From the Department of Biochemistry, School of Medicine and
Biomedical Sciences, State University of New York at Buffalo,
Buffalo, New York 14214
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INTRODUCTION |
Research over the past 6 years has identified the primary
components of the high affinity iron uptake system in the yeast Saccharomyces cerevisiae (1-8). One of the striking
features of this pathway is the involvement of a protein, encoded by
FET3, that is homologous to the multicopper oxidases,
ceruloplasmin, and ascorbate oxidase (5, 9). The Fet3 protein is
localized to the yeast plasma membrane (7, 8) in association with the
FTR1 gene product, the putative iron permease (8). Ftr1 contains a REGLE motif that is homologous to a functionally essential feature of mammalian ferritin thought to be involved in the trafficking of Fe(III) from the ferroxidase center to the site of iron core formation in that protein (10, 11). The apparently obligate role of
Fet3 in high affinity iron accumulation in yeast directly links the
iron status of the cell to its copper status: copper-deficient yeast
are also iron-deficient because the apo-Fet3 found in copper-deficient cells is inactive with respect to its essential role in iron uptake (4,
5, 8). This insight has provided significant clues to the molecular
interactions between iron and copper in mammals (12, 13). That is, the
identification of the role that Fet3 appears to play in iron
accumulation in yeast and its homology to ceruloplasmin has provided
support for the hypothesis that the ferroxidase activity exhibited by
ceruloplasmin is integral to this protein's role in mammalian iron
metabolism (8, 9, 12, 13).
The initial step in iron uptake in yeast is the reduction of medium
Fe(III) to Fe(II) by plasma membrane metal reductases (1-3). The major
reductase is encoded by FRE1. This Fe(II) is suggested to be
the substrate for Fet3, which reoxidizes it to Fe(III) coupled to a
four-electron reduction of O2 (7, 9, 13). In this model,
the Fe(III) is taken into the cell via Ftr1 (8, 13). Evidence for this
model is that yeast plasma membranes containing active Fet3 are
reported to consume O2 in an Fe(II)-dependent manner with an Fe(II)/O2 stoichiometry of 4:1, consistent
with Equation 1 (7). This equation describes the ferroxidase reaction previously demonstrated to be catalyzed by ceruloplasmin (14). Fet3
also possesses a ferroxidase activity (9).
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(Eq. 1)
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This model postulates dioxygen as an obligate co-substrate in
iron accumulation in yeast. In this report we test the hypothesis that
the state of cell aerobiosis also regulates the expression of iron
uptake activity in yeast, as for example, it regulates the expression
of respiratory function in this organism (15). Indeed, we show that
anaerobically grown yeast do not express genes that encode proteins
associated with high affinity iron uptake in yeast. The data indicate
that this regulation by culture anaerobiosis involves Aft1, a putative
trans-activator known to regulate the expression of genes associated
with high affinity uptake in an iron-dependent manner (16,
17). These genes are proposed to constitute the iron "regulon" in
yeast (17). Although the mechanism by which Aft1 modulates expression
of these genes is not precisely known, in vivo DNA
footprinting does indicate Aft1 binds to a consensus cis
element in the 5'-untranslated region of these several loci in
iron-depleted cells and not in iron-replete ones. This suggests,
although does not prove, the simple model that iron binding to Aft1 in
iron-replete cells blocks Aft1-DNA binding and resulting
trans-activation (17). We show also that the mRNA
species corresponding to the proteins of the high affinity iron uptake
pathway accumulate within 5 min following reoxygenation of an anaerobic
culture, or addition of a membrane-permeant Fe(II) chelator to
anaerobic cells. This result, together with the fact that this
expression pattern is modulated in part by Aft1 activity, suggests that
a small, labile Fe(II) pool in the cell mediates this regulation also.
These observations indicate that high affinity iron uptake by yeast is
regulated in two ways by dioxygen. Not only is O2 a
substrate essential for this uptake, but its absence or presence in the
growth medium modulates the expression of this uptake activity.
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EXPERIMENTAL PROCEDURES |
Yeast Strains and Culture Conditions--
Wild type strain
DEY1457 (MAT
ade6 can1 his3 leu2 trp1 ura3),
DEY1422 (DEY1457 fet4::LEU2), and DEY1422T2
(DEY1457 fet4::LEU2 GAL4p-FET4) were obtained from
D. Eide (18). The latter strain expresses Fet4 protein from the
GAL promoter when grown in galactose but is
Fet4
when grown in glucose (18). ARY1458
(ftr1
) was constructed in DEY1457 using a fragment of
FTR1 disrupted by TRP1 (8) while ARY1459
(aft1
) was constructed in DEY1457 using a fragment of AFT1 disrupted by TRP1 (16). Cultures of these
strains were grown in YPD (2% yeast extract, 1% peptone, and 2%
glucose) to early log phase (A660 nm = 1.0).
Also, DEY1457(pT14) and ARY1458(pT14) were prepared by transformation
of the two parental strains with plasmid pT14, a
CEN-containing vector (YCp50) that expresses a genetically
dominant, gain-of-function iron-independent allele of Aft1 protein
designated Aft1-1up (16). Experiments using these
transformants were performed in synthetic selective medium
(minus uracil) containing 10 µM each copper
and iron (19). Cultures grown anaerobically under argon or nitrogen
were supplemented with 2 µg/mL ergosterol and 0.2% Tween 80 (20).
Anaerobic cultures for experiments were prepared using stocks that were
themselves grown anaerobically.
Analytical Measurements and Northern
Analysis--
59Fe uptake and Fe(III) reductase
measurements were made at 30 °C as described previously (19) in 0.1 M MES1 at pH 6.0 which contained 20 mM citrate and 2% glucose. When present, ascorbate was added to 1 mM. This buffer contained
<15 nM contaminating iron as determined by flameless
atomic absorption spectrophotometry (fAAS). All uptake velocities were
from the linear portion of the time versus 59Fe
accumulation curve. Uptake was measured at [59Fe] = 0.1 µM. This iron concentration is essentially the
Km value for high affinity iron uptake, which has
been estimated to be 0.15 µM (3). Thus, the uptake rates
reported here represent second-order
Vmax/Km values.
Total RNA isolation and Northern analysis were by standard techniques
(21). All samples were taken from early log phase cultures. fAAS
analyses were performed using a Perkin-Elmer model 1100B with a model
700 graphite furnace on extracts prepared from whole cells digested at
75 °C in 6 M perchloric acid. Accumulation of the
complex between 2,2'-bipyridyl (BIP) and Fe(II) in yeast cells was
established by extracting cells pretreated with BIP (100 µM for 1 h) with benzyl alcohol (22). The
rose-colored BIP3·Fe(II) complex was quantitated in the
benzyl alcohol phase by its absorbance at 520 nm (
= 11,000 M
1 cm
1 as determined from a
standard curve). Statistical analyses were carried out using INSTAT
(GraphPad, San Diego, CA), while 59Fe uptake
versus time data were graphed and fit by linear least squares using Cricket Graph (Cricket Software, Malvern, PA).
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RESULTS |
High Affinity Iron Uptake Activity Is Not Expressed in
Anaerobically Adapted Cells--
The proposed role of dioxygen as
substrate for high affinity iron uptake in yeast suggested that
expression of iron uptake activities might be regulated by the state of
aerobiosis of the culture. Indeed, the transcripts encoding all three
members of the high affinity iron accumulation pathway, Fre1 (metal
reductase), Ftr1, and Fet3 were undetectable in anaerobically grown
cells (Fig. 1, panels A and
B, t = 0). Consequently, these cells
exhibited essentially no Fe(III) reductase activity (Fig.
2, panel A, t = 0) nor 59Fe uptake (Fig. 2, panel B,
t = 0). On the other hand, these activities quickly
recovered when anaerobically grown cultures were resuspended and then
grown in air-saturated medium (Fig. 2). This recovery was preceded by
the accumulation of the iron-associated mRNA species absent in the
initial anaerobic culture (Fig. 1). This recovery of the steady-state
transcript level was rapid, occurring within 15 min, with some recovery
5 min (Fig. 1, panel B). This rapid increase indicates
that the state of aerobiosis as well as the medium iron concentration
(2, 5, 8) modulates the expression of these genes, although it
does not suggest the mechanism of this apparent
transcriptional regulation.

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Fig. 1.
Anaerobic and aerobic growth and expression
of genes of the iron regulon. A, wild type strain DEY1457
was grown in YPD to early log phase under nitrogen. After an initial
cell sample was removed (t = 0), the remaining cells
were resuspended in air-saturated medium and allowed to double twice (4 h total) diluting the culture by one-half with fresh medium after each
doubling. Samples were taken at the times indicated. Total RNA was
prepared from all cell samples, size-fractionated on agarose, and
probed with random-primed DNA fragments taken from the respective
genes. Even sample loading was confirmed by analysis of mRNA due to
ACT1. A composite of two blots is shown; the lower half
shows the 1.7- and 2.1-kb transcripts for FTR1 and
FET3, respectively, while the upper one shows the 3-kb
mRNA for FRE1. B, in a separate experiment, cell samples were taken at short time points following the switch to aerobic growth.
The Northern analysis for FTR1 and FET3
transcripts only is shown.
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Fig. 2.
Anaerobic and aerobic growth and Fe(III)
reductase and 59Fe-uptake activities. Early log phase
cell samples from anaerobically grown cultures of DEY1457 (wild type)
were used for measurement of Fe(III) reduction (panel A,
t = 0) and 59Fe uptake (panel B,
t = 0). The remainder of the culture was resuspended in
air-saturated medium and allowed to double twice while maintaining the
culture density by dilution with fresh medium. Samples were taken for
reductase and uptake measurements at the times indicated. 59Fe accumulation was measured under air in the absence
(open symbols) and presence (filled symbols) of
ascorbate. Ascorbate renders iron uptake reductase-independent. The
t = 0-4 h values are from two experiments with samples
in triplicate; the error bars represent 1 S.E.
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We next assessed the sensitivity of this transcriptional control to
[O2]. To do so, cultures were grown anaerobically and at
t = 0, air-saturated medium was added to achieve a
final concentration of 0.24, 2.4, or 12.2 µM dissolved
O2. Cell samples were taken at intervals for isolation of
total RNA. This RNA was then analyzed by Northern analysis as above.
The results of this analysis are shown in Fig.
3. Addition of O2 to 0.24 µM caused little increase in the FET3 and
FTR1 transcript abundance. In contrast, 2.4 and 12.2 µM O2 caused an increase in these species
within 5 min with a maximum increase at 15 min. At this time, both
transcripts began to decline in the cells exposed to 2.4 µM O2, a decline that was apparent in the
cells exposed to 12.2 µM O2 only after 30 min. Our interpretation of this pattern is that the limiting dioxygen added to these latter cultures was consumed by the cells reducing the
dissolved [O2]
0.24 µM (or that
concentration below which no induction occurred). These results
indicate that the mechanism that triggers the transcriptional response
to O2 functions in the concentration regime of 1 µM dissolved O2. The results also indicate
that both transcripts are relatively unstable since their steady-state
level rapidly declines following the apparent down-regulation of
transcription (see also below).

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Fig. 3.
Dioxygen dependence of expression of
FET3 and FTR1 mRNA. Wild type strain
DEY1457 was grown anaerobically in YPD to early log phase. Separate
cultures were exposed to the concentrations of dissolved O2
as indicated by addition of air-saturated medium. Cell samples were
removed at the times indicated, and total RNA was prepared,
size-fractionated, transferred to nitrocellulose, and probed with
32P-labeled DNA fragments from FET3 and
FTR1. The autoradiograph of the resulting blot is shown.
Even sample loading was confirmed by analysis of mRNA due to
ACT1.
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Regulation of Iron Uptake Activities by Aerobiosis Is Linked to the
Aft1 Protein--
We next addressed the mechanism of the
down-regulation of FTR1 and FET3 expression (the
high affinity iron permease complex) in anaerobic cells. We considered
two explanations for this expression pattern which are not, however,
mutually exclusive. The first was that O2 was a direct
effector of expression of these genes through a pathway that was
independent of Aft1 and perhaps iron as well. The second possibility
was that the state of oxygenation of the cells modulated the
sensitivity of the Aft1-dependent iron-sensing pathway to
medium iron concentration. The latter explanation was reasonable and
had precedent in the case of the transcriptional regulators in
Escherichia coli, Fur, and Fnr (23). Thus, this possibility
was tested first by making the Aft1 trans-activation of iron
uptake activities relatively iron-independent and determining whether
this negated the anaerobic down-regulation. This was accomplished by
using transformed strains DEY1457(pT14) and ARY1458(pT14). The plasmid
pT14 carries an Aft1 gain-of-function allele,
AFT1-1up (16), while, as a control, ARY1458 carries
a deletion in FTR1, encoding the high affinity iron
permease. Because of a Cys to Phe mutation, the DNA binding activity of
Aft1up protein at otherwise iron-repressible genes,
e.g. FTR1 and FET3 is less strongly suppressed by
medium iron (16, 17). That is, in an Aft1up-carrying
strain, the expression of high affinity iron uptake activity is only
weakly repressed by 40 µM ferrous sulfate in comparison
to the 50% repression observed in wild type (16).
Wild type transformant DEY1457(pT14) was grown both aerobically and
anaerobically in synthetic (SC) medium, which is normally repressing
for expression of high affinity iron uptake (17, 18). The two cultures
were washed, maintaining their respective states of aerobiosis, and
were then resuspended in air-saturated buffer to initiate aerobic
uptake. Samples were taken also for preparation of total RNA, which was
subjected to Northern analysis. The data showed (Fig.
4, Northern analysis) that in air-grown wild type strain DEY1457, as expected, the Aft1up protein
supported a 4-5-fold higher level of expression of the "iron
regulon" (17), which resulted in a comparable fold increase in iron
uptake (Fig. 4, uptake values). Consistent with the model being tested,
the Aft1up protein supported significant expression of this
activity in anaerobically grown cells, as well, although the
suppression of the anaerobic phenotype was not complete. That is, these
cells recovered ~20% of the transcript abundance and uptake activity seen in the air-grown culture of the pT14 transformant, while, as shown
above, the untransformed cells exhibited no detectable uptake activity
(Fig. 4). In the same experiment, the ftr1
strain expressing Aft1up, ARY1458(pT14), exhibited essentially no
high affinity 59Fe uptake, demonstrating that the uptake
seen in the wild type transformant grown under nitrogen or air was
Ftr1-dependent (data not shown). These results are
consistent with the suggestion that the lack of expression of high
affinity iron uptake in anaerobic cultures was due at least in part to
the putative iron-dependent suppression of Aft1 protein DNA
binding (and subsequent trans-activation) (16, 17), a degree
of suppression that would not occur in an aerobic culture at a
comparable medium iron concentration (e.g. in YPD which is
partially derepressing; cf. Fig. 7). The conclusion that
Aft1 was involved in this expression pattern was also supported by the
fact that the up-regulation of the iron-associated genes upon
oxygenation of an anaerobic culture was absent in strain ARY1459, a
construct carrying a deletion of AFT1 (data not shown). This
observation was consistent with the fact that these genes are not
expressed in aerobically grown cultures of an
aft1
-containing strain (16, 17).

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Fig. 4.
Anaerobic repression of high affinity uptake
is mediated through the Aft1 protein. Cultures of DEY1457 and
DEY1457(pT14), which plasmid expresses the gain-of-function
Aft1up protein, were grown either anaerobically and
aerobically as indicated in synthetic media (which is repressing for
iron uptake in AFT1 wild type). Samples were then taken for
Northern analysis and aerobic 59Fe uptake measurement as
described in Figs. 1-3. Even sample loading was confirmed by analysis
of mRNA due to ACT1. The 59Fe uptake values
are shown under the respective mRNA samples.
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Regulation of Iron Uptake Activities by Aerobiosis Is Linked to
Cellular Fe(II)--
If this anaerobic repression were due to an
iron-dependent modulation of Aft1 activity, then
manipulation of iron availability by chelation would also be expected
to alter this anaerobic expression pattern. One iron chelator,
bathophenanthroline disulfonate (BPS) is well documented to induce
expression of aerobic iron uptake in yeast by activating the Aft1
protein-dependent expression of FTR1 and
FET3, as well as the other genes in the iron "regulon" (16, 17). This activation is linked to the depletion of cellular iron
caused by an inhibition of iron uptake. That is, addition of BPS to an
59Fe-uptake mixture caused an immediate inhibition of
uptake, which was relieved if the BPS was washed out (Fig.
5, filled circles). Similarly,
addition of BIP to yeast caused a rapid and strong inhibition of
59Fe uptake, also (Fig. 5, filled triangles).
However, BPS, which is anionic, is likely to be impermeant to yeast
cells as it is to mammalian cells (22). BIP, in contrast, is
membrane-permeant (22) and, unlike BPS, does enter yeast cells as
indicated by a distinct rose coloration of the BIP-treated cells due to
the (BIP)3·Fe(II) complex (
max 520 nm,
= 11,000 M
1 cm
1). This complex,
which was retained by the cells for at least 2 h following the
washout of the extracellular BIP (filled triangles), was
extracted from the cells by benzyl alcohol treatment (22) and
quantitated spectrophotometrically (Fig. 5, inset).
BPS-treated yeast cells did not accumulate (BPS)3·Fe(II)
as indicated by the lack of any cell-associated absorbance due to this
Fe(II) complex (
max 533 nm,
= 25,000 M
1 cm
1, data not shown). Note
that the presence of the (BIP)3·Fe(II) complex in the
cells had little effect on resumption of 59Fe uptake after
removal of the BIP remaining in the uptake buffer (filled
triangles).

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Fig. 5.
Inhibition of high affinity aerobic
59Fe-uptake by iron chelators and recovery. Early log
phase cell samples from aerobically grown cultures of DEY1457 (wild
type) were used for measurement of 59Fe uptake at an iron
concentration = 0.2 µM. BPS (filled
circles) or BIP (filled triangles) was added (to 100 µM) to one each of two separate uptake mixtures; the
chelators were then washed out and the cells resuspended in fresh
59Fe without chelator at the times indicated. The control
uptake mixture had no added chelator (open circles). The
data points are the average of triplicate samples and are
representative of two separate experiments. Inset, visible
absorption spectrum of a benzyl alcohol extract (1 ml) of YPD-grown
cells (109 cells) treated with BIP (100 µM)
for 1 h in iron uptake buffer. The cells were then washed free of
metal and chelator prior to extraction of the BIP3·Fe(II)
complex from the cells with benzyl alcohol. The spectrum shown was
obtained versus an extract from a control cell culture that
had not been treated with BIP.
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This difference in permeance of BPS and BIP allowed for a test of the
hypothesis that a pool of intracellular Fe(II) provided the signal for
repression of Aft1-dependent trans-activation of Aft1-target genes, particularly in anaerobic cells. We reasoned that
BIP, being membrane-permeant, had the potential to pass into the cells
and alter the intracellular iron distribution, perhaps causing a
partitioning of iron away from Aft1 protein or from the iron pool
sensed by Aft1, leading to trans-activation of Aft1 protein
target genes. BPS, on the other hand, could not affect this pool in
this manner, since this chelator was membrane-impermeant. The results
were consistent with this reasoning in that while addition of BPS to
anaerobic cultures caused only a limited induction of the various
iron-regulated genes over 30 min (Fig. 6,
panel A, lanes 1, 3, 5, and
7), upon BIP addition the quantity of these transcripts
showed a significant increase within 5 min and a strong increase by 15 min (Fig. 6, panel A, lanes 2, 4,
6, and 8). This result is consistent with the
hypothesis that the regulation of expression of the
iron-dependent target genes in yeast is due at least in
part to an intracellular pool of iron that is accessible to BIP and
which is possibly redox active, as suggested by the effects of
anaerobiosis. The similar effect of aerobiosis on the one hand and
presumed Fe(II) chelation by BIP on the other suggests it is the Fe(II)
in this pool that is directly or indirectly repressing with respect to
Aft1 trans-activation.

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Fig. 6.
Regulation of expression of FTR1
and FET3 by iron chelation and supplementation.
Panel A, anaerobically grown (YPD) cultures of wild type strain
DEY1457 were treated with BIP or BPS (100 µM), and cell
samples were withdrawn at the times indicated. Total RNA was prepared
from all cell samples, size-fractionated on agarose, and probed with
random-primed DNA fragments taken from the respective genes. Even
sample loading was confirmed by analysis of mRNA due to ACT1.
Panel B, aerobically grown (YPD) cultures of wild type strain
DEY1457 pretreated for 1 h with BPS or BIP (100 µM)
were washed and resuspended in 10 µM FeCl3 in YPD (t = 0). Cell samples were withdrawn and analyzed
by Northern blots as in panel A. As shown, transcript
abundance was decreased > 90% within 10 min upon exposure to
iron.
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For this pool to be sensitive to relatively small changes in available
iron concentration and for this sensitivity to be translated into tight
control of the expression of high affinity iron uptake activities, the
pool itself would have to be small, and the level of these activities
would have to be controlled by their rate of expression. This latter
condition requires that the transcripts for FTR1 and
FET3, for example, be short lived, as suggested above (Fig.
3), and that the permease complex be turned over rapidly or its
activity be tightly regulated. The first of these requirements was
tested by adding iron back to cells induced to express high affinity
iron uptake by pretreatment with either BPS or BIP. As shown in Fig. 6
(panel B) FTR1 and FET3 transcripts
were >90% degraded within 10 min of exposure of the induced cells to
10 µM iron. This result shows that these mRNAs do, in
fact, have relatively short half-lives; for comparison, the mean
half-life of mRNA species in yeast is 20 min (24). The fact that
the steady-state level of these transcripts falls to <10% of that in
the initial induced state indicates that, at a minimum, the expression
of these transcripts is repressed within 5 min of cell exposure to Fe(III). Clearly, the amount of increase of cell iron in this period
would be small, suggesting that the iron pool that regulates this
expression would also be small and likely to be located early in the
cellular iron utilization pathway. This pool could be similar in kind
to the transferrin iron-dependent labile iron pool
postulated in mammalian cells that may be involved in ferritin and
transferrin receptor regulation (22, 25). As to second of these
requirements, the fact that O2 is a substrate for
Fet3-dependent high affinity iron uptake ensures the tight
"regulation" of uptake activity per se.
The data above are consistent with the hypothesis that a pool (or
pools) of iron within the cell exists which interacts directly or
indirectly with Aft1. One elaboration of this model is that Aft1
activity is negatively modulated by the Fe(II)/Fe(III) ratio in this
pool, which ratio depends on the state of aerobiosis of the culture.
Another possibility is that the total [Fe(II) + Fe(III)] in the pool
negatively regulates Aft1 and that anaerobiosis redistributes iron into
this pool. Either model requires that anaerobic cells do accumulate
sufficient iron into this pool(s) despite the fact that the high
affinity iron uptake pathway in these cells is repressed. In fact, fAAS
analysis of anaerobically grown, log phase cells showed that they
accumulated equivalent iron from the YPD medium as did aerobically
grown ones, 2.9 versus 3.5 pmol iron/106 cells,
respectively (values ± 0.5; the difference in means was not
significant at p < 0.1). This result indicated that at
the medium iron concentration (9 µM as determined by
fAAS, data not shown) and growth rate in YPD (doubling time, 90 min),
iron uptake in anaerobic cells was sufficient to maintain cellular iron
levels. Furthermore, this demonstrated that there was (sufficient) iron in an anaerobic cell to act as repressor of Aft1 activity upon BIP
treatment or addition of O2. On the other hand, these data did not reveal the redox state of this iron nor its intracellular distribution and thus did not directly test either of the models suggested above.
A Potential Role of the Low Affinity Fe(II) Transporter, Fet4, in
Anaerobic Iron Uptake--
Thus, despite the lack of high affinity
iron uptake, anaerobically adapted cells accumulated equivalent iron
stores in comparison to air grown ones. One possible explanation of
this apparent paradox was that this anaerobic iron accumulation was due
to the low affinity iron transporter, encoded by the FET4
gene (18). FET4 encodes a protein required for a low
affinity iron uptake in S. cerevisiae that is independent of
Fet3 (and Ftr1) and which is specific for Fe(II). The
Km value for iron in Fet4-mediated iron uptake is
~30 µM. An additional distinction between the low and
high affinity iron uptake systems is that FET4 expression is
Aft1-independent. The following results supported this possible
explanation.
First, in contrast to the transcripts encoding the components of the
high affinity iron uptake pathway, the level of FET4 mRNA was equivalent in aerobic and anaerobic cells as indicated by
Northern analysis of the same RNA preparations used above (data not
shown). Thus, anaerobically grown cells did have the capacity to
accumulate iron via the Fet4 protein in that these cells did express
this activity.
Second, if this uptake were in fact due to Fet4, it should be
inhibitable by iron chelation, i.e. by BPS (18). In the
experiments above, the effects of BPS and BIP were followed for short
times after chelator addition. If accumulated iron stores were involved in the anaerobic repression, growing the cells in BPS should result eventually in iron depletion and expression of high affinity iron uptake even in anaerobic cultures. This proved to be the case. Growth
of cells in the presence of BPS did lead to expression of this activity
as demonstrated by Northern blot analysis and iron uptake measurement
(Fig. 7). As the results show, there was an ~90% recovery of both FTR1 mRNA and high affinity
iron uptake in the BPS-treated anaerobic culture in comparison to the
air-grown one. That BPS treatment did lead to cell depletion of iron
was confirmed by fAAS analysis of these cells; growth in BPS reduced the iron content in the anaerobic culture by 70% (from 2.9 to 0.9 ± 0.4 pmol iron/106 cells). The percentage inhibition by
BPS of iron accumulation in an aerobic culture was the same; cell iron
was reduced from 3.5 to 1.1 ± 0.5 pmol iron/106
cells.

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Fig. 7.
Cell iron depletion does lead to anaerobic
expression of high affinity iron uptake. Cultures of DEY1457 (wild
type) were grown either anaerobically and aerobically in YPD in the absence or presence of BPS (100 µM) as indicated. Samples
were then taken for Northern analysis and aerobic 59Fe
uptake measurement. Even sample loading was confirmed by analysis of
mRNA due to ACT1. The 59Fe uptake values are
shown under the respective mRNA samples.
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Third, previous data had indicated that Fet4 protein levels did
modulate the activity of the high affinity uptake system (18). We
wished to build upon these observations with the express purpose of
demonstrating a role for Fet4-mediated iron uptake more directly in the
regulation of high affinity uptake. Thus, DEY1457 and mutant strains
DEY1422 (fet4
) and DEY1422T2 were evaluated for this latter activity. DEY1422T2 is fet4
but in addition
carries an integrated copy of a GAL4
promoter:FET4 fusion, putting FET4 expression under control of medium galactose (18). The hypothesis that Fet4-dependent iron uptake modulated the expression of
Ftr1/Fet3-dependent uptake in some fashion was indicated by
the fact that a fet4
strain did exhibit an 3-fold
increase in Ftr1-dependent uptake in comparison to wild
type (18). In an extension of this previous observation, we reasoned
that if the proposed pool of Fe(II) that regulated expression of this
activity was at least partially dependent on Fet4, high affinity iron
uptake by DEY1422T2 would be galactose-dependent, while in
DEY1422, the expression of FTR1, for example, would be less sensitive to medium iron concentration than in wild type, since this strain lacked a functional Fet4 protein. These predictions were confirmed. Thus, DEY1457, DEY1422, and DEY1422T2 were grown aerobically on both glucose and galactose and then assayed for high
affinity iron uptake. As the data in Fig.
8 (panel A) show, on glucose
(open bars), when both mutant strains are effectively Fet4
, they exhibited a 2-fold greater high affinity iron
uptake in comparison to wild type. On the other hand, while
galactose-grown DEY1422 also exhibited this increased high affinity
iron uptake in comparison to wild type, this uptake (hexagonal
bars) was strongly repressed in DEY1422T2 in which Fet4 protein
(and low affinity iron uptake) was overexpressed (18). These uptake
results, which confirm what has been observed (18), were consistent
with the hypothesis that the regulation of Aft1 function is linked at
least in part to low affinity iron uptake mediated by Fet4.

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Fig. 8.
Fet4 modulates expression of high affinity
iron uptake. In panel A, strains DEY1457 (wild type)
and DEY1422 and 1422T (both fet4 ) were grown aerobically
in YP medium (30% derepressing, Fig. 7) with glucose (open
bars) or galactose (hexagonal bars) as a carbon source.
DEY1422T2 carries an integrated copy of FET4 under control
of the GAL4 promoter (18). Log phase cultures were prepared
for 59Fe uptake as described. In panel B,
DEY1457 and DEY1422 (fet4 ) were grown anaerobically
(shaded bar) in YPD and then measured for 59Fe
uptake under air as described. Values for aerobically grown cultures
are shown as control (open bars).
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A prediction that follows from this model is that the suppression of
the expression of high affinity iron uptake in anaerobically grown
cells would be less strong in a fet4
strain in comparison to wild type. To test this prediction, strains DEY1457 (wild type) and
DEY1422 (fet4
) were grown under air and nitrogen and then assayed (under air) for high affinity iron uptake as above. The data
showed (Fig. 8, panel B) that in anaerobiosis (shaded
bar) lack of low affinity iron uptake (in strain DEY1422) led to
recovery of 40% of the high affinity iron uptake seen in the aerobic
culture of wild type strain DEY1459 (open bars). Although
some repression of uptake activity in DEY1422 persisted under
anaerobiosis, the significant recovery observed was consistent with the
proposal that iron entering the cell via Fet4, an uptake activity that is apparently independent of the state of aerobiosis (or medium iron
concentration), is linked to a cellular pool of iron that modulates the
Aft1-dependent expression of the high affinity iron regulon.
 |
DISCUSSION |
We initiated this study of the expression of high affinity iron
accumulation in S. cerevisiae to assess how the state of
culture aerobiosis might modulate this activity. We proposed that,
since dioxygen was an obligate substrate for this uptake via the
ferroxidase reaction catalyzed by Fet3 (6, 7, 9, 13), its level might
also regulate the level of this activity in the cell. The data clearly
show that the expression of FTR1 and FET3 (and
FRE1) was down-regulated in anaerobically grown cultures.
The expression of these genes was known to be regulated by iron, a
regulation that was modulated by the putative
trans-activator, Aft1; neither of these loci is expressed in
an aft1
-containing strain (16, 17). The feedback
inhibition due to iron appears to arise from the fact that cellular
iron inhibits the binding of Aft1 to a specific target sequence in the
5'-noncoding region of the several genes that are iron-regulated
through Aft1 (17). The simplest model, therefore, is that iron binding
to Aft1 inhibits or alters its binding to these sequences. In this
simplest model, Aft1 is itself also a trans-activator when
bound to these cis elements whose consensus sequence is
PyPuCACCCPu (17). Only part of this model has been experimentally
confirmed; however, while Aft1-DNA binding in an
iron-dependent manner has been demonstrated (17), neither
the trans-activation by Aft1 nor the mechanism by which the
DNA binding is modulated by iron has been characterized.
One model that links the iron-dependent expression of the
genes encoding the high affinity uptake activity via Aft1 to the affect
of anaerobiosis is that the state of aerobiosis alters the sensing of
the cellular iron level or distribution, or redox state as has been
suggested in the case of Fur (23, 26), an iron-regulated repressor
protein in E. coli (23, 26, 27). Fnr, a positive regulator
of many of the same bacterial genes, is postulated also to be an
iron-binding protein whose activity is redox sensitive (23, 28).
However, the mechanism of action of Aft1 is likely to be the reverse of
the postulated mechanism for Fur, for example. Fur is thought to bind
Fe(II) and in this metal-bound form binds to a "Fur" box in target
genes as a repressor. Raising the redox potential of the cell
(making it more oxidizing, presumably displacing the
Fe(II)/Fe(III) redox equilibrium toward Fe(III)) or chelating the iron
leads to iron dissociation from Fur, relieving the DNA binding and the
repression of transcription (23, 26).
Nonetheless, this model of Fur action appears highly relevant to our
finding that anaerobically grown yeast did not express the components
of the high affinity iron uptake pathway, the expression of which is
known to be iron- and Aft1-dependent. Our data showed that
the regulation of expression of FTR1 and FET3 by
the state of aerobiosis was similarly Aft1-dependent and
iron-mediated. This fact was shown most dramatically by two results.
First, expression of the gain-of-function AFT1-1up
allele effectively although not completely reversed the down-regulation seen in an anaerobic culture of the AFT1 wild type strain.
The AFT1-1up allele is known to largely (although
not completely) uncouple expression of these genes from the cellular
iron level (16, 17). Although the mechanism of this uncoupling is not
known, a reasonable hypothesis is that the mutation in Aft1 associated with the AFT1-1up allele, a Cys to Phe substitution,
inhibits or reduces the binding of iron to Aft1 protein. Consequently,
the protein's activity as a trans factor is rendered
relatively insensitive to cellular iron levels, since the
Aft1up protein binds constitutively to the DNA at medium
iron concentration
100 µM (16, 17). This is similar to
a Fur protein that is mutant at the potential Fe(II) ligands
Cys92 and Cys95 and is inactive with respect to
DNA binding and transcriptional repression, presumably because it
exists only in an apo-form (27). On the other hand, evidence of Aft1
protein phosphorylation has been shown, although not with regard to
iron-dependent gene regulation (29). That is, the link
between cellular iron levels and Aft1 trans-activity might
well be more complex than a simple metal-protein binding
equilibrium, for example, and remains to be experimentally delineated.
Second, expression of FTR1 and FET3 increased
rapidly in anaerobic cells following chelation of intracellular iron
specifically. That is, our data showed that in anaerobic cells this
rapid chelator effect was seen only with a chelator that was
membrane-permeant. Thus, BIP treatment caused a strong induction of
expression of FTR1 and FET3 in anaerobically
grown cells, while treatment with BPS, which can only work
extracellularly, did not. As shown here, chelation of extracellular
iron in aerobic cultures by either reagent blocked high affinity
uptake, which effect is known to up-regulate these loci (16). However,
chelation of extracellular iron would have a limited impact on iron
accumulation within the 5-30-min time frame that with BIP treatment
led to induction of expression of the iron-dependent genes
(Fig. 6, panel A); over short time periods, intracellular
iron levels in such cultures would be relatively insensitive to
addition of BPS, for example. At long treatment times, however, BPS did
cause a level of cellular iron limitation that led to expression of the
high affinity iron uptake, even in anaerobic cultures (Fig. 7). This
result is consistent with the demonstrated inhibition of iron uptake
due to BPS.
Bipyridyl is a chelating agent that can diffuse into the cell and
thereby have the potential to chelate intracellular "labile" iron.
What is significant about BIP, too, is that it is an Fe(II) chelator
(22). Therefore, strong and rapid up-regulation of the iron- and
Aft1-dependent loci in anaerobic cells treated with BIP (in
contrast to those treated with BPS) suggests that these cells contain a
chelatable pool of Fe(II) that is directly or indirectly involved in
the equilibria that regulate Aft1 protein-DNA binding or
trans- activation (17). This pool would be similar to the
chelatable or labile iron pool postulated in mammalian cells that plays
a significant role in the regulation of expression of ferritin and
transferrin receptor biosynthesis (22, 25) or the iron pool in E. coli that regulates Fur activity (26). Our results do not directly
show how large this pool in yeast is; however, based on the 520-nm
absorbance of the benzyl alcohol cell extract and assuming a cell
volume of 60 µm3 (30), cellular
[BIP3·Fe(II)] ~ 10 µM can be
calculated, or ~ 0.5 pmol of chelatable Fe(II)/106
cells. This value compares to the steady-state level of iron in yeast
determined in this work, 3 pmol/106 cells, suggesting that
~15% of the total cell iron is chelatable by BIP. This result also
can be compared with the chelatable iron pool measured in rat
hepatocytes of 18% of the total transferrin iron accumulated by the
cells in 30 min (22). On the other hand, these values for "labile"
Fe(II) are to be taken with caution, since they are determined by use
of a chelator that very likely displaces some equilibrium distribution
of iron in the cell. This could involve both the physical
redistribution of Fe(II) as well as the distribution between Fe(II) and
Fe(III).
Although the data consistently support a model of regulation of
FTR1 and FET3 in anaerobiosis via the iron- and
Aft1-dependent signal transduction pathway, this support
is, in general, qualitative not quantitative. For example, the
Aft1up protein supported only 20% recovery of expression
of these genes in anaerobic cells (Fig. 4). Similarly, in the test of
the hypothesis that Fet4 contributed to the iron pool sensed by Aft1 in
anaerobic cells, the data showed that deletion of FET4
resulted in the recovery of only 40% of the expression of high
affinity uptake seen in an aerobic culture (Fig. 8, panel
B). While it is perhaps not surprising that the repression of
expression of these genes observed in anaerobic cells is not completely
suppressed by any one of these genetic manipulations, the results do
leave open the possibility that an additional pathway contributes to
the anaerobic repression. Certainly, the expression pattern could
implicate a repression mechanism that functions in anaerobic cells and,
in one model, is relieved by oxygen either directly or indirectly.
However, no such anaerobic repressor has been identified in yeast. In
comparison, the Rox1 protein is a well characterized repressor of
otherwise anaerobically expressed genes; the expression of
Rox1, which is a short lived protein, is positively regulated by heme
and oxygen, so that Rox1 is produced in aerobic cells but absent in
anaerobic ones (31). This pattern of Rox1 level (repressor activity)
leads to the opposite expression pattern than that seen for the genes encoding high affinity iron uptake with respect to culture aerobiosis. Nonetheless, an anaerobic repressor function that contributes to the
modulation of the expression of these loci cannot be ruled out on the
basis of the results presented here.
On the other hand, the data do indicate that the level of high affinity
iron uptake activity in yeast is controlled by the rate of expression
of this activity at least at the level of transcription. This is shown
here by the fact that the mRNAs encoding the high affinity iron
uptake proteins are relatively short lived, thus their steady-state
level will be determined by their synthesis. This transcriptional
activation appears very sensitive to the change in intracellular iron
concentration (or the iron concentration in a specific pool), since it
is strongly inhibited within 5 min following cell exposure to medium
iron. This result is consistent with the notion that the cellular iron
pool controlling this expression is small and labile, and may contain
Fe(II), as indicated by the effects of BIP.
This latter inference raised an apparent dichotomy between the models
presented here and elsewhere (8, 13), namely that Fe(III), following
its generation by the ferroxidase reaction catalyzed by Fet3, is taken
into the cell by Ftr1 protein, but Fe(II) is proposed as the species
that is sensed by Aft1. It is also likely that the major storage form
of iron in yeast is Fe(III), probably as a polyphosphate in the yeast
vacuole (32). The question was, then, where does the Fe(II) in the
putative regulatory pool come from? One possibility is that all of the
Fe(III) brought into the cell via Ftr1 is subsequently reduced, with
this Fe(II) being sensed by Aft1. In this model, iron targeted to the
vacuole is reoxidized, comparable to the Fe(II)/Fe(III) conversion in the formation of the iron core in ferritin (10, 11). None of our data
excludes this mechanism as contributing to Aft1-dependent gene regulation. Another possibility was that the Fet4 iron transporter was a source of the regulatory Fe(II); Fet4 is specific for ferrous iron (18). Our data do support a role for Fet4 iron as part of this
regulatory mechanism, although they do not prove it.
This proposition is kinetically appealing. The Km
for Fe(II) in Fet4-dependent uptake is 30 µM
(18), so that in the iron concentration regime that regulates
expression of FTR1, FET3, and FRE1
(the ferrireductase), 1-10 µM (1, 18), the rate of
Fe(II) uptake by Fet4 is strictly first-order in iron concentration.
This kinetic situation predicts that the iron concentration in the
intracellular pool(s) to which this Fe(II) is targeted is directly
proportional to the iron concentration in the medium. Furthermore, the
expression of Fet4 uptake is independent of medium iron concentration
(18), that is, Fet4-dependent uptake is constitutive. This
expression pattern is consistent with what one would predict for a
component of a signal transduction pathway. Last,
Ftr1-dependent, high affinity iron uptake is induced
2-3-fold in a fet4
strain in comparison to wild type
(18) (Fig. 8). This fact and the new data on anaerobically grown
cultures that we show here (Fig. 8) are all consistent with a model in
which Fet4 iron is likely to be an important source of the
intracellular Fe(II) pool that regulates expression of high affinity
uptake activity via Aft1.
This discussion of the iron uptake capacity of Fet4 relates also to an
apparently paradoxical result, namely, the equivalent iron accumulation
in wild type cells grown aerobically or anaerobically, 3.5 versus 2.9 pmol of iron/106 cells, respectively,
despite the lack of high affinity iron uptake in the latter condition.
This result indicates that, in a YPD-grown anaerobic culture, Fet4 iron
accumulation alone is equivalent to the iron accumulation under air due
to Fet3 and Fet4. This result is actually quite informative about the
interplay of gene expression regulation and modulation of enzyme
activity by substrate concentration. First, Fet4-mediated iron uptake
is likely to be 2-3-fold greater from anaerobic in comparison to
aerobic medium. Fe(II) will predominate in the former condition while
Fe(III) will predominate in the latter one. Fet4 iron uptake is
strongly dependent on the redox state of the exogenous metal as is
indicated by the nearly 3-fold increase in Fet4-dependent
uptake upon prereducing the metal with ascorbate (18). Second, while
aerobic cultures do express high affinity iron uptake activity, in YPD
this uptake is about one-third of maximum, as, for example, is
indicated by the data in Fig. 7. These data showed that BPS treatment
(inducing conditions) resulted in an increase in the rate of high
affinity uptake from 3.9 to 11.4 pmol/106 cells/min. Thus,
although when fully expressed, Fet3 and Fet4 exhibit similar
Vmax values (18), in YPD Fet4, which is not regulated by medium iron, is fully expressed, while Fet3 is not. In
other words, this difference in expression indicates that the loss of Fet3 uptake in anaerobiosis would not have the
dramatic impact on iron accumulation that might be considered likely at first glance. Last is the fractional saturation of the two systems by
iron at the iron concentration present in YPD, 0.5 mg/liter or 9 µM, as determined by fAAS (data not shown). Assuming that some fraction of this iron in a complex medium like YPD was not biologically available for uptake, for example, 50% of the total, even
the available "free" iron concentration = 4.5 µM
would be 30 times the Km value for Fet3 uptake (0.15 µM) (3) and 0.15 × Km for Fet4
uptake (Km = 30 µM) (18). Given the
relative level of expression of Fet3 and Fet4 activity in YPD (above),
at this iron concentration, aerobic Fet3 uptake (97% saturation of
30% of expressible transport activity) would be ~3 times Fet4 uptake
(13% saturation of 100% of expressible transport activity). In going
to the anaerobic state, the loss of the uptake contribution due to Fet3
would be compensated for by the anticipated 2-3-fold increase in Fet4
uptake due to the predominance of Fe(II) in the medium under this
condition.
This analysis does not suggest the implication that Fet3 is
redundant to Fet4. Aerobic cultures growing in iron-limited medium become iron-starved if they lack Fet3-dependent high
affinity iron uptake (5). That is, Fet3 plays an essential role under conditions of iron deprivation that cannot be rescued by Fet4 uptake;
it is simply too inefficient at medium iron concentration <1
µM, particularly in aerobic cultures in which condition
iron is essentially present only as Fe(III). What the results presented do suggest is that, with a two-tiered mechanism of regulation of high
affinity iron uptake, yeast can very tightly control the rate of iron uptake, particularly when going from an
anaerobic to an aerobic growth condition. That is, in the yeast system
dioxygen, in effect, regulates high affinity iron uptake in two ways.
First, it appears to modulate the expression of the genes encoding the components of this pathway, perhaps by altering the sensitivity of the
cellular iron-sensing pathway that functions in this gene regulation
via the transcription factor, Aft1. Second, as substrate for Fet3, it
is an obligate co-substrate for high affinity iron uptake. The first of
these modulation effects is somewhat longer term, while the latter is
immediate to the process of iron accumulation by the cell.
That this two-tiered regulatory mechanism presumably has selective
advantage indicates that repressing the expression of high affinity
iron uptake in anaerobiosis has selective advantage. What this
advantage is to a facultative anaerobe like yeast remains to be
elucidated, but that it is linked to environmental dioxygen level
suggests that it may well relate to suppressing
metal-dependent oxidative stress (33). It may appear
paradoxical that high affinity iron uptake by an anaerobic cell has
pro-oxidant character; iron accumulation by aerobic cells would appear
to have more potential for cytotoxicity. However, a facultative
anaerobe like yeast is not unlike an obligately aerobic tissue that has
become ischemic. That such tissues are at risk for significant cell
damage upon reoxygenation and that iron, as Fe(II), released during the
ischemia appears to be a significant contributor to this reperfusion
damage are both well documented phenomena. For example,
desferrioxamine-chelatable iron is released by isolated, ischemic
lungs, which exhibited several indices of oxidative damage following
reperfusion (34). Similar damage was shown in brain tissue following
hypoxia-ischemia reperfusion; a marked intracellular translocation of
iron during the ischemia could be linked to the oxidative damage caused
by the subsequent reoxygenation (35). A delocalization of intracellular iron occurs in ischemia in skeletal muscle, also; furthermore, the
oxidative reperfusion injury sustained by this tissue is attenuated by
the application of iron chelators in the reperfusate (36). As noted
above, in yeast the data suggest that, in the transition from anaerobic
to aerobic growth, suppression of an influx of iron via Fet3 is
important for cell homeostasis. In the yeast system, this type of
prediction can be tested readily. Indeed, understanding the
corresponding, albeit implied toxic relationship between dioxygen and
iron in yeast indicated by the results reported here may well clarify
the role of intracellular, labile iron in a variety of human
pathologies (37).
We thank Drs. Andrew Dancis and David Eide
for yeast strains, and Dr. Dancis for DNA reagents for constructing
deletions in FTR1 and AFT1 and for the
AFT1-1up expression plasmid. We thank Drs. Edward
Niles, Mark O'Brian, and Cecile Pickart for critical review of this
manuscript and helpful comments.