
utilize siderophores synthesized by fungi and bacteria. Siderophores
are imported by four siderophore transporters (Arn1-4p) with distinct
substrate specificities (25-27), or their ferric iron can be released,
reduced, and transported by the high affinity iron uptake system (25). A triple deletion strain YOR382
YOR383
YDR534 had no obvious growth defect (data not shown). When a
FET3 mutation, which
abolishes the high affinity iron uptake, was introduced in the triple
deletion strain, the colonies grown in the presence of limiting amounts of a siderophore such as ferroxiamine B (2 µM) were
slightly smaller than in a simple
FET3 strain (Fig.
4A). No difference was
observed in the presence of saturating concentrations (100 µM) of ferroxiamine B (data not shown). These data
suggest that the loss of the three GPI-anchor proteins decreases
siderophore-mediated iron utilization. We also observed that a
YFH1
strain grew more poorly on 100 µM ferroxiamine B than on
2 µM of this siderophore (Fig. 4, B and C, first row). This indicates that ferroxiamine B is an
efficient iron source for the cell which becomes toxic at high
concentrations in a frataxin-deficient strain. This growth defect was
partially alleviated by the
YOR382
YOR383
YDR534 deletion (Fig.
4C, first and second rows). In
addition, while mitochondrial free iron reached 3.8 nmol/mg of protein
in the simple
YFH1 strain, it was decreased to 1.1 nmol/mg of
protein in the quadruple deletion strain. This strongly suggests that
in the latter strain less iron penetrates the cell. Deletion of the
FET3 gene in a
YFH1 strain substantially improved
cellular growth on 100 µM ferroxiamine B (Fig.
4C, first and third rows) and
decreased mitochondrial free iron concentration to 0.4 nmol/mg of
protein, indicating that a large part of the iron contained in
ferroxiamine B was imported by the high affinity iron uptake system.
Introduction of the
YOR382
YOR383
YDR534 deletion in the
YFH1
FET3 strain slightly increased cellular growth on 100 µM ferroxiamine B (Fig. 4C, third and
fourth rows). Altogether, these data suggest that the
GPI-anchor proteins facilitate siderophore-mediated iron uptake both in
a FET3-dependent and -independent manner.
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YLR136c bears homology to mammalian TIS, an RNA-binding protein
that promotes deadenylation and degradation of the tumor necrosis factor- mRNA, thereby, regulating tumor necrosis factor-
mRNA half-life (41, 42). TIS and tumor necrosis factor-
genes are induced by the same agents. By analogy, we reasoned that mRNA half-life of the iron regulon genes might be decreased by YLR136c. As
expected for an iron-replete glucose-rich medium the expression of
YOR382w was low, yet, it was slightly higher in the
YLR136 mutant
than in the wild-type strain (Fig. 5,
No BPS). In the presence of BPS which results in iron
deprivation and Aft1p activation, the expression of YOR382w was induced
but the mRNA levels were significantly higher in the
YLR136
strain (Fig. 5, +BPS). Some increase in FET3
mRNA levels was also observed in the
YLR136 mutant (Fig. 5). By
analogy with the mammalian TIS protein, these data suggest that the
mRNAs of some genes of the iron regulon could be less stable in the
wild-type than in the
YLR136 strain. YLR136c could act by binding
either directly to iron regulon mRNAs or to another factor
regulating the expression of the iron regulon. It must be noted that
the expression of AFT1 or YFH1 was not modified in a
YLR136 mutant (data not shown).
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Expression of the Iron Regulon in Glycerol Grown Cells--
We
have previously reported that the phenotypes observed in null
yfh1 mutants are strongly dependent on the laboratory strain (7). In contrast with several other S. cerevisiae strains, W303-1BYFH1 does not significantly lose mitochondrial DNA and grows
well in a rich medium containing glycerol, an obligatory respiratory
carbon source. In the presence of glycerol, W303-1B and W303-1B
YFH1
strains had similar high respiration rates, and moreover, the mutant
cells did not accumulate free iron in mitochondria (data not shown). In
addition, we found that in both strains the expression of typical
members of the iron regulon such as FET3, YOR382w, YOR383c,
and to a much lesser extent, YLR136c, was high (Fig. 1, lanes
3 and 4, and see below, Fig.
6).
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We analyzed the evolution of FET3 and YOR382w gene
expression after a shift from glycerol to fresh glucose and glycerol
media. At the time of the shift, cells were in pre-stationary phase of growth with low levels of FET3 and YOR382w mRNAs both in
wild-type and YFH1 strains (Fig. 6, time zero). After
wild-type cell transfer to glucose medium YOR382w expression decreased
slowly (Fig. 6B) while FET3 expression decrease
was preceded by a transient increase (Fig. 6C). In contrast,
a rapid induction of these genes that was complete within 2 h was
observed in the wild-type and
YFH1 strains incubated in the presence
of glycerol (Fig. 6, A and D), and in the
YFH1
strain cultivated in the presence of glucose (Fig. 6, A and
C). These data show that both in wild-type and
YFH1
strains grown on glycerol Aft1p encounters low iron concentrations, implying that little iron is extruded from mitochondria.
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DISCUSSION |
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Mitochondria are the subcellular compartment which utilizes most
of the cellular iron, mainly in the heme of the cytochromes and in
iron-sulfur cluster proteins. Mitochondria are also the site of
iron-sulfur cluster biosynthesis (for reviews, see Refs. 13 and 14).
Using DNA microarray hybridization analysis in a frataxin-deficient
mutant we found that all known iron sources were mobilized in an
AFT1-dependent manner, implying that cells are
starved for iron (Fig. 7). Thus, we have
substantially extended previous findings showing that YFH1
deletion elicits increase in the expression of the high affinity iron
uptake genes, FET3 and FTR1 (4, 8). Our data
suggest that iron is not only actively mobilized from the external
medium via the high affinity iron uptake system and siderophore
transporters but also from intracellular compartments (Fig. 7). Iron
could be mobilized from the vacuole, a presumed iron storage
compartment (48), via the Fth1p/Fet5p (28) and Smf3p (37) transporters.
YLR205c, a predicted heme oxygenase, could also provide cells with
iron, in agreement with the observation that this enzyme is induced in
mammalians and plants during iron deprivation (49, 50). The altered
iron homeostasis resulting from frataxin deficiency might be associated with a more general redistribution of ion fluxes since the expression of several genes involved in copper, manganese, or cobalt homeostasis is significantly increased in a YFH1 strain. A possible explanation would be that iron starvation increases the expression of those metal
transporters with broad specificity that are also able to transport
iron (36). Consequently, heavy metal uptake and homeostasis would be
altered.
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However, the most impressive increase in gene expression concerned
YOR382w, YOR383c, and YDR534c, three predicted cell-wall GPI-anchor
protein genes of unknown function that are regulated by
AFT1. The triple deletion of these genes results in a less efficient utilization of ferroxiamine B, a hydroxamate-type
siderophore (23, 24). Thus, a YFH1 strain harboring the triple
YOR382
YOR383
YDR534 deletion accumulates less mitochondrial
iron and grows better in the presence of high ferroxiamine B
concentrations than a simple
YFH1 strain. More work will be
necessary to find the specific conditions under which these GPI-anchor
proteins become important and which function they have.
The iron regulon is regulated at the transcriptional level by AFT1. Our data suggest that it could also be regulated at the post-transcriptional level by YLR136c. YLR136c deletion elicits increased mRNA levels of YOR382w, YOR383c, and to a lesser extent, FET3. By analogy with the mammalian TIS protein which regulates the mRNA half-life of specific genes by promoting RNA deadenylation and degradation (41, 42), it is possible that YLR136c is a post-transcriptional regulator of the iron regulon genes, perhaps acting as a safeguard gene ensuring a faster response to new iron conditions.
Iron homeostasis has mostly been studied in cells cultivated in the
presence of glucose. Under these conditions, Aft1p encounters high iron
levels released from mitochondria and the expression of the iron
regulon is low. In contrast, we found that in glycerol-grown wild-type
cells with high respiratory activity, the iron regulon is highly
expressed, implying that Aft1p encounters low iron levels and that
little iron is released from mitochondria (8). Moreover, glycerol-grown
YFH1 cells show high respiration and no mitochondrial iron
accumulation. These data indicate that frataxin has a less essential
role in glycerol-grown cells, when respiratory activity is high. It has
recently been reported that yeast frataxin is an iron-binding protein
(12). The authors have found that in yeast mitochondria frataxin is
present as monomers that contain no iron and multimers of high
molecular weight which contain more than 16 iron atoms per frataxin
molecule. An interesting hypothesis would be that in glycerol-grown
cells with high respiratory needs, iron is mainly used for
mitochondrial heme and iron-sulfur cluster biosynthesis and does not
bind much to frataxin. Conversely, in glucose-grown cells with low
respiration and low iron needs, frataxin would be essentially present
as iron binding multimers, mediating iron export to the cytosol. This
favors the idea that frataxin controls iron partitioning between
mitochondria and cytosol to regulate mitochondrial iron supply. A
recent report suggests that the cAMP-dependent protein
kinase Tpk2 plays a role in this regulation (51) by connecting
respiratory and iron metabolism pathways. However, mitochondrial
control seems restricted to genes responsible for iron mobilization.
Ribonucleotide reductase, ferrochelatase, or Sur2p and Erg25p
desaturases, are maintained outside of this regulatory pathway.
Although the strongest signals were obtained for the iron regulon
genes, the expression of several other genes was altered in a YFH1
strain. We can mention the mating pathway, sulfur metabolism, protein
recycling in the vacuole and proteasome, and a number of genes of
unknown function, such as the seven-transmembrane helix protein Hsp30p,
an opsin-related protein (29). These data point out to new unexpected
links between frataxin deficiency and remote metabolic pathways.
Surprisingly, none of those genes that protect cells against oxidative
stress, and in particular the mitochondrial manganese superoxide
dismutase SOD2 gene, were significantly induced in our
YFH1 strain. Currently, we have no satisfactory explanation but lack
of SOD2 induction might contribute to the dysfunction of
iron-overloaded mitochondria.
No AFT1 homologue has been found in humans. However, human
genes involved in iron metabolism are post-transcriptionally regulated by cytosolic iron regulatory proteins which are iron sensor proteins (for a review, see Ref. 52). It can be predicted that in humans also
frataxin deficiency results in cytosolic iron deprivation and this
opens the possibility that Friedreich ataxia causes a profound
alteration in ionic homeostasis.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. J. Friesen for help. Many thanks to Dr. B. McNeil and N. Winegarden for the construction and gift of yeast DNA chips (Toronto Gene Chip Consortium, OCI, Toronto, Canada). We thank A. Goffeau for reading the manuscript and constant interest and T. Roganti for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by grants from Human Frontier, the Belgian National Fund for Scientific Research, the European Commission, and the Interuniversity Poles of Attraction Program of the Belgian Government Office for Scientific, Technical, and Cultural Affairs.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 all correspondence should be addressed: Unité de Biochimie Physiologique, Place Croix du Sud, 2-20, 1348 Louvain-la-Neuve, Belgium. Tel.: 32-10474691; Fax: 32-10473872; E-mail: foury@fysa.ucl.ac.be.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M005804200
2 F. Foury, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: BPS, bathophenantroline sulfonate; GPI, glycosylphosphatidylinositol; TIS, tristetraproline.
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