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INTRODUCTION |
Iron is required as a cofactor for critical proteins that mediate
diverse processes such as cellular respiration and synthesis of
metabolic intermediates (1). However, iron is also extremely toxic,
capable of generating free radicals that damage proteins, lipids, and
DNA (2). Thus, eukaryotic cells such as yeast regulate iron uptake into
cells in homeostatic fashion, inducing uptake in response to iron
starvation, and repressing uptake in response to iron overload (3).
Several mutant strains of yeast have been identified that exhibit
dysregulation of cellular iron uptake. In these mutants, iron uptake
into cells continues without homeostatic repression, and the
assimilated iron is sequestered in mitochondria. Furthermore, in these
mutants, Fe-S1 cluster
proteins are defective, suggesting a link between iron trafficking and
the status of Fe-S clusters in the cell (4-10).
Fe-S clusters are modular units in which iron and sulfur are
coordinated in various combinations and linked to the peptide backbone
of proteins via cysteine sulfurs (11). Because of their ability to
donate or accept electrons with tremendously varied range of
potentials, these clusters are involved in many fundamental biological
processes from cellular respiration to metabolic conversions (12).
Although the clusters can form spontaneously under laboratory conditions, recent genetic and biochemical evidence suggests that their
in vivo formation is catalyzed by specific proteins (13). In
bacteria, several of these proteins are associated together on the
isc operon, and their precise biological roles in Fe-S cluster synthesis are being determined in ongoing biochemical work
(14-16). A cysteine desulfurase (IscS) mediates release of sulfur from
cysteine for formation of Fe-S clusters (17). Fe-S cluster
intermediates assembled onto a protein template (IscU) may be
transferred to an apoprotein recipient (15). A protein with conserved
cysteines (IscA) may bind and donate iron. A chaperone belonging to the
Hsp70 family (HscA) and a co-chaperone (HscB) likely play roles in
folding or unfolding synthetic intermediates, such as the intermediates
assembled on the IscU template (14, 16). Finally, a ferredoxin which
itself contains a 2Fe-2S cluster (Fdx) is encoded by the
isc operon and facilitates iron-sulfur protein synthesis
when overexpressed, but its precise function has not been ascertained
(18).
In the yeast Saccharomyces cerevisiae, homologs of the
bacterial proteins of the isc operon are nuclear encoded
mitochondrial proteins. Mutations leading to decreased expression of
the Hsp70 chaperone Ssq1p (homologous to HscA) (5), and Nfs1p
(homologous to IscS) (8), have been shown to result in dysregulated
cellular iron uptake, mitochondrial iron accumulation, and deficient
Fe-S cluster proteins. The synthetic phenotype resulting from
loss-of-function of Isu1p (homologous to IscU) and Nfu1p (homologous
the C terminus of the bacterial NifU), has been characterized by iron
accumulation in mitochondria and deficient Fe-S proteins (10). In
addition, mutants in Nfs1p, Isu1p, Jac1p, and Ssq1p have been selected
for their ability to escape the oxygen-dependent lysine
auxotrophy that occurs as a consequence of Sod1p deficiency (6). These mutants manifest Fe-S protein deficiencies and iron trafficking abnormalities. Finally, experiments depleting ferredoxin, Yah1p (homologous to Fdx), by turning off a regulated promoter, have uncovered a role for this protein in synthesis or maintenance of Fe-S
clusters and in control of mitochondrial iron levels (9). Other yeast
genes without homologs on the isc operon that produce similar phenotypes when mutated or depleted are YFH1, the
yeast frataxin homolog (4), and ATM1, an ABC transporter of
the mitochondrial inner membrane (19). YFH1 deletion results
in mitochondrial iron accumulation (4) and deficient mitochondrial
iron-sulfur protein activities (20). Atm1p depletion results in iron
trafficking abnormalities, deficient cytoplasmic Fe-S proteins but
normal mitochondrial Fe-S proteins (7).
Arh1p is an essential yeast protein with reductase activity localized
to the inner mitochondrial membrane (21, 22). Arh1p lacks a bacterial
homolog on the isc operon. In this work, we demonstrate a
role for Arh1p in cellular and mitochondrial iron homeostasis.
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EXPERIMENTAL PROCEDURES |
Growth Media--
Rich medium consisted of 1% yeast extract,
2% peptone, and various carbon sources. To induce the GAL1
promoter, 2% raffinose and 0.5% galactose served as the carbon
source. Expression from the GAL1 promoter was turned off by
shifting the cultures to identical medium without galactose. In some
experiments, 2% glucose or 3% ethanol was used as carbon source. For
experiments with different concentrations of iron, standard defined
medium with 2% raffinose or 2% glucose was modified by addition of
ferric ammonium sulfate.
Plasmids and DNA Constructions--
The open reading frame of
Arh1p was amplified by PCR using Pfu polymerase and genomic DNA as a
template. The primers added XbaI and NdeI sites
on the 5' end and BglII and XhoI sites on the 3'
end. The PCR product was cloned into the XbaI and
XhoI sites of pBluescript II, and the resulting plasmid
pBS-Arh1 was linearized by BglII and desphosphorylated with
calf intestinal phosphatase (Roche Molecular Biochemicals). The
BamHI-BglII fragment containing three tandemized
copies of the hemagglutinin epitope (HA3) (23) was subcloned into the
dephosphorylated BglII site, creating plasmid pBS-Arh1HA3.
The correct orientation of the tag was confirmed by sequencing. The
XbaI-XhoI fragment containing the open reading
frame and tag (Arh1p-HA3) was subcloned into the corresponding sites of
the integrating expression vector pEMBlyex4i to create pGal-Arh1HA3. In
this vector, there is a GAL1 promoter, CYC1
transcription start site and terminator, and URA3 marker for
selection of yeast transformants. A unique StuI site in the URA3 gene of the plasmid is present for directing
integration into the ura3-52 locus. For knockout of
ARH1, an interruption/deletion cassette was constructed. A
1.2-kilobase DNA fragment containing the HIS3 gene
with BamHI ends was amplified by PCR using pRS413 as
template, digested with BamHI to generate cohesive ends, and ligated into the BamHI and BclI sites of the
ARH1 open reading frame of pBS-Arh1. The
interruption/deletion cassette was released by digestion with
XbaI and XhoI.
Strains--
The parental diploid strain YPH501 (24) was
transformed with the XbaI-XhoI fragment of the
ARH1 knockout cassette and transformants were selected for
histidine prototrophy. Correct integration was confirmed by genomic
PCR. Plasmid pGal-Arh1HA3 was linearized at the unique StuI
site and integrated into the ura3-52 site of the
heterozygous ARH1/
arh1 knockout strain. The
transformants were sporulated and tetrads were germinated on plates
containing galactose as a carbon source. A tetrad clone that was
His+ (marker for
arh1) and Ura+
(marker for pGal-Arh1HA3) was identified and referred to as the Gal-Arh1 strain.
Bacterial Expression and Antibodies--
For expression in
bacteria, the ACO1 and YAH1 open reading frames
were amplified by PCR from yeast genomic DNA and inserted into the
NdeI and XhoI sites of pET21b (Novagen). The
overexpressed proteins with C-terminal His6 tags were found
to be sequestered in inclusion bodies. These proteins were solubilized
in 8 M urea, purified on Ni-NTA columns (Qiagen), and
eluted with 0.4 M imidazole. The eluates were further
purified by preparative SDS-polyacrylamide gel electrophoresis and used
to immunize rabbits for generation of polyclonal antibodies. Antibodies
to Cyc1p were generated by immunizing rabbits with yeast holocytochrome
c purchased from Sigma. Anti-HA (Roche Molecular
Biochemicals) and anti-Cox3p (Molecular Probes) are commercially
available mouse monoclonal antibodies.
Fractionation of Mitochondria--
Mitochondria were isolated as
described (25). Intact mitochondria (equivalent to 200 µg of protein)
were resuspended in 20 mM Tris-HCl, pH 7.5, 0.6 M sorbitol. For releasing the intermembrane space contents,
mitochondria were subjected to hypotonic shock by diluting to 0.1 M sorbitol and incubating at 0 °C for 10 min. The
mitoplasts were recovered by centrifuging at 12,000 × g and then solubilized in 0.5% Triton X-100. The lysate was
centrifuged for 10 min at 15,000 × g and supernatant
and pellet fractions were separated (26). For quantitation of the
55Fe label, fractions were exposed to 2% SDS in 200 µl
for 15 min at room temperature and suspended in 1 ml of scintillation
mixture for counting in a Beckman scintillation counter.
Assays--
High-affinity cellular iron uptake was measured as
described (27). Aconitase was assayed by measuring the formation of
cis-aconitate at 240 nm. The reaction mixture of 1 ml
contained of 20 mM iso-citrate, 90 mM Tris-HCl,
pH 8.0, 25 µl (100 µg) of purified mitochondria solubilized in
1.5% n-octyl-
-D-glucopyranoside containing 1 mM sodium citrate. The
A240 was
measured for 2 min at room temperature, and the activity was expressed
as nanomoles of cis-aconitate formed per mg of mitochondria
protein per min (28). Succinate dehydrogenase was assayed on
mitochondrial lysate in 0.5% Triton X-100 by following the reduction
of p-iodonitrotetrazolium violet to the
p-iodonitrotetrazolium-formazan as described (29). Leu1p
(3-isopropylmalate dehydratase) activity was measured in concentrated
cytoplasmic lysates (~10 mg/ml). The assay mixture of 500 µl
consisted of 100 mM potassium phosphate buffer, pH 7.0, 2 mM citraconate, and 20 µl (200 µg) of cell extract. The
mixture was transferred to a 2 mm quartz cuvette and the decrease in
A235 was recorded over 3 min at room
temperature. Activity was expressed as
A235/min/mg of protein (30). For detection of
heme, mitochondrial proteins (100 µg) separated by polyacrylamide gel
and blotted to nitrocellulose were incubated with peroxide developer
and chemiluminescent substrates (Blaze, Pierce) for 5 min prior to
exposing the blot to film (31).
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RESULTS |
Gal-Arh1 Strain with Regulated Arh1p Expression--
To
characterize the function of Arh1p, we replaced a portion of the open
reading frame with a HIS3 marker gene in the diploid strain
YPH501. Sporulation of this diploid strain resulted in 2+:2
segregation for viable colonies with all the viables showing auxotrophy
for histidine (not shown). This result is consistent with the
previously reported essential nature of the ARH1 gene (21,
22). To further study cellular effects of Arh1p, we generated a strain
in which the only functional ARH1 gene was replaced with a
gene fusion. In this construct, the open reading frame was fused at the
C terminus to 3 copies of the HA epitope and placed under control of
the GAL1 galactose-inducible promoter. Growth of the strain
in galactose medium, inducing for the promoter, resulted in high level
expression of the protein as monitored by monoclonal anti-HA
antibodies. In the absence of inducer, as expected, Arh1p was
undetectable (Fig. 1, panel
A).

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Fig. 1.
Panel A, regulated expression of Arh1p.
Mitochondria were isolated from a Gal-Arh1 strain grown under inducing
conditions (+Gal) or noninducing conditions
( Gal). Mitochondrial proteins were separated by
polyacrylamide gel, transferred to nitrocellulose, and probed with
antibody to the HA epitope. In the Gal-Arh1 strain, the endogenous
ARH1 gene was deleted and replaced with a construct
containing the GAL1 promoter driving the open reading frame
followed by three tandemized HA epitopes (Arh1p-HA3, 66 kDa).
Panel B, growth properties of Gal-Arh1 strain. YPH499
(wild-type parent) or Gal-Arh1 cells were grown on YPAD plates. Cells
were suspended in water, counted, and serial 10-fold dilutions of
105 cells were placed on agar plates with different carbon
sources. Galactose plate contains 2% raffinose and 0.5% galactose.
Glucose plate contains 2% glucose. Ethanol plate contains 3% ethanol.
Plates were photographed at 48 h (Galactose and
Glucose) or 72 h (Ethanol).
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The growth properties of this strain were then evaluated. The growth of
the Gal-Arh1 strain on galactose containing media was indistinguishable
from the wild-type parent YPH499 (Fig. 1, panel B, top).
However, suprisingly, when incubated in the absence of inducer and in
the presence of glucose, which acts as a strong repressor for the
GAL1 promoter, growth and colony formation were minimally
affected. Colonies were only slightly smaller than wild-type controls
after 2 days (Fig. 1, panel B, middle) but then
caught up after another day incubation (not shown). The implication of this observation is that a small amount of "leaky" Arh1p protein expression, undetectable by immunoblotting, was sufficient to support
growth. Cells with repressed Arh1p expression were not normal, however.
Plating efficiency of the Gal-Arh1 cells on ethanol agar plates was
decreased by more than 2 logarithms, and colony size was markedly
diminished compared with the wild-type control cells (Fig. 1,
panel B, bottom). The ethanol carbon source is noninducing for the GAL1 promoter and furthermore, is
nonfermentable. Failure to utilize nonfermentable carbon sources may
reflect mitochondrial dysfunction resulting from Arh1p depletion. Such
a phenotype would be consistent with a nonredundant mitochondrial
function for this protein.
Role for Arh1p in Regulating Cellular Iron Uptake--
We next
examined cellular iron uptake in the Gal-Arh1 strain and found that it
was dysregulated. In the wild-type YPH499 strain grown in the presence
of iron chelator (bathophenanthrolene disulfonate) the rate of
high-affinity iron uptake was induced, whereas the same strain exposed
to iron during growth showed repressed iron uptake. Mutations of
several mitochondrial genes implicated in mitochondrial iron
homeostasis exhibit altered responses of cellular iron uptake to iron
exposures. In the MA14 mutant, carrying a mutation in the
NFS1 gene, cellular iron uptake was induced and incompletely
repressed by iron exposures in the medium (Fig.
2). Similarly, the Gal-Arh1 mutant grown
in the absence of inducer showed a lack of response of cellular uptake
to medium iron. At most media iron concentrations, cellular iron uptake
was nonrepressed in the Gal-Arh1 strain compared with wild-type levels.
At 10 µM medium iron concentration, uptake was completely
repressed to the wild-type level (Fig. 2).

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Fig. 2.
Dysregulation of cellular iron uptake in
Gal-Arh1 strain. High-affinity cellular iron uptake was measured
for strains YPH499 (wild-type parent), Gal-Arh1 (engineered strain with
regulated Arh1p expression), and MA14 (containing nfs1-14
allele) grown in defined media with different concentrations of iron.
The defined media contained 2% glucose, which is repressing for the
GAL1 promoter controlling Arh1p expression.
Bathophenanthrolene disulfonate (BPS) refers to the iron
chelator bathophenanthrolene disulfonate. Iron was added to the
cultures as ferric ammonium sulfate at the indicated concentrations, or
bathophenanthrolene disulfonate was added at 10 µM.
Cultures were grown for 14 h and then diluted 10-fold into media
of the same composition and allowed to reach logarithmic growth.
Cellular iron uptake was measured over 1 h (pmol/106
cells/h) by incubation with 55Fe and collection of the
cells on glass fiber filters.
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Role for Arh1p in Regulating Mitochondrial Iron
Homeostasis--
Arh1p is localized to the mitochondrial inner
membrane (22) and might affect mitochondrial iron usage and
distribution. To directly test this possibility, we exposed the
wild-type YPH499 strain or the Gal-Arh1 strain to various iron
concentrations in defined growth medium lacking the galactose inducer.
Radioactive 55Fe was added as a tracer. After 16 h of
growth during which the labeling of intracellular iron pools reached a
steady state, mitochondria were isolated and the iron content and
distribution were ascertained by analysis of the radionuclide. The
Gal-Arh1 mutant showed a striking inability to control mitochondrial
iron levels (Fig. 3). At the lowest
medium iron concentration (0.1 µM, Fig. 3, panel A), wild-type and Gal-Arh1 mitochondrial iron levels were
comparable and less than 1 pmol/µg of protein. A 10-fold increase in
medium iron exposure had minimal effect on the mitochondrial iron of the wild-type but resulted in more than a 20-fold increase in mitochondrial iron of the mutant (1.0 µM, Fig. 3,
panel B). An additional 5-fold increase in medium iron
exposure had small effects on the wild-type but resulted in another
more than a 5-fold increase in mitochondrial iron in the mutant (5.0 µM, Fig. 3, panel C). The exposure to still
higher levels of medium iron did not result in further increase in
mitochondrial iron in the Gal-Arh1 mutant (50 µM, Fig. 3,
panel D).

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Fig. 3.
Dysregulation of mitochondrial iron levels
and distribution in Gal-Arh1 strain. The wild-type parent strain
(YPH499, open bars) and the engineered Arh1 mutant strain
(Gal-Arh1, filled bars) were exposed to varying iron
concentrations (0.1, 1.0, 5.0, and 50 µM for panels
A-D, respectively), and the iron contents of mitochondria were
determined. The strains were grown in defined media with the carbon
source, raffinose, which was noninducing for the GAL1
promoter. Iron was added at the indicated concentrations and
55Fe radionuclide was used as a tracer. After 16 h of
growth, mitochondria were isolated and an aliquot equivalent to 200 µg of protein was fractionated according to standard protocols (26).
The iron content for each fraction was determined by scintillation
counting of radionuclide in the samples. The fractions were
mitochondria (Mito), intermembrane space (IMS),
mitoplasts (Mitoplast), Triton X-100 supernatant
(Sup), and Triton X-100 insoluble pellet
(Pellet). The iron content of the fractions is displayed as
picomole of iron/µg of mitochondrial protein.
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Within mitochondria, the characteristics of iron were also strikingly
altered in the Gal-Arh1 strain. At the lowest iron concentration, total
mitochondrial iron was comparable in the wild-type and Gal-Arh1 strain,
as was iron in the intermembrane space and mitoplast fractions. However, when mitoplasts were sonicated disrupting the inner membrane, a significant portion of the iron label was pelleted by low speed centrifugation (not shown). We considered that this iron pool might
represent adherence of iron to membrane lipids or accumulation of
aggregated iron proteins. Triton X-100 almost quantitatively solubilizes most mitochondrial proteins, and therefore we evaluated the
distribution of iron after Triton X-100 solubilization of mitoplasts.
In the Gal-Arh1 mutant a significant fraction of mitoplast iron was
pelleted even after Triton X-100 treatment (40% versus 13%
in the wild-type) consistent with the possibility that the iron label
may be present in aggregated proteins (Fig. 3, panel A,
TX-100 pellet). Increases in total mitochondrial iron in the Gal-Arh1 strain were associated with marked increases in the Triton X-100-insoluble fraction (38-, 80-, and 10-fold increased compared with
wild-type in Fig. 3, panels B-D). Although not to the same degree, iron was also increased in the intermembrane space and in the
Triton X-100 soluble fraction of the mitoplasts under these iron
loading conditions. In summary, the Gal-Arh1 strain with repressed
Arh1p expression showed a loss of mitochondrial iron homeostasis. A
distinct Triton X-100-resistant form of iron appeared in the absence of
an increase in the total mitochondrial iron level; in addition, after
iron exposure during growth, mitochondrial iron accumulated to a
remarkable degree.
Iron-Sulfur Protein Deficiencies in the Gal-Arh1 Strain--
In
view of the marked alterations of iron distribution to mitochondria,
iron proteins of the mitochondria were evaluated. Aconitase (Aco1p), a
soluble enzyme of the mitochondrial matrix depends on an intact
iron-sulfur cluster for its enzymatic activity. Aconitase activity was
equivalent to the wild-type level in the Gal-Arh1 strain under inducing
conditions for Arh1p expression (not shown), but activity was markedly
decreased in the same strain grown in the absence of inducer (Fig.
4). The deficiency was not due to altered
protein expression or turnover, because Aco1p could still be detected
in normal abundance (Fig. 4). The discrepancy between normal protein
levels and decreased activity indicates that enzymatically inactive
protein was present. The residual aconitase activity in the Gal-Arh1
strain varied between 16 and 34% of wild-type levels despite more than
100-fold changes in the mitochondrial iron contents (ranging from 0.4 to 54 pmol/µg of protein). The deficiency was not improved by iron
starvation as might be expected if it were a toxic effect of iron
overload. Conversely, the Aco1p activity was not recovered in the iron
loaded mitochondria.

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Fig. 4.
Deficiency of aconitase activity in the
Gal-Arh1 cells. Aconitase activity was assayed in mitochondria
isolated from the wild-type parent (YPH499, open bars) or
engineered Arh1 strain (Gal-Arh1, filled bars) grown under
noninducing conditions and exposed to different iron concentrations in
the growth medium (0.1, 1.0, 5.0, and 50 µM). The
specific activity is defined as nanomole of cis-aconitate
formed per mg of mitochondrial protein per min. The aconitase protein
level (Aco1p, 85 kDa) was assayed by immunoblotting with monospecific
antibody.
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Heme Protein Deficiencies in the Gal-Arh1
Strain--
Mitochondrial heme proteins were examined. By immunoblot
analysis, cytochrome oxidase subunit 3 (Cox3p) was found to be
virtually absent from the Gal-Arh1 mitochondria with elevated iron
content (isolated from 1.0, 5.0, and 50 µM iron
containing media). By contrast, the protein was present and
undiminished in Gal-Arh1 mitochondria with normal iron content
(isolated from 0.1 µM iron containing medium) (Fig.
5). Cox3p is mitochondria encoded, and therefore, mtDNA damage might account for the observed effects on
Cox3p. However, staining with the fluorescent dye
4',6-diamidino-2-phenylindole dihydrochloride showed that mtDNA was
present in Gal-Arh1 mitochondria grown in the absence of inducer (not
shown). In addition, a similar trend was observed for cytochrome
c protein, which is nuclear encoded. This protein was
markedly decreased in the Gal-Arh1 mitochondria, with the exception of
the low iron point. Covalently bound c-type heme groups of Cyc1p
(cytochrome c) and Cyt1p (cytochrome bc1) can be
visualized by their intrinsic peroxidase activities (Fig. 5), and these
were found to be markedly decreased in the Gal-Arh1 mutant. In
mitochondria isolated from low iron growth conditions, the signals from
heme in Cyc1p and Cyt1p were increased compared with iron-loaded
mitochondria; however, complete normalization was not achieved. Thus,
the mutant phenotype characterizing cells depleted of Arh1p included
deficiency of heme proteins. The hemoprotein deficiency was mitigated
in the Gal-Arh1 mutant mitochondria isolated from low iron medium. In
accord with this last observation, these mitochondria were pink in
color as distinguished from the paler color of their counterparts
isolated from iron replete media.

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Fig. 5.
Deficiency of heme proteins in the Gal-Arh1
mitochondria with increased iron content. Mitochondria were
isolated from wild-type parent (YPH499) or mutant strain (Gal-Arh1)
grown under noninducing conditions and exposed to different iron
concentrations (0.1, 1.0, 5.0, and 50 µM).
Delta-1-pyrroline-5-carboxylate dehydrogenase (Put2p), an
unrelated matrix protein was evaluated as a control. Heme proteins,
cytochrome oxidase subunit 3 (Cox3p, 24 kDa), and cytochrome
c (Cyc1p, 12 kDa) were assayed by immunoblotting.
Heme groups in Cyc1p (Cyc1p heme) and the bc1 heme
(Cyt1p heme, 31 kDa) were evaluated separately by heme
blotting.
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Time Course of Arh1p Depletion--
We examined the appearance of
iron regulatory phenotypes after shifting the Gal-Arh1 strain from
galactose (inducing) conditions to raffinose (noninducing) conditions,
in rich media containing ~1 µM iron. At the time of the
switch, expression of HA-tagged Arh1p could be easily detected with
anti-HA monoclonal antibody (Fig. 6,
panel A). A more rapidly migrating band reactive with anti-HA antibody on immunoblots was frequently observed (* in Fig. 6,
panel A). However, the amount of this species varied from experiment to experiment and most likely represents proteolytic clipping at the N terminus of Arh1p. A similar pattern of proteolysis has been described for the Drosophila homolog of Arh1p (32). Subsequently, after growing under noninducing conditions for 12 h,
the protein declined to undetectable levels and remained undetectable for the 24-, 36-, 48-, and 60-h time points. During this 60-h time
period, cell growth was normal in the absence of detectable Arh1p. At
the end of 60 h, galactose was added back to the medium, and the
Arh1p protein expression was turned back on for 10 h (Fig. 6,
panel A, +10). Control proteins Put2p, a matrix protein
involved in proline utilization, and Por1p, a structural outer membrane protein, were present in equivalent amounts in mitochondria isolated from all time points (Fig. 6, panel A).

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Fig. 6.
Time course of Arh1p depletion. The
Gal-Arh1 strain was grown under inducing conditions in rich medium
containing 2% raffinose and 0.5% galactose. At time 0, the cells were
collected by centrifugation and transferred to medium lacking
galactose. At 12-h intervals, cells were diluted and aliquots were
removed for measurements of high-affinity iron uptake and cellular
fractionation. After the cells had grown for 60 h in galactose
minus medium, galactose was added back to the culture and cells were
grown for an additional 10 h. The density of the culture was
maintained between A600 of 0.2 and 2.0 by
additions of fresh medium. Panel A, Arh1p expression.
Mitochondria from the time course were evaluated for Arh1p expression
(Arh1p-HA3, 66 kDa) using anti-HA monoclonal antibody.
Control proteins (Put2p, Por1p) were also evaluated. The
lower band in the Arh1p blot (*, 55 kDa) was present in
variable amounts in different experiments and likely represents the
effect of proteolysis during isolation. Panel B, cellular
iron uptake. Cells were evaluated for high-affinity iron uptake by 20 min incubation with 55Fe and filtering on glass fiber
filters. Panel C, aconitase. Aconitase activity and protein
were measured on mitochondria from the various time points. Aconitase
specific activity is displayed on the graph (nanomole of
cis-aconitate formed per mg of mitochondrial protein per
min) and the Western blot showing the corresponding Aco1p levels is
shown above the graph. Panel D, succinate
dehydrogenase (SDH activity) was measured on mitochondria
from the various time points (filled squares). The units are
nanomole of p-iodonitrotetrazolium violet reduced per mg
mitochondrial protein per min. Isopropylmalate dehyratase activity
(Leu1p activity) was measured on cytoplasmic fractions
(filled circles). The units are change in absorbance per mg
of cytoplasmic protein per min. Panel E, cytochrome
c. Cyc1p was evaluated by immunoblotting using monospecific
polyclonal antibodies (top) or heme blotting
(bottom). Panel F, Yah1p. The ferredoxin, Yah1p,
was evaluated in mitochondria from the Arh1p depletion time course.
Yah1p migrated anomalously at 30 kDa although the predicted size was
less than 20 kDa; this anomalous mobility may have been due to the
acidic charge of the protein.
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Cellular iron uptake was examined during the course of Arh1p depletion
and repletion. When assessed at the time of the shift to noninducing
conditions and 12 h later, cellular iron uptake was
indistinguishable from wild-type (not shown), although Arh1p protein
was already undetectable at the 12-h time point. Cellular uptake then
increased 10-fold at the 24-h time point (Fig. 6B). The iron
uptake level remained elevated throughout the rest of the experiment,
failing to recover with the restoration of the Arh1p expression after
galactose readdition (Fig. 6, panel B). The delayed
appearance of the mutant phenotype (increased iron uptake) after
apparent disappearance of Arh1p was observed for the other mutant
phenotypes as well, which appeared in coordinated fashion between 12 and 24 h after removal of the galactose inducer. Aconitase
activity, normal at the first two time points, declined to less than
half of wild-type levels, remaining at this low level until it
recovered with restoration of Arh1p at the last time point (Fig. 6,
panel C). As noted previously, deficient aconitase activity
was not attributed to lack of protein expression or increased turnover,
because immunoblotting with monospecific rabbit antisera demonstrated a
uniform abundance of protein at all time points (Aco1p in
Fig. 6, panel C). In this experiment, activities of other
Fe-S proteins were also assayed. Succinate dehydrogenase, a complex of
four subunits of the inner mitochondrial membrane, contains Fe-S, heme,
and FAD cofactors. Succinate dehydrogenase activity declined and
recovered in parallel with the changes in aconitase activity (Fig. 6,
panel D). Leu1p, 3-isoproplmalate dehydratase, is a
cytoplasmic protein with strong sequence homology to the 4Fe-4S protein
aconitase. Leu1p has been shown to contain iron (7), and this most
likely is in the form of a 4Fe-4S cluster. Leu1p activity also changed
in parallel with aconitase activity, indicating that both mitochondrial
and cytosolic Fe-S proteins were affected by Arh1p depletion (Fig. 6,
panel D).
As regards hemoprotein deficiency, cytochrome c protein and
heme were independently examined and found to decline together at the
24-h time point (Fig. 6E). The nadirs of protein and
cofactor, however, did not occur until the 36-h time point and thus
were slightly delayed with respect to the nadir of aconitase activity. During the recovery time point (+10), cytochrome c heme
recovered to 20% of wild-type, whereas cytochrome c protein
was present at 50% of wild-type; the results suggest that some
apoprotein may be present (Fig. 6, panel E). Significantly,
the recovery of cytochrome c, like the recoveries of iron
uptake, aconitase, and Leu1p were incomplete and delayed after recovery
of Arh1p expression. Perhaps the delayed return of these phenotypes to normal results from toxic effects of accumulated mitochondrial iron
which take time to reverse, or other downstream effects of Arh1p
activity that require time to manifest themselves.
Arh1p might work via effects on Yah1p, analogous to the way human
homologs of these proteins are thought to function (33). Therefore, we
evaluated Yah1p protein during the time course of Arh1p depletion.
Yah1p detected by immunoblotting with specific antibody was unchanged
(Fig. 6, panel F) indicating that Arh1p has no effect on
Yah1p protein levels.
 |
DISCUSSION |
Arh1p is an essential protein of mitochondria with reductase
activity (21, 22), and we show here that it is required for iron
homeostasis of the cell and mitochondria. A yeast strain depleted of
Arh1p exhibited a multifaceted phenotype characterized by inability to
repress cellular iron uptake appropriately in response to environmental
iron exposure. The iron assimilated by these cells accumulated in
mitochondria. On the other hand, iron proteins were deficient,
including Fe-S proteins and heme proteins.
Apparently distinct threshold levels of Arh1p expression were required
for viability and correct iron regulation. During the time course
experiment in which expression of Arh1p from the GAL1 promoter was turned off, the level of protein declined, and the cells
became dysregulated in terms of iron metabolism. Even then, growth
continued unimpeded. Most likely, the Arh1p protein would need to
decrease still further to arrest growth, and this decrease was not
achieved using the GAL1 promoter. Different threshold levels
of protein needed for viability and for correct iron regulation were
also noted for Nfs1p, the cysteine desulfurase homolog of mitochondria
(8). The implication is that a very minute amount of Arh1p protein
suffices for viability. Furthermore, the essential functions of Arh1p
are likely to be directly related to its functions in iron metabolism.
What is the function of Arh1p in iron metabolism? Depletion of Arh1p
from cells was correlated with a decline in aconitase activity, without
alteration in aconitase protein level. This combination of findings
suggests that there might be a problem with synthesis, assembly, or
maintenance of the Fe-S cluster, which is required for interaction of
aconitase with its substrate. Alternatively, aconitase in the
Arh1p-depleted cells might be inactive because of improper folding or
lack of critical interactions with other proteins. What is clear is
that the effects on aconitase do not result from iron toxicity, because
Arh1p depleted mitochondria with more than 100-fold differences in iron
content exhibited invariant deficiency of aconitase activity.
A further hint about the function of Arh1p in iron metabolism may be
derived from its sequence. Arh1p is a reductase of the mitochondrial
inner membrane which contains NADPH and FAD binding motifs, enabling
pairs of electrons to be received and then released one at a time to a
substrate (21, 22). A possible substrate of the reductase is Yah1p,
another essential mitochondrial protein (34). Biochemical studies on
adrenodoxin reductase, the human homolog of Arh1p, suggest that
transient interaction with a 2Fe-2S ferredoxin, homologous to Yah1p,
occurs via a patch of negatively charged residues on the ferredoxin
(33). The ferredoxin might undergo a single electron reduction during
such an interaction and the reduced ferredoxin, in turn, might transfer
reducing equivalents to downstream electron acceptors. A role for the
ferredoxin, Yah1p, as the major proximal substrate for the ferredoxin
reductase, Arh1p, is supported by the similar phenotypes resulting from
depletion of either protein. The cellular depletion of Yah1p has been
thoroughly characterized (9) and exhibits strong similarities with
Arh1p depletion phenotypes presented here. However, the ultimate
substrates in this mitochondrial electron transport chain are unknown.
Possibilites are iron or iron chelates, intermediates of Fe-S cluster
assembly, or proteins that require reduction as part of their folding
pathway. Iron may need to be reduced prior to incorporation into Fe-S
clusters. It is not known what form of iron is delivered to the
interior of the mitochondria where Arh1p and/or Yah1p might act on it. The iron might be bound as a ferric chelate analogous to an
iron-siderophore complex, which could then be reduced and mobilized by
the reductase. In fact, the inner mitochondrial reductase resembles the
ferric chelate reductase of the cell surface (Fre1p) in that it also uses FAD and NADPH cofactors (35). Alternatively, the Arh1p reductase
might be required for release of an intermediate Fe-S cluster
transiently assembled on a protein template. Such a mechanism has been
described for release of a cluster formed between NifU dimers (15).
Finally, the reductase might be involved in protein folding or
unfolding related to cluster insertion. Further work is needed to
determine which of these possibilities is correct.
In addition to Aco1p, other Fe-S proteins were affected by the Arh1p
depletion. Deficiency of succinate dehydrogenase and isopropylmalate
dehydratase activities were also observed. This is important because
these proteins are Fe-S proteins that reside in distinct cellular
compartments. Succinate dehydrogenase is a multisubunit enzyme of the
inner membrane and carries heme, FAD, and three Fe-S prosthetic groups
(36, 37). Leu1p is a cytoplasmic protein homologous to aconitase and
thought to contain a 4Fe-4S cluster involved in isomerization of
ispropylmalate as part of the leucine biosynthetic pathway (7, 30). The
fact that both of these proteins were deficient and dependent on
mitochondrial Arh1p is consistent with a role of mitochondria in
synthesis of clusters for varied mitochondrial and extramitochondrial
proteins as has been described (7).
Another facet of the role of Arh1p in iron metabolism relates to iron
trafficking. Arh1p-depleted cells failed to repress cellular uptake
appropriately in response to iron exposure. The excess iron accumulated
within mitochondria. The most straightforward explanation for these
findings is that one or more iron-sulfur protein regulators control
iron trafficking to mitochondria. Regulation might occur at the level
of mitochondrial import, storage, or export. Others have proposed that
the proteins Yfh1p and Atm1p are involved in mitochondrial iron export,
and Arh1p might influence this process (38). However, an iron-sulfur
protein regulator of iron trafficking to mitochondria has not yet been identified.
The effect of Arh1p depletion in producing deficiencies of heme
proteins was likely a toxic effect resulting from iron overload, because the deficiencies correlated with increased iron content of
mitochondria. Cytochrome oxidase subunit 3 was present in the Arh1p-depleted mitochondria with normal iron content but was virtually absent in the iron overloaded mitochondria. Cytochrome c
likewise appeared in iron-depleted mitochondria but not in the
iron-loaded mitochondria. This iron toxic effect on heme proteins could
be the result of repressed heme synthesis or increased heme turnover. Alternatively, the biosynthesis or turnover of the apoproteins prior to
heme insertion or after heme removal might be affected. An effect of
Arh1p depletion on hemoproteins was not anticipated and was not
observed with similar levels of iron overload in the nfs1-14 mutant (not shown). The reason for the difference is not clear.
Detailed biochemical studies on adrenodoxin reductase, the human
homolog of Arh1p, have demonstrated a role in steroid synthesis. In
humans, an electron transport chain exists in mitochondria of
steroidogenic tissues. Reducing equivalents are transferred from NADPH
to the reductase and then to a ferredoxin termed adrenodoxin. The
adrenodoxin in turn donates electrons to P450 cytochromes, which
participate in hydroxylation reactions involved in steroid synthesis
(39). In yeast, homologs of the reductase and ferredoxin exist (Arh1p
and Yah1p, respectively), but cytochrome P450 proteins have not been
identified in mitochondria, and a role for this electron transport
chain in sterol or steroid synthesis has not been demonstrated (22).
Conversely, the adrenodoxin reductase and adrenodoxin are expressed
ubiquitously outside of steroidogenic tissues in humans (39). In light
of our results, it is therefore possible that these human proteins also
function in regulation of iron trafficking and in activation of Fe-S
cluster proteins, perhaps through aiding in synthesis or maintenance of
the clusters.