From the Liver Diseases Section, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892-1800, the
§ Department of Chemistry and Biochemistry, University of
Oklahoma, Norman, Oklahoma 73019, and the
Department of
Chemistry, University of Wisconsin, Oshkosh, Wisconsin 54901
Received for publication, November 6, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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Saccharomyces cerevisiae takes up
siderophore-bound iron through two distinct systems, one that requires
siderophore transporters of the ARN family and one that requires the
high affinity ferrous iron transporter on the plasma membrane. Uptake
through the plasma membrane ferrous iron transporter requires that the
iron first must dissociate from the siderophore and undergo reduction
to the ferrous form. FRE1 and FRE2 encode cell
surface metalloreductases that are required for reduction and uptake of
free ferric iron. The yeast genome contains five additional
FRE1 and FRE2 homologues, four of which are
regulated by iron and the major iron-dependent transcription factor, Aft1p, but whose function remains unknown. Fre3p
was required for the reduction and uptake of ferrioxamine B-iron and
for growth on ferrioxamine B, ferrichrome, triacetylfusarinine C, and
rhodotorulic acid in the absence of Fre1p and Fre2p. By indirect
immunofluorescence, Fre3p was expressed on the plasma membrane in a
pattern similar to that of Fet3p, a component of the high affinity
ferrous transporter. Enterobactin, a catecholate siderophore, was not a
substrate for Fre3p, and reductive uptake required either Fre1p or
Fre2p. Fre4p could facilitate utilization of rhodotorulic acid-iron
when the siderophore was present in higher concentrations. We propose
that Fre3p and Fre4p are siderophore-iron reductases and that the
apparent redundancy of the FRE genes confers the capacity
to utilize iron from a variety of siderophore sources.
Virtually every organism on earth requires iron as an essential
nutrient. Although iron is the second most abundant metal in the crust
of the earth, the bioavailability of iron can be extremely low. This
poor bioavailability occurs because iron is rapidly oxidized in an
aerobic environment to the ferric form (Fe(III)),1 which is poorly
soluble in water and forms precipitates of oxyhydroxides. Microorganisms have the capacity to scavenge iron from insoluble precipitates by secreting and taking up siderophores, low molecular weight compounds that bind to Fe(III) with very high affinity and
specificity. Siderophores are synthesized and secreted in the iron-free
form, which then binds and solubilizes Fe(III) in the extracellular
environment. The Fe(III)-siderophore complex is then recognized and
selectively taken up by specific transport mechanisms. Many
microorganisms synthesize one or a few types of siderophores, yet have
the capacity to take up iron from a variety of siderophores secreted by
other species of bacteria and fungi (1). Budding and fission yeast
appear to be an exception; they neither synthesize nor secrete these
compounds (2, 3). Saccharomyces cerevisiae can, however,
recognize and take up iron from a variety of structurally distinct
siderophores (4-10).
S. cerevisiae has two genetically separable systems for the
uptake of siderophore-bound iron. One system depends on a family of
homologous transporters of the major facilitator superfamily that is
expressed as part of the AFT1 regulon and are termed
ARN1, ARN2 (also TAF1), ARN3 (also
SIT1), and ARN4 (also ENB1) (6-11). These transporters are expressed in intracellular vesicles. The individual ARN transporters exhibit specificity for
different siderophores of the hydroxamate and catecholate classes;
however, some siderophores, such as rhodotorulic acid, are not
substrates of the ARN transporters (9). A second system of
uptake for siderophore-bound iron depends on the high affinity ferrous
iron (Fe(II)) transport complex, which is encoded by FET3
and FTR1 and is located on the plasma membrane (12-15). A
low affinity Fe(II) transporter encoded by FET4 is also
expressed on the plasma membrane (16). For the siderophore-bound
Fe(III) to become a substrate for the Fe(II) transporter, the iron must
be both reduced and dissociated from the siderophore. This is
accomplished in a single step by the activity of plasma membrane
reductase systems, which contain flavocytochromes and have the capacity
to reduce siderophore-bound iron (4, 5, 17). FRE1 and
FRE2 encode plasma membrane metalloreductases that can
reduce oxidized forms of both iron and copper (18-23). Strains deleted
for FRE1 exhibit only 10% of the Fe(III)-citrate reductase
activity that is inducible in wild-type strains. Deletion of both
FRE1 and FRE2 results in cells that are
completely lacking Fe(III)-citrate reductase activity and fail to grow
on iron-poor media. The completed sequence of the S. cerevisiae genome revealed the presence of five additional genes
with striking similarity to FRE1 and especially to
FRE2. Four of these (FRE3, FRE4,
FRE5, and FRE6) are greater than 35% identical
to FRE2 and are regulated at the transcriptional level by
Aft1p (24). The fifth homologue (FRE7) is regulated by
exogenous copper ions through the Mac1p transcription factor. The
functions of these new FRE family members have not been identified.
We have investigated the role of the FRE family of genes in
the uptake of siderophore-bound iron. Although the FRE genes
appeared to have no role in the ARN-dependent
uptake of siderophores, they were required for the uptake of
siderophore-bound iron through the high affinity Fe(II) transport
system. Although FRE1 and FRE2 encoded the
majority of siderophore reductase activity, Fre3p could specifically
facilitate reduction and uptake of iron bound to the trihydroxamate
siderophores ferrioxamine B (FOB), ferrichrome (FC), and
triacetylfusarinine C (TAFC) and to the dihydroxamate rhodotorulic acid
(RA). Fre3p was expressed on the plasma membrane in a pattern
consistent with its role in iron uptake through the plasma membrane
Fe(II) transport system. Uptake of iron bound to the catecholate
siderophore enterobactin (ENT) also occurred through the Fe(II)
transport system and required either Fre1p or Fre2p. Expression of
Fre4p was sufficient to facilitate the utilization of RA-bound iron
when the siderophore was present in higher concentration.
Yeast Strains, Plasmids, and Media--
All strains were
constructed in YPH499 MATa ura3-52 lys2-801(amber)
ade2-101(ochre) trp1- Siderophores--
Iron-free forms of FC, RA, and the mesylate
salt of FOB (Desferal) were purchased from Sigma. Ferric and desferric
forms of TAFC and ENT were prepared as described (9, 29). Ferric RA, FC, and FOB were prepared by incubating the desferri-siderophores with
an equimolar amount of FeCl3 in 50 mM sodium
citrate, pH 6.5, and 5% glucose.
Surface Reductase Assay--
Cells were grown to mid-log phase
in YPD medium or defined iron media containing 10 µM
ferrous ammonium sulfate, and the assay was performed as described (18)
with the following modifications. Ferrozine was substituted for
bathophenanthroline disulfonate (BPS) as the Fe(II) acceptor, and 1 mM Fe(III)-FC, -FOB, -TAFC, and -RA were the reductase
substrates. Absorbance of the cell-free assay mixture was measured at
562 nm.
Plate Assay and Uptake Assay--
For the plate assay, modified
synthetic complete media were used in which copper and iron were
omitted, and 1 µM copper sulfate and 100 µM
BPS were added. Trace amounts of iron are supplied by agar (Difco).
Plate assays for low concentrations of FC, FOB, TAFC, ENT, and RA were
performed as described (10) using 10 µM desferri-FC, -FOB
and -TAFC, 5 and 10 µM ferric-ENT, and 15 µM ferric-RA (a concentration with iron-chelating
capacity equivalent to 10 µM FC, FOB, TAFC, and ENT).
Plate assays for high concentrations of FOB and RA were performed using
100 µM of the desferri-siderophore. Because of ligand
exchange between the BPS and the siderophores, the exact concentration
of the ferric-siderophore complex in the plate assay is not known.
Uptake assays were performed as described (30) in Multiscreen
Filtration plates (Millipore) using 1 µM each of
55Fe(III) and desferri-FOB and-ENT.
Immunofluorescence--
Strains YPH499 pFRE3-HA and YPH499
FET3-HA FTR1-Myc were grown to mid-log phase in defined iron media
containing 5 µM ferrous ammonium sulfate and 1 mM ferrozine to induce the expression of Fre3p. Cells were
washed and prepared for immunofluorescence microscopy as described
(15). Primary antibody was affinity-purified HA.11 (Babco) at 1:300,
and secondary antibody was Cy3-conjugated polyclonal anti-mouse IgG
from donkey (Jackson ImmunoResearch) at 1:500. Cells were visualized
with a Zeiss 63X/1.4NA objective. Images were acquired with a cooled
CCD camera (Cooke) using IP Labs software (Scanalytics).
Reduction of Ferric Siderophores by FRE Reductases--
FOB-bound
iron can be taken up at the plasma membrane by a reductive mechanism
that requires heme (5). The metalloreductase encoded by FRE1
is a plasma membrane heme protein of the cytochrome b-type,
and strains deleted for FRE1 exhibit low Fe(III)-citrate reductase activity (17-19). We tested whether other
ferric-siderophores of the hydroxamate class were substrates for cell
surface reductases (Fig. 1). In addition
to FOB, both the trihydroxamates FC and TAFC and the dihydroxamate RA
were substrates for plasma membrane reductases. RA-bound Fe(III), which
is only taken up through the Fe(II) transport system, displayed the
highest rate of reduction, whereas Fe(III) bound to FC, which is taken
up by both the Fe(II) transport system and the Arn1p and Arn3p
transporters, displayed the lowest rate of reduction. FRE1
and FRE2 encoded 90-98% of the siderophore reductase
activity, but there were measurable differences in the siderophore
reductase activities of a fre1 The Role of FRE3 in Ferrioxamine B-mediated Iron Uptake and
Growth--
FOB-bound iron can be transported into the cell by both an
Arn3p-dependent system and a Fet3p-dependent
system that also involves an Fe(III) to Fe(II) reduction step. We
tested the role of FRE1, FRE2, and
FRE3 in the uptake of FOB-iron in strains both expressing Arn3p and deleted for Arn3p (Fig.
2A). When cells were grown in rich media, deletion of FRE3 alone resulted in an increased
uptake of FOB-iron when compared with the congenic parent strain. This effect was consistently reproducible and suggested that deletion of
FRE3 resulted in changes in the intracellular iron pool.
Deletion of FRE1 and FRE2 resulted in dramatic
decreases in FOB-iron uptake, both in an ARN+ and an
arn3
To clarify further the role of FRE1, FRE2, and
FRE3 in the utilization of FOB-iron, we examined the growth
of FRE-deleted strains on iron-limited media containing FOB
as an iron source (Fig. 2C). We used strains in which
ARN-dependent FOB uptake was inactivated by
deletion of ARN3; therefore, iron uptake was dependent on
Fet3p and the high affinity Fe(II) uptake system. An arn3 The Role of Fre3p in the Uptake of Ferrichrome, Triacetylfusarinine
C, and Rhodotorulic Acid--
Because Fre3p could facilitate the
reductive uptake of FOB-iron, we questioned whether structurally
related siderophores were also substrates for Fre3p. The iron-binding
sites of FC and TAFC are similar in structure to that of FOB in
that they each consist of three bidentate hydroxamate ligands that bind
Fe(III) in a six-coordinate complex. We examined the capacity of the
FRE homologues to facilitate growth when FC and TAFC in low
concentration (10 µM) were provided as iron sources (Fig.
3, A and B). Again,
these experiments were performed using strains in which the
ARN-dependent siderophore uptake systems were
inactivated by deletion of ARN1 and ARN3 to test
FC and by deletion of ARN2 to test TAFC. Deletion of either
FRE3 alone or FRE1 and FRE2 together
had no effect on FC-mediated growth (Fig. 3A). Deletion of
FRE1, FRE2, and FRE3 in combination, however,
resulted in a failure to grow on FC, indicating that Fre3p can
facilitate reductive iron uptake from both FC and FOB. As was the case
for FOB, deletion of FRE1, -2, and -3 in an ARN+
background did not impede FC-mediated growth, further supporting the
role of the FRE genes in siderophore-iron uptake through the
Fe(II) transport system but not through the ARN system.
Similar results were obtained when a strain deleted for FRE1,
-2, and -3 was tested for TAFC-mediated growth (Fig. 3B), indicating that Fre3p can facilitate reductive iron
uptake from each of the three trihydroxamate siderophores tested.
RA-mediated iron uptake in S. cerevisiae significantly
differs from FOB-, FC-, and TAFC-mediated uptake in that RA is not a
substrate for the ARN transporters and RA-iron uptake occurs exclusively through the Fe(II) transport system (9). Also, RA is a
dihydroxamate siderophore that coordinates Fe(III) in a
RA3Fe2 complex (31). Despite these differences,
Fre3p also facilitated RA-iron-mediated growth (Fig. 3C), as
the fre1 Localization of Fre3p to the Plasma Membrane--
Both components
of the high affinity Fe(II) transport system, Fet3p and Ftr1p, are
expressed on the plasma membrane (10, 13-15). In contrast, the Arn1p,
Arn3p, and Arn4p transporters are largely confined to intracellular
vesicles that comigrate on density gradients with Pep12p, a protein of
the late endosome (9, 10) (data not shown). To determine the cellular
localization of Fre3p, we constructed a strain in which the chromosomal
copy of FRE3 carries a triple copy of the HA epitope at the
carboxyl terminus. We confirmed that the epitope-tagged Fre3p was
functional by expressing the HA-tagged Fet3p from a plasmid in a
FRE3-deleted strain and observing restoration of FOB-iron
uptake to wild-type levels. We grew the Fre3-HAp strain and a strain
expressing an HA-tagged version of Fet3p in media containing limiting
amounts of iron (to induce Fre3-HAp expression) and performed indirect
immunofluorescence (Fig. 4). Fre3p was
detected at the periphery of the cell in a pattern that was similar to
that of Fet3p, indicating that both Fre3p and Fet3p were expressed on
the plasma membrane. Fre3p was not detected in intracellular vesicles.
This localization to the plasma membrane was consistent with a role for
Fre3p in reductive iron uptake at the plasma membrane through the high
affinity Fe(II) system. The absence of detectable Fre3p in
intracellular vesicles is also consistent with genetic data indicating
that Fre3p is not required for ARN-dependent siderophore
uptake.
Reductive Uptake of Fe(III)-Enterobactin--
ENT is a siderophore
of the catecholate class that is synthesized and secreted by species of
Gram-negative bacteria, such as E. coli. S. cerevisiae was
recently reported to take up ENT through the ARN4
transporter (7), but it is not known whether ENT-bound iron can be
taken up via a reductive mechanism that requires the high affinity
Fe(II) transport system. We examined the uptake of ENT-bound iron in
strains bearing a deletion of ARN4 alone and deletions of
all four ARN genes, both in strains with intact high
affinity Fe(II) transport (FET3+) and in strains lacking
high affinity Fe(II) transport (fet3
To evaluate ENT-iron uptake via the high affinity Fe(II) system, we
tested the capacity of ENT to stimulate growth of strains on
iron-limited media (Fig. 5B). Strains individually deleted for ARN4 or FET3 grew as well as the wild-type
parent strain in the presence of ENT. Deletion of both ARN4
and FET3 resulted in a strain that did not grow in the
presence of ENT, indicating that ENT is a substrate for both the
ARN-dependent and
FET3-dependent systems of iron uptake. To
determine the role of the FRE genes in
FET3-dependent ENT-iron uptake, we tested the
capacity of ENT to stimulate growth of FRE-deleted strains
that were also deleted for ARN4 (Fig. 5C). In
this experiment, the arn4 The Role of Fre4p in Rhodotorulic Acid Iron
Utilization--
Fre4p, Fre5p, and Fre6p did not appear to have a role
in the reductive uptake of low concentrations of siderophore-bound
iron, because endogenous levels of expression of these proteins in
fre1 Uptake of siderophore-bound iron through the high affinity Fe(II)
transport system requires the activity of plasma membrane reductases
that are encoded by the FRE family of genes. Here we have
shown that Fre1p and Fre2p can facilitate the reduction of ferric-siderophores of the trihydroxamate and dihydroxamate classes. This finding was consistent with previous observations that the plasma
membrane reductase system of budding yeast exhibits an extremely broad
range of substrate specificity that includes both ferric and nonferric
electron acceptors (32). We also demonstrated that Fre3p could
facilitate the reduction of FOB and that Fre3p and Fre4p could
facilitate the reductive uptake and utilization of siderophore-iron,
presumably by acting as ferric-siderophore reductases. Although Fre4p
could facilitate utilization of RA-iron, we were not able to detect
significant Fre4p-dependent RA-iron reductase or uptake
activities. The most likely explanation for these observations is that
S. cerevisiae can grow in the face of extremely low rates of
iron uptake, and it is less likely that Fre4p facilitates iron uptake
through an activity other than that of a reductase. Fre3p and Fre4p
exhibit the most similarity to Fre2p with 72 and 56% identity,
respectively, at the amino acid level (24). Fre5p and Fre6p exhibit
lesser degrees of similarity to Fre2p (38 and 35%, respectively), and
the function of these gene products remains unknown. Although the
siderophore reductase activity exhibited by Fre3p is 40-fold lower than
that of Fre1p and Fre2p, FRE3 and FRE4 mRNA
transcripts are present in quantities roughly comparable to those of
FRE1 and FRE2 (24). This observation suggests
that the differences in activity are due less to differences in the
rate of transcription and more to differences in the intrinsic properties of the proteins. We observed that overexpression of Fre3p
did not result in increased Fre3p-specific reductase activity when
compared with the activity associated with endogenous levels of
expression. This observation suggests the possibility that the plasma
membrane reductase system has additional components that may be present
in limiting quantities.
Although Fre1p, Fre2p, Fre3p, and Fre4p could facilitate the reductive
uptake of siderophore-iron, they were not functionally interchangeable.
Fre3p and Fre4p did not facilitate growth on low concentrations of
ferric-ENT, but Fre3p could facilitate growth on low concentrations of
di- and trihydroxamates. Fre4p did not facilitate growth in the
presence of low concentrations of catecholate siderophore or
hydroxamate siderophores nor on high concentrations of the
trihydroxamate FOB. Yet Fre4p did facilitate growth on high
concentrations of the dihydroxamate RA. These results suggest that the
FRE reductases differ both in their specificities and affinities for different siderophores. Further evidence of differing specificities is seen in the case of the FRE3 deletion
strain. Deletion of FRE3 in cells grown in rich media leads
to an increased rate of uptake of FOB-iron, which might occur if Fre3p
facilitated uptake of a significant amount of iron from a specific (but
as yet unknown) source. Thus, in the absence of Fre3p, cells would respond to the loss of iron uptake from this specific source by up-regulating remaining systems of iron uptake. An alternative explanation for the increase in iron uptake in the absence of Fre3p is
that Fre3p is acting as both a reductase and a sensor of iron. Although
externally directed environmental iron sensors have not been reported
in budding yeast, a two-component Fe(III) sensor has recently been
identified in Salmonella enterica (33).
The differences in specificity exhibited by the FRE reductases may be
based on differences in the structure of the iron coordination site in
the siderophores. Alternatively, differences in specificity may be
accounted for by the reduction potentials of the siderophores. In Table
I, the capacities of siderophores to
stimulate growth of FRE-deleted strains and the reduction potentials
(31) of these siderophores are presented. ENT had both the most
negative reduction potential and the most stringent reductase
requirements, stimulating Arn4p-independent growth only when Fre1p or
Fre2p was expressed. Although the data presented here suggest that
Fre1p or Fre2p can reduce ENT-iron, ENT, with a reduction potential of
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
63 his3-
200
leu2-
1. To generate a FRE1 deletion,
strains were transformed with BamHI- and
HindIII-digested pUMG2 and transformants screened by surface
ferric reductase activity. For deletion of FRE2, a
1.4-kilobase pair fragment of genomic DNA from a fre1 fre2
double deletion strain (17) was PCR-amplified, and the product was used
to transform to histidine prototrophy. Transformants were screened by
PCR. PCR-mediated gene disruption was used to generate deletions of the
other FRE genes (25). The following primers were used to
amplify the HISG-URA3-HISG cassette from the plasmid pMPY-ZAP.
For FRE3, 5'-
TGCTTGTCAGGAGCAAGCGCCTCCCCTGCTAAGACAAAAATGTACGGCAAGTTctcactatagggcgaattgg-3' and
5'-TACCAACTTTGGTATTCTTCAAAGTATTCGATTGCCTTTGCAGGCTTCTCTActaaagggaacaaaagctgg-3'; for FRE4,
5'-AGCTTTCGGCCGCAAAGGCCCCACCCAGTAAAACGTCTCTAATAAATACTCActcactatagggcgaattgg-3' and
5'-AGGCTTGGTATTCCTCCAAGTATTCAATCATTCTTGATGGGTTTCTGATAACctaaagggaacaaaagctgg-3'; for FRE5,
5'-AGGTTCTTTAGCAAAACCAGCATCAACTAAGAAAAGAACGCAATGGGACCAGctcactatagggcgaattgg-3' and
5'-ATTCTGGAATTCCTCCACGTATTCTATCCATTTGTCACCGTGTTCAAGAACTctaaagggaacaaaagctgg-3'; and for FRE6,
5'-ACGTGGCTGATATCTTTAACAAAGGCTTTTAATATAAAATTACCACACACTGctcactatagggcgaattgg-3' and
5'-ACTGATATTCCTCGAAATACTCTATAATTCTCTCTGGGTAACCGAGGAGTTTctaaagggaacaaaagctgg-3'. Deletions were confirmed by PCR, and FOA-resistant clones were selected. Construction of multiply deleted FRE mutants was
performed by repeated PCR-based gene disruption. Construction of the
ARN deletion strains and the strain YPH499 FET3-HA
FTR1-Myc was previously described (10). The FRE3-HA strain, which
expresses a triple copy of the HA epitope fused to the carboxyl
terminus of Fre3p, was constructed by PCR epitope tagging (26) using
the plasmid pMPY-3xHA and the following primers:
5'-CTAGAGAAGCCTGCAAAGGCAATCGAATACTTTGAAGAATACCAAAGTTGGGAACAAAAGCTGGAGCTCCAC-3' and
5'-AATATATACTTGTGATAGGTAAAATAGTGAGGAAATAAATAAGGTAATTGACTATAGGGCGAATTGGGTACC-3'. Integration of the HA epitope was confirmed by PCR and by Western blotting. The genomic clone of FRE3-HA was constructed by
isolating genomic DNA from the FRE3-HA strain and digesting
it with HpaI and PstI. After electrophoresis, DNA
fragments from 4.0-5.0 kilobase pairs were extracted (GELase,
Epicentre Technologies), ligated into PstI- and
SmaI-digested Yep351, and transformed into
Escherichia coli DH5-
. Clones were screened by colony
hybridization using a PCR-amplified FRE3 open reading frame
as a probe, and positive clones were analyzed by restriction
mapping. Plasmid-encoded expression of Fre3p-HA was confirmed by
Western blotting. Rich media (YPD), synthetic defined media (SD), and
defined-iron media were prepared as described (27, 28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
fre2
strain
and a fre1
fre2
strain that also carried a
deletion of FRE3 (Fig. 1, inset). Deletion of
FRE3 in the fre1
fre2
strain resulted in a 73% decrease in the reduction of FOB. This loss of FOB
reductase activity was completely restored when the
fre1,2,3
strain was transformed with a plasmid expressing
Fre3p, indicating that Fre3p can facilitate the reduction of FOB-iron.
These data did not indicate whether the very low level of residual
reductase activity associated with expression of Fre3p, Fre4p, Fre5p,
and Fre6p could significantly affect iron uptake and utilization by the
cell. For this reason, we tested the capacity of FRE-deleted strains to take up Fe(II)-FOB and to grow when FOB was the iron source.
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Fig. 1.
Capacity of the FRE gene
products to reduce Fe(III)-siderophores in FRE+ and
fre deletion strains. After culture in rich
media or defined iron media containing 10 µM ferrous iron
(inset), cells of the indicated genotype in the exponential
phase of growth were collected, washed, and assayed for Fe(III)-
siderophore reductase activity in 1 mM Fe(III)-ferrichrome,
-ferrioxamine B, -fusarinine, and -rhodotorulic acid with ferrozine as
the Fe(II) acceptor. Absorbance at 562 nm was measured. The experiments
were repeated twice, and data from a representative experiment are
shown. Inset, the fre1,2,3
and the
fre1,2
strain were transformed with either a high copy
number plasmid bearing the FRE3 gene (pFRE3) or the empty
parent plasmid (pRS424) and the Fe(III)-ferrioxamine B reductase
activity measured as described. Samples were prepared in triplicate,
and the experiment was repeated three times. Error bars
indicate the S.D.
background, indicating that Fre1p and Fre2p
reductase activities contribute to FOB-iron uptake. Deletion of
FRE3 in addition to FRE1 and FRE2
resulted in a small, additional decrease in FOB-iron uptake in both the
ARN+ and the arn3
backgrounds, so that there
was no detectable FOB-iron uptake in the arn3
fre1
fre2
fre3
strain. To
determine whether Fre3p contributed significantly to FOB-iron uptake,
Fre3p was overexpressed from a high copy-number vector in the
arn3
fre1
fre2
fre3
strain (Fig. 2B). Fre3p overexpression
resulted in a slight increase in uptake when FOB-iron was present at 1 µM and a larger increase in uptake when FOB-iron was
present at 10 µM. These data indicated that Fre3p could
facilitate the reductive uptake of FOB-iron.
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Fig. 2.
Requirement of Fre1p, Fre2p, or Fre3p for
reductive uptake of ferrioxamine B-iron. A, absence of
FOB-55Fe uptake in a strain deleted for ARN3,
FRE1, FRE2, and FRE3. Congenic strains
of the indicated genotype were grown to the exponential phase in rich
media, and uptake of 55Fe bound to 1 µM FOB
was measured. B, uptake of FOB-55Fe when Fre3p
is overexpressed. A strain deleted for ARN3,
FRE1, FRE2, and FRE3 was transformed
with either a high copy number plasmid containing the FRE3
gene (pFRE3) or the parent vector (pRS414) and grown to exponential
phase in defined iron media containing 10 µM ferrous
ammonium sulfate to induce expression of Fre3p. Uptake of
55Fe in the presence of either 1 µM FOB
(gray bars) or 10 µM FOB (black
bars) was measured. C, failure of FOB to stimulate
growth in yeast deleted for ARN3, FRE1,
FRE2, and FRE3. Congenic strains of the indicated
genotype were plated in serial dilutions on synthetic iron-poor media
containing 100 µM BPS ( ) or 100 µM BPS
and 10 µM desferri-FOB (+). Plates were incubated at
30 °C for 3 days.
fre1
fre2
strain did not grow in the
absence of FOB, but grew as well as the FRE+ parent strain
in the presence of FOB, indicating that the reductase activities
encoded by FRE1 and FRE2 are not absolutely
required for utilization of FOB-iron. An arn3
fre1
fre2
fre3
strain,
however, failed to grow in the presence of FOB, confirming that Fre3p
can facilitate the uptake and utilization of FOB-iron. This result also
indicated that endogenous levels of expression of Fre4p, Fre5p, Fre6p,
and Fre7p could not support the reductive uptake of FOB-iron. Deletion
of FRE3 alone had no effect on growth on FOB, and
transformation of the arn3
fre1
fre2
fre3
strain with a low copy number
plasmid carrying either the FRE1 or FRE3 gene
completely restored growth on FOB (data not shown). Fre1p, Fre2p, and
Fre3p are not required for uptake of FOB through the
ARN-dependent system, as an ARN+ fre1
fre2
fre3
strain grew well on FOB (Fig.
2C).
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Fig. 3.
Requirement of Fre1p, Fre2p, or Fre3p for
growth on ferrichrome, triacetylfusarinine C, and rhodotorulic
acid. Congenic strains of the indicated genotype were plated in
serial dilutions on synthetic iron-poor media containing 100 µM BPS and either 0 µM ( ) or 10 µM (+) desferri-FC (A), desferri-TAFC
(B), or 15 µM (+) ferric-RA (C).
Plates were incubated for 4 days at 30 °C.
fre2
FRE3+ strain grew
as well as the congenic parent strain on ferric-RA, whereas the
fre1
fre2
fre3
strain did not grow.
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Fig. 4.
Localization of Fre3p to the plasma
membrane. Indirect immunofluorescence microscopy was performed on
cells expressing Fre3p-HA (A), Fet3p-HA (B), and
the untagged parent strain (C). HA.11 was the primary
antibody, and Cy3-conjugated donkey anti-mouse was the secondary
antibody. Images are in pairs with fluorescence on the left and DIC on
the right.
, Fig.
5A). Deletion of
ARN4 in both FET3+ and fet3
backgrounds resulted in decreases in ENT-iron uptake to undetectable
levels, confirming that uptake of ENT-iron is facilitated by Arn4p.
Uptake of ENT-iron was increased over 10-fold in a fet3
strain, and this was associated with a large increase in the level of
expression of the Arn proteins, especially Arn4p, in the
fet3
strain (data not shown). This increase likely
reflects the relative iron deprivation and subsequent Aft1p activation
that results from the loss of high affinity Fe(II) uptake in
fet3
strains. These results suggested that the majority of ENT-iron uptake occurs through Arn4p, but whether ENT-iron could
also be taken up through a reductive mechanism remained unclear.
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Fig. 5.
A, uptake of enterobactin-Fe through
ARN4. Congenic strains of the indicated genotype were grown
to exponential phase in rich media and assayed for uptake of
55Fe-enterobactin. B, requirement of either
FET3 or ARN4 for growth on enterobactin;
C, failure of enterobactin to sustain growth in a strain
deleted for ARN4, FRE1, and FRE2. Congenic
strains of the indicated genotype were plated in serial dilutions on
synthetic iron-poor media containing 100 µM BPS and
either 0 µM ( ), 5 µM (+) (B)
or 10 µM (+) (C) ferric-ENT. Plates were
incubated for 3 days at 30 °C.
FRE+ and the arn4
fre1
strains grew well on iron-limited
media in the presence of ENT and very slowly in the absence of ENT. In
contrast, the arn4
fre1
fre2
and the arn4
fre1
fre2
fre3
strains exhibited virtually no growth in the
presence or absence of ENT. Expression of FRE1 from a low
copy number plasmid restored ENT-mediated growth to the
arn4
fre1
fre2
strain (data
not shown), indicating that either Fre1p or Fre2p is also required for
the FET3-dependent utilization of ENT-iron.
fre2
fre3
strains did
not provide sufficient activity to result in growth on low
concentrations of siderophore. This apparent lack of function could
occur because of the following: 1) FRE4, FRE5, and
FRE6 are not expressed as functional enzymes on the plasma
membrane; 2) the siderophores we tested were not substrates for Fre4p,
Fre5p, and Fre6p; or 3) the tested siderophores were substrates but
exhibited lower affinity for Fre4p, Fre5p, and Fre6p. We tested the
last hypothesis by examining the growth of FRE-deleted
stains on a higher concentration of RA and FOB. In Fig.
6, each of the six iron-regulated
FRE genes was serially deleted, and the resulting strains
were tested for the capacity to grow in the presence of 100 µM RA and FOB. Deletion of FRE1, FRE2, and FRE3 had no effect on the capacity of a
high concentration of RA to stimulate growth on iron-limited plates
(Fig. 6A). Deletion of FRE1, FRE2, FRE3, and
FRE4, however, resulted in a strain that grew very slowly on
iron-limited media in the presence or absence of 100 µM
RA. In contrast, a high concentration of FOB did not stimulate growth
of the arn3
fre1
fre2
fre3
strain. These data indicate that Fre4p could
specifically facilitate the reductive uptake of RA-iron, but not
FOB-iron, through the Fe(II) transport system.
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Fig. 6.
Fre4p stimulation of growth in the presence
of higher concentration RA but not FOB. Congenic strains of the
indicated genotype were plated in serial dilutions on synthetic
iron-poor media containing 100 µM BPS
( rhodotorulic acid (A),
ferrioxamine
B (B)) and media containing 100 µM BPS
and either 100 µM desferri-RA (+rhodotorulic
acid (A)) or 100 µM desferri-FOB
(+ferrioxamine B (B)). Plates were incubated for
4 days at 30 °C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
750 mV, is thought to be beyond the range of physiological reductants
(31). This may not be the case in an in vivo setting, however, as a positive shift in the reduction potential of the siderophore may occur when the pH is lowered, the concentration of the
reduced species is very low, or the hydrophobicity of the microenvironment is altered. In contrast to ENT, RA exhibits both the
least negative reduction potential and the least stringent reductase
requirements, serving as a substrate for Fre1p, Fre2p, Fre3p, and, at
higher concentrations, Fre4p. Whether a particular ferric-siderophore
can be reduced by one of the FRE reductases may depend more on whether
the reductase can generate sufficient reduction potential than on the
capacity of the reductase to "recognize" a structural motif.
Comparison of growth stimulation capacity and reduction potential of
siderophores
, minimal growth). Growth on plates containing
100 µM RA is reported in parentheses. Reduction
potentials (31) are listed in mV. Each reduction potential was measured
at pH 7 in dilute aqueous solution versus a standard
hydrogen electrode. Variations in activity coefficients, due to
differences in buffer type or ionic strength, are generally assumed to
be <10 mV.
Reductive uptake of iron at the plasma membrane requires the function
of both an Fe(III)-reductase complex (containing a FRE gene
product and possibly other components (34)) and an Fe(II) transport
complex (containing Fet3p and Ftr1p). Fet3p and Ftr1p are physically
associated, and their intracellular association is required for
efficient expression on the plasma membrane (13-15). Although deletion
of the FRE genes did not impede the expression of the
Fet3p-Ftr1p complex on the plasma
membrane,2 the reductase and
transport complexes may be associated as part of a plasma
membrane-based iron uptake "machine."
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ACKNOWLEDGEMENTS |
---|
We thank Jerry Kaplan for helpful discussions and critical reading of this manuscript and Robert Stearman and Richard Klausner for generous gifts of plasmids.
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FOOTNOTES |
---|
* 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.
¶ Supported by National Institutes of Health Grant GM53836 and National Science Foundation Grant MCB9708737.
** To whom correspondence should be addressed: Liver Diseases Section, NIDDK, Bldg. 10, Rm. 9B16,National Institutes of Health, 10 Center Dr., Bethesda, MD 20892-1800. Tel.: 301-435-4018; Fax: 301-402-0491; E-mail: carolinep@intra.niddk.nih.gov.
Published, JBC Papers in Press, December 18, 2000, DOI 10.1074/jbc.M010065200
2 C.-W. Yun, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: Fe(III), ferric iron; Fe(II), ferrous iron; FOB, ferrioxamine B; FC, ferrichrome; TAFC, triacetylfusarinine C; ENT, enterobactin; RA, rhodotorulic acid; BPS, bathophenanthroline disulfonate; HA, hemagglutinin; PCR, polymerase chain reaction.
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