From the Department of Pathology, University of Utah, Salt Lake City, Utah 84112
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ABSTRACT |
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Mutations in the nuclear gene encoding the
mitochondrial protein frataxin are responsible for the neurological
disorder Friedreich ataxia (FA). Yeast strains with a deletion in the
frataxin homologue YFH1 accumulate excess iron in
mitochondria and demonstrate mitochondrial damage. We show that in the
absence of YFH1, mitochondrial damage is proportional to
the concentration and duration of exposure to extracellular iron,
establishing mitochondrial iron accumulation as causal to mitochondrial
damage. Reintroduction of YFH1 results in the rapid export
of accumulated mitochondrial iron into the cytosol as free, non-heme
bound iron, demonstrating that mitochondrial iron in the yeast FA model
can be made bioavailable. These results demonstrate a mitochondrial
iron cycle in which Yfh1p regulates mitochondrial iron efflux.
Friedreich ataxia (FA)1
is a neurodegenerative disease transmitted as an autosomal recessive
trait with a prevalence of 1 in 50,000 individuals (1). The FA gene was
identified by positional cloning and found to encode a 210-amino acid
mitochondrial protein designated frataxin (2). Most cases of FA are
because of the expansion of a polymorphic GAA trinucleotide repeat
located in the first intron of the frataxin gene, resulting in reduced
frataxin mRNA levels (3, 4). The defect responsible for FA also
affects non-neuronal organs, and patients usually succumb to a
cardiomyopathy in the fourth decade. Gait ataxia is the most common
presenting symptom, and most patients eventually manifest dysarthria,
areflexia, pyramidal weakness of the legs, extensor planar responses,
and distal loss of joint position and vibration sense (1). The frataxin
protein is localized to the mitochondria (5), but its function has not
been determined. The frataxin protein is highly expressed in neuronal
and heart tissue (2), both of which are postmitotic and highly
dependent upon mitochondrial respiration (1). Iron deposits have been
found in the myocardium of FA patients, and myocardial mitochondrial
respiration has been found to be defective (6).
Yeast disrupted for YFH1 (yeast frataxin homologue)
accumulate iron in mitochondria (7). Mitochondrial DNA (mtDNA) is
damaged and mitochondrial respiratory activity is impaired (8, 9). Mitochondrial iron accumulation in yeast with YFH1 deletions
is associated with subnormal cytosolic iron concentrations (7). Lowered
cytosolic iron concentrations induce transcription of FET3,
a component of the plasma membrane high affinity iron uptake system
(10). The increased rate of iron uptake results in a doubling of
cellular iron content relative to wild-type cells, but the excess iron
is abnormally localized to mitochondria.
Mitochondrial defects in patients with FA and in the yeast model could
be a direct result of mitochondrial iron accumulation. Alternatively, a
deficiency of frataxin protein could result in mitochondrial damage,
and iron overload may be one manifestation of the mitochondrial damage.
This phenomenon has been observed in two patients with acquired
idiopathic sideroblastic anemia (11). To determine whether iron
accumulation is the cause or consequence of the mitochondrial defect,
we constructed a yeast strain that contains respiration-competent
mitochondria and regulatable YFH1. Using this approach, we
found that YFH1 maintains mitochondrial iron homeostasis at
the level of iron efflux. This result indicates that under normal
conditions there is a dynamic flux of iron through mitochondria, which
is disrupted by the loss of YFH1.
Generation of MET-YFH Strain--
pMET-YFH was generated by
polymerase chain reaction using YFH1 open reading
frame-flanking primers (5' cga gat atc tag agt gta gca atg att aa 3'
and 5' ccc gag ctc tta gcg gcc gcg acc tcc ttg gct ttt aga aat ggc ct
3') and was cloned behind a MET25 promoter in the vector
pTf63. This vector has origins of replication for yeast (2µ) and
Escherichia coli (ori), the URA3 gene
to allow selection for yeast transformants, and the ampicillin
resistance gene for selection in E. coli (12). Constructs
were checked by DNA sequencing. yfh1 Gradient Fractionation--
MET-YFH cells and wild-type cells
containing pTf63 were grown to log phase in CM-URA(+met), then washed
and resuspended in CM-URA(+met) supplemented with
59FeCl3 to a final iron concentration of 3.3 µM, and incubated for 20 min at 30 °C. Cells were then
washed, resuspended in nonradioactive CM-URA(+met), and incubated with
shaking at 30 °C for 3 h to allow the 59Fe to label
the cellular iron pool. Cell samples were then grown for indicated
times either in CM-URA(+met) or in CM-URA( To determine whether iron accumulation is the cause or consequence
of the mitochondrial defect in yfh1
INTRODUCTION
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Abstract
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EXPERIMENTAL PROCEDURES
cells (7) were mated
either to wild type or to the fet3
strain DY1397-6
(12). The resulting diploids were transformed with the pMET-YFH plasmid
and sporulated onto complete media lacking uracil and containing 2%
glucose (CM-URA) and lacking methionine to maintain expression of
YFH1. Spores were analyzed for the presence of the
disruptions by polymerase chain reaction and Southern analysis. Plasmid
was maintained by growth on media lacking uracil throughout all experiments.
met) and placed on ice for
the remainder of the experiment. Growth curves indicated that the cells
were in log phase growth at all times, and time courses of wild-type
and yfh1
cells indicated that incubation on ice did not
affect cellular distribution of radioactive iron (data not shown).
Washed cell samples were spheroplasted and Dounce-homogenized.
Postnuclear supernatants were centrifuged (12,000 × g,
30 min) to generate organelle pellets, which were resuspended and
layered onto preformed 0-25% iodixanol gradients. These gradients
were spun at 10,000 × g for 2 h and then
fractionated. Radioactivity in each fraction was measured, and the
position of mitochondria in the gradients was determined by Western
blotting and probing with monoclonal anti-porin antibody (Molecular Probes).
RESULTS
cells, we constructed the MET-YFH strain, which contains respiration-competent mitochondria, a deleted chromosomal copy of YFH1, and a plasmid-based,
methionine-regulated YFH1. Cells grown in the absence of
methionine expressed abundant YFH1 message, whereas addition
of methionine produced complete repression of YFH1
transcription within 2 h (Fig.
1A). Cells grown in the
presence of methionine completely repressed YFH1 message, whereas removal of methionine resulted in full transcription of YFH1 (Fig. 1B). Correlated with YFH1
induction was a suppression of FET3 transcription (Fig.
1B), whereas repression of YFH1 resulted in
increased expression of FET3 mRNA within 4 h (Fig.
1A). Increased iron uptake correlated with the expression of
FET3 transcripts (Fig. 2).
When cells were maintained in the absence of methionine, iron uptake
remained low, whereas cells moved to media containing methionine
rapidly induced the high affinity iron uptake system. This demonstrates
that the cytosol becomes iron-depleted when YFH1 is
repressed, because FET3 transcription and the high affinity iron transport system are positively regulated by iron deficiency (12).
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Fig. 1.
Methionine-dependent expression
of YFH1 in MET-YFH cells. A, Northern
blot of MET-YFH cells grown overnight in the absence of methionine and
then transferred to media containing methionine for 2, 4, 6, or 8 h. B, Northern blot of MET-YFH cells grown overnight in the
presence of methionine and then transferred to media lacking methionine
for 2, 4, 6, or 8 h. Northern analysis was performed and the blots
were probed with YFH1, FET3, and actin using
techniques described previously (19).
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Fig. 2.
Repression of YFH1 produces
a rapid increase in iron transport. Iron uptake of MET-YFH cells
first grown in the absence of methionine and then transferred to media
containing or lacking methionine is shown. Iron uptake was assayed in
the presence of 0.5 µM 59Fe and 1 mM ascorbate as described previously (12).
To determine the relationship between cellular iron accumulation and mitochondrial damage, MET-YFH cells were grown in media lacking methionine and then incubated in media containing or lacking methionine that was supplemented with increasing concentrations of iron. Cell samples were then washed and plated on media that lacked methionine and contained glucose as the carbon source. The resultant colonies were then replica-plated onto media containing 1% glycerol and 1% ethanol as carbon sources. Mitochondrial damage was assessed as the percentage of colonies that were petite, i.e. unable to grow when glycerol and ethanol were the sole carbon sources. In the absence of YFH1, mitochondrial damage was proportional both to the amount of iron in the growth media and to the time cells were exposed to iron (Fig. 3A). MET-YFH cells lacking FET3, which are defective for high affinity iron acquisition, demonstrated no iron-dependent mitochondrial damage even in the presence of 500 µM iron (Fig. 3B). These results indicate that the mitochondrial damage that follows YFH1 depletion is dependent upon cellular iron uptake.
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Cells with a deleted YFH1 gene demonstrated increased
mitochondrial iron content. This accumulation could have resulted from either increased iron uptake or decreased iron efflux. To distinguish between these possibilities we monitored mitochondrial iron content following reintroduction of Yfh1p in yfh1 cells. MET-YFH
cells grown in the presence of methionine were incubated with
59Fe for 20 min and then grown in the presence of
methionine for 3 additional hours to label cellular iron stores.
Following this, cells were either maintained in media containing
methionine or transferred to media lacking methionine to induce
expression of YFH1. Cells were then homogenized and
organelles were fractionated on iodixanol gradients. Virtually all
59Fe was found in fractions that also contained the
mitochondrial protein porin (Fig. 4).
When maintained in media containing methionine, there was only a
marginal, time-dependent decrease in
mitochondria-associated iron (Fig. 4A). When cells were
incubated in medium lacking methionine to induce expression of
YFH1, there was a rapid decrease in mitochondria-associated iron (Fig. 4B). Iron mobilized from the mitochondria was
immediately available as cytosolic free iron as FET3
transcription was rapidly repressed (Fig. 1B). Although
these data do not rule out an effect of YFH1 on
mitochondrial iron uptake, they do establish that YFH1 mediates the efflux of iron from mitochondria.
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DISCUSSION |
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In this study, we demonstrate a role for Yfh1p in maintaining mitochondrial iron homeostasis. Yfh1p is unlikely to be the actual transporter, as sequence analysis indicates that it contains no transmembrane sequences. Yfh1p may mediate iron efflux by Atm1p, an ABC transporter located in the mitochondrial inner membrane (13). Although yeast strains deleted for ATM1 show a mitochondrial iron accumulation phenotype similar to YFH1 deletion strains (14), a direct biochemical interaction between Yfh1p and Atm1p has yet to be determined.
Our results suggest that the mitochondrial defect in tissues from
patients with FA is the consequence of iron accumulation. These results
are consistent with the hypothesis that mitochondrial damage results
from iron-mediated oxygen radical production. Reintroduction of
YFH1 resulted in the efflux of iron from mitochondria,
suggesting that the excess mitochondrial iron in the similar FA
syndrome may potentially also be mobilized. If this proves to be the
case then perhaps chelation therapy should be considered for the
treatment of FA, if a strategy for selectively chelating mitochondrial
iron could be devised. Many drugs have been administered to patients with FA in an attempt to alleviate progression of the disease, including choline chloride, lecithin, thyrotropin-releasing hormone, -vinyl,
-aminobutyric acid, 5-hydroxytryptophan, and benserazide. None has had any beneficial effects, and current therapy of FA is
limited to supportive care (1). Patients with hereditary aceruloplasminemia exhibiting basal ganglion dysfunction because of
iron accumulation in neuronal tissues have been successfully treated by
chelation with desferrioxamine (15), indicating that it can chelate
iron in neuronal tissues. Excess iron in cardiac myocytes is also
accessible to desferrioxamine as demonstrated in patients with cardiac
iron overload because of thalassemia (16).
The observation that Yfh1p affects iron efflux indicates that, under
normal conditions, iron can both enter and exit yeast mitochondria.
This suggests the existence of a mitochondrial iron cycle. A mechanism
must exist to regulate mitochondrial iron accumulation in response to
iron need, either for mitochondrial proteins or for heme synthesis, the
final step of which occurs within the mitochondrial matrix. Increased
mitochondrial iron demand could be met by either increased influx or
reduced efflux. A priori we expected regulation to occur at
the level of uptake, but there is no theoretical reason why the site of
regulation could not be at the level of efflux. Excessive mitochondrial
iron accumulation is found in erythroid precursors of patients with
sideroblastic anemia. Iron deposition results from deficiencies in heme
synthesis, due either to mutations in heme biosynthetic enzymes or to
pharmacologic inhibition of porphyrin synthesis (17). These
observations have led to the suggestion that iron may only exit
mitochondria as heme (18). As our results suggest the existence of a
yeast mitochondrial iron efflux pathway independent of heme, we propose
that the homologous frataxin-mediated pathway may be inactivated or
absent in cells of erythroid lineage. Inhibition of a pathway allowing
efflux of free iron from erythroid mitochondria may support the high rate of heme biosynthesis required by this cell type.
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ACKNOWLEDGEMENTS |
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We thank J. Leslie and D. Ward for advice on gradient fractionation and J. Kushner for numerous helpful discussions.
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FOOTNOTES |
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* This research was supported by National Institutes of Health (NIH) Grant DK52380 with additional support from NIH Cancer Center Grant CA42014 and NIH Center of Excellence in Hematology Grant DK49219.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.
These authors contributed equally to this work.
§ Supported by National Institutes of Health Training Grant GM07464.
¶ Current address: Dept. of Genetics, University of Washington, Seattle, WA 98175.
To whom reprint requests should be addressed. E-mail:
kaplan{at}bioscience.biology.utah.edu.
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ABBREVIATIONS |
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The abbreviation used is: FA, Friedreich ataxia.
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