From the Department of Pathology, School of Medicine, University of Utah, Salt Lake City, Utah 84132
Received for publication, September 19, 2002, and in revised form, October 28, 2002
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ABSTRACT |
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A yeast mutant was found to have defective
growth on low iron medium despite a normal high affinity iron transport
system. The phenotype results from a gain of function mutation in
PDR1, which encodes a transcription factor that acts as a
regulator of pleiotropic drug resistance in Saccharomyces
cerevisiae. The mutant allele, PDR1(R821H), was found
to result in increased expression of at least 19 genes, three of which
are ATP-binding cassette (ABC) transporters. Expression of at least six
genes was required to show the low iron growth defect. Wild type cells
transformed with the PDR1(R821H) allele or a
PDR1 dominant allele (PDR1-3) showed the low iron
growth defect as well as increased resistance to drugs such as
cycloheximide and oligomycin. Transformation of PDR1(R821H)
into Investigations of the budding yeast Saccharomyces
cerevisiae have provided many insights into general principles of
eukaryotic transition metal metabolism (for review see Ref. 1).
Analyses of yeast mutants that show defective growth under iron-limited conditions have shown that growth on low iron medium requires a
functioning high affinity iron transport system (2, 3). The structural
components of the high affinity transport system are the products of
the FET3 and FTR1 genes, which are induced in
response to iron deprivation by the iron-sensing transcription factor
Aft1p (4). Fet3p mediates iron transport by acting as a ferroxidase,
converting ferrous iron to ferric iron, which is then transported by
the permease Ftr1p (5). Many mutants have been found to be unable to
grow on low iron as a consequence of defects in genes that encode
components of the high affinity iron transport system or in genes
required for assembly of the system (6).
Other mutants have been found to be unable to grow on low iron media,
despite having a functional high affinity iron uptake system. The gene
for one such mutant was identified to encode a methyl sterol oxidase,
Erg25p, an oxo-diiron containing enzyme that catalyzes an
oxygen-dependent step in ergosterol biosynthesis (2). The
gene was cloned because the erg25 mutant enzyme has a low
affinity for iron. When cellular iron levels were low, the mutant
enzyme lost activity resulting in sterol deficiency.
Because little is known regarding intracellular iron metabolism, we
have pursued the identification of genes that lead to defective
intracellular iron metabolism. In this article we describe a mutant
that is unable to grow on low iron media despite normal high affinity
iron uptake. We show that the mutation is a gain of function allele of
the transcription factor
PDR1,1 which
regulates a diverse set of genes, many of which are multidrug resistance genes. We suggest that expression of this dominant allele
results in transition metal efflux and storage.
Strains and Media--
The S. cerevisiae strains used
in this study were derived from DY150 and DY1457 of the W303
background. MS35 is a mutant derived from DY150 that was isolated in a
screen that selected for resistance to streptonigrin (3). The mutant
was backcrossed two times to ensure that the phenotypes were caused by
a single gene. The
Luria-Bertani medium was used to propagate Escherichia coli
strain DH5
The Iron Transport Assay--
Cells in early exponential phase were
grown in either CM or CMBPS(0) for 6-8 h. The cells were washed and
iron transport was assessed as previously described (3), with the
following modifications. To measure iron uptake, 5 × 106 cells were mixed with 1 mM ascorbate and
0.5 µM 59Fe, supplied as
59FeCl3. Cells were incubated at 30 °C for
10 min, placed on filters (Whatman GF/C), and washed with EDTA
containing buffer to remove unincorporated iron. The filters were air
dried and associated radioactivity was determined. The uptake activity
is expressed as femtomole of 59Fe uptake per min per
106 cells.
Assays to measure iron retention were done as described previously
(14). Briefly, cells growing exponentially in CMBPS(0) were harvested
and incubated in LIM-EDTA (i.e. LIM prepared without EDTA)
(12) with 0.5 µM 59Fe at 30 °C for 10 min.
Then, 1 mM BPS was added to aliquots of cells to chelate
the free 59Fe. The cells were then incubated for up to
5 h. Associated radioactivity was measured at various time points
throughout the 5-h incubation.
Microarray Analysis--
RNA was isolated from DY150 and MS35 in
early exponential phase grown in YPD. Total RNA was isolated using
standard techniques (15). Purification of mRNA was done using the
Promega PolyATtractTM mRNA isolation system.
Fabrication of DNA microarrays, synthesis of fluorescent-labeled
cDNA, hybridization to the microarrays, and subsequent scanning
were performed at the Huntsman Cancer Institute Microarray Core Facility.
Northern Analysis--
Total RNA was isolated and analyzed using
standard techniques (15). All samples were isolated from mid-log phase
cultures grown in either CM or CMBPS(0). A Cloning of PDR1, PDR3--
DNA transformations of E. coli and S. cerevisiae were performed by standard
procedures (16). The PDR1 gene was cloned by transformation
of MS35 with a yeast genomic library and selection for complementation
of the low iron phenotype using techniques described previously (3). A
single unique plasmid was isolated and mapped to chromosome VII by
sequencing the ends of the plasmid insert. Subcloning mapped a minimum
MS35 complementing activity to an AvrII-NheI
fragment containing only PDR1. This fragment complemented
MS35 when cloned into both high and low copy yeast expression vectors.
The laboratory of Karl Kuchler (Biocenter, Vienna, Austria) generously
provided the PDR1-3 allele.
PDR3 was amplified via PCR using genomic DNA extracted from
DY150 as a template and the following reaction conditions:
denaturation, 94 °C, 40 s; annealing, 55 °C, 40 s; and
elongation, 68 °C, 4 min. The following primers were used:
5'-ATTCTCACTGCCCTCTATGCC-3', 5'-TTCCTCAGTATGAGTAGGGGG-3'. The PCR
product was cloned using the TOPO XL PCR CloningTM Kit from
Invitrogen. The PDR3 open reading frame was isolated from
the pCR-XL-TOPO cloning vector using BamHI and
NotI. The BamHI-NotI fragment
complemented the MS35 low iron phenotype when cloned into both high and
low copy yeast expression vectors.
Identification of PDR1(R821H)--
The mutated gene in MS35 was
identified by constructing a library from the MS35 genome as outlined
in standard protocols (15). Genomic DNA was isolated from mid-log phase
MS35 cultures grown in YPD. Partial enzyme digestions on the genomic
DNA were done using Sau3A1. The partially digested DNA was
fractionated on a 10-40% sucrose gradient, and centrifuged at 25,000 rpm for 22 h. Once fractionated, the DNA was ligated to
pRS416-zero, a centromeric plasmid, digested with BamHI.
Ligations were transformed by electroporation into
ElectromaxTM DH10BTM E. coli cells
purchased from Invitrogen (17, 18). Once the amplified MS35
genomic library was extracted from bacteria, it was transformed into
DY150 and screened for genes that conferred cycloheximide resistance to
the wild type cells. Approximately 15,000 colonies were screened and
six colonies were found to be cycloheximide resistant. Plasmids were
recovered from the six positive candidates, and when sequenced were
found to contain the same gene: PDR1 with a mutation of the
arginine at position 821 to a histidine. The allele is referred to as
PDR1(R821H).
Atomic Absorption Assay--
Cells were grown to mid-log phase
in the indicated media. For whole cell readings, 2.0 × 108 cells were collected, washed by centrifugation four
times with 50 mM Tris (pH 6.5), 10 mM EDTA, and
once with sterile deionized water. The washed cells were frozen at
Mutant MS35 Has a Defect in Intracellular Iron Metabolism--
The
yeast mutant MS35 was identified by its reduced growth on low iron
medium. This defect may be caused by inactive high affinity iron
transport, resulting from mutations in genes directly involved in iron
transport or in genes involved in assembly of the transport system.
Measurements of 59Fe transport were performed to determine
the rate of iron uptake in wild type and MS35 cells. For both strains,
59Fe uptake increased when cells were incubated in
iron-poor media, a condition that induces the transcription of
FET3 and FTR1 (Fig. 1A). The MS35 strain did show
a lower rate of uptake, but this decrease is not expected to result in
a growth defect, as we have previously observed that much more
substantial reductions (>90%) in high affinity iron transport
activity are required to show a phenotype of poor growth on low iron
media (22). Thus, increased levels of Fet3p and Ftr1p are synthesized
in response to low iron in MS35.
An inability to grow on low iron can also result from defective copper
loading of apoFet3p (20). As apoFet3p is copper-loaded in a post-Golgi
intracellular compartment, defects in either copper homeostasis or
vesicular traffic result in the appearance of apoFet3p on the cell
surface. Cell surface apoFet3p can be copper-loaded by addition of high
copper and chloride, restoring iron transport (6, 21). Consequently,
mutations in genes required for the assembly of Fet3p can be identified
through suppression of the low iron growth defect by growth in high
copper media in the presence of chloride. MS35 showed poor growth in
iron-deficient media that was not suppressed by adding copper and
chloride (Fig. 1B). Taken together, these results show that
the defect in MS35 is not because of a mutation in the assembly or
function of the high affinity iron transport system. Thus, the
inability to grow on low iron must be caused by a defect in
intracellular iron metabolism.
With the exception of the dominant allele
AFT1up, all other known mutations in iron
transport have been found to be recessive. A diploid from a cross
between MS35 and wild type cells, however, showed that the inability to
grow on low iron media was semidominant (Fig. 1C), as growth
of the heterozygote on low iron media was greater than that of a
homozygous mutant diploid but less than that of a wild type diploid.
Sporulation of the heterozygote showed a 2:2 segregation pattern for
the low iron phenotype (data not shown), indicating that this phenotype
is because of a defect in a single gene.
Mutant MS35 Shows Increased Multidrug Resistance--
We performed
a transcript analysis of mRNA isolated from mutant and wild type
cells grown in YPD using microarrays. Results from two independent
experiments demonstrated that MS35 cells showed increased transcription
of at least 19 genes (Table I). Some of
these genes are notable as belonging to the ATP-binding cassette (ABC)
protein transporter family (23). This transcription pattern is similar
to that of the transcription factor PDR1, which regulates a
large family of ATP transporters (24). These ATP-driven membrane
transporters are homologous to the mammalian multidrug resistance proteins and confer resistance to a variety of
structurally and functionally unrelated agents (23). For two of the
up-regulated genes, PDR5 (pleiotropic drug resistance) and
ICT1 (increased copper tolerance), we verified increased
expression in mutant cells using Northern analysis (Fig.
2A), and showed that increased expression of these genes was seen in both high and low iron media. We
note that Northern analysis suggests a much greater induction of these
genes than microarray. Consistent with their multidrug-resistant transcriptional expression pattern, MS35 cells showed a marked resistance to both cycloheximide and oligomycin, two drugs that are
unrelated in structure and function (Fig. 2B).
Similar to the low iron phenotype, increased drug resistance was
semidominant in a heterozygotic diploid of MS35 and wild type yeast
(Fig. 2C). Sporulation of 15 heterozygotic diploids showed a
2:2 cosegregation of cycloheximide resistance with the phenotype of
poor growth on low iron, suggesting that both phenotypes are because of
mutations in a single gene.
The Low Iron Phenotype of MS35 Is Caused by a Mutation in
PDR1--
Transcription of ABC genes is primarily controlled by two
homologous transcription factors, Pdr1p (25) and Pdr3p (26, 27), which
belong to a large family of zinc finger-containing transcription
factors (28). Pdr1p and Pdr3p have an overlapping pattern of regulation
in that they control many of the same genes (27). Semidominant gain of
function point mutations in PDR1 and PDR3 have
been characterized in yeast that, like MS35, are resistant to
abnormally high concentrations of various drugs (29). In these mutants,
as in MS35, levels of Pdr1p- and Pdr3p-regulated ABC genes are
increased at least 2-fold (24).
Several experiments indicated that MS35 has a gain of function mutation
in Pdr1p. First, expression of PDR1 or PDR3 from
a high copy plasmid partially suppressed the low iron growth defect although the cycloheximide resistance phenotype was less affected (Fig.
3A), consistent with studies
showing that expression of the normal allele reduces the phenotype of
PDR gain of function alleles (10, 30). Second, deletion of
PDR1 in MS35 confers growth on low iron medium and abrogates
cycloheximide and oligomycin resistance (Fig. 3B).
Third, transformation of wild type cells with a genomic library derived
from MS35 cells and cloned into single copy plasmids resulted in the
identification of plasmids that conferred both cycloheximide resistance
and the low iron growth phenotype. Sequence analysis of the genes
encoded on two of the plasmids revealed a PDR1 gene with a
mutation that resulted in the substitution of histidine for arginine at
amino acid position 821 (referred to henceforth as
PDR1(R821H)). That PDR1(R821H) is mutated in the
same region as other gain of function mutations (28) suggests that the
mutation in MS35 is a gain of function allele of PDR1. Further support was provided when transformation of the gain of function allele PDR1-3 also conferred cycloheximide
resistance and a low iron defect to wild type cells (Fig.
3C). Finally, transformation of PDR1(R821H) into
wild type cells leads to a reduction in high affinity iron transport
(Fig. 3D).
The PDR1(R821H) Allele Causes a Decrease in Cytosolic
Iron--
Whereas we hypothesized that the decrease in iron transport
activity in MS35 cells or in cells transformed with
PDR1(R821H) was not great enough to lead to the observed low
iron growth defect, we devised a genetic experiment to test this
hypothesis. If PDR1(R821H) causes a low iron growth defect
by affecting iron uptake, we would not expect the growth defect to
occur in cells with a deletion in a component of the high affinity iron
transport system. Cells containing a deletion in FET3 and
transformed with a PDR1(R821H) containing plasmid were found
to have a decided growth disadvantage when plated on low iron medium
(Fig. 4A). This result
suggests that the low iron growth defect is independent of iron uptake activity.
We considered the possibility that the low iron growth defect results
from export or sequestration of cytosolic iron through genetic
manipulation of CCC1, which encodes a transporter that effects the transport of iron from cytosol to vacuole (7). Deletion of
CCC1 produces sensitivity to high medium iron concentrations because of an inability to sequester excess iron in the vacuole. Transformation of PDR1(R821H) into
We attempted to measure iron efflux through the use of 59Fe
pulse-chase experiments. Neither wild type cells nor MS35 cells, when
incubated with 59Fe for 10 min, showed loss of
radioactivity over a subsequent 5-h period (data not shown). These
experiments were performed after cells had been incubated in low iron
medium for 6 h, and under such conditions, it may not be possible
to easily observe iron efflux because of low intracellular iron pools
and/or the possible rapidity of the efflux process. We therefore
examined the effect of PDR1(R821H) on cells incubated in
high iron medium. Initially, we measured the total cellular iron
content in wild type and Expression of at Least Six Genes Is Required for the Low Iron
Phenotype--
As determined by microarray analysis, the effect of
the PDR1(R821H) allele is to increase transcription of at
least 20 different genes. To address which of these genes is
responsible for the low iron phenotype, we took advantage of the
collection of homozygous diploid deletion strains purchased from
Research Genetics. This collection consists of 4,600 yeast strains,
each of which has a targeted deletion in a specific gene.
We transformed PDR1-3 or PDR1(R821H)
into strains carrying a single deletion in 15 of the genes that were
identified as overexpressed by microarray analysis. The strains that
were not tested were not included in the deletion collection.
Transformation of either of these PDR1 alleles into wild
type diploids (BY4743) reproduced the mutant phenotype of reduced
growth on low iron medium and increased cycloheximide resistance (Fig.
6A). A
We found that deletion of any of six of the top 19 genes
shown to be up-regulated by microarray analysis resulted in the loss of
the low iron growth defect while still retaining cycloheximide resistance. An example of the phenotype of transformed deletions and
their phenotypes is given in Fig. 6C and the names or
designations of the relevant genes are listed in Table
II. These results suggested that all six
genes must be overexpressed for the low iron phenotype, as loss of any
of the six was sufficient to complement expression of
PDR1(R821H).
PDR1(R821H) Confers Resistance to Copper and Manganese
but Increased Sensitivity to Cobalt--
The observation that
PDR1(R821H) can protect cells from high iron toxicity led us
to examine whether this allele provides protection from toxicity of
other transition metals. Wild type cells transformed with
PDR1(R821H) showed increased resistance to Mn2+
and Cu2+ but increased sensitivity to Co2+
(Fig. 7A). The increased
resistance to Cu2+ was associated with decreased
accumulation of Cu2+ as measured by atomic absorption
spectroscopy (Fig. 7B). We did not observe a statistically
significant difference in Mn2+ content between
vector-transformed and PDR1(R821H)-transformed cells (Fig.
7C).
We took advantage of the homozygous deletion collection to ask whether
the same genes that were responsible for iron sensitivity were
responsible for copper resistance. As shown in Fig.
8 at least five genes are required to
show the copper phenotype. These genes, however, are different from
those that lead to the iron sensitivity (Table II). Thus, transition
metal response because of PDR1(R821H) is a multigenic
trait.
Most previously characterized yeast mutants unable to grow in low
iron are because of either defects in iron transport or mutations in
iron-requiring proteins. Invariably, these mutants are recessive. MS35
is extraordinary in that it is semidominant, has a low iron growth
defect, and yet has no defect in the assembly of the iron transport
system. Identification of the mutant gene in MS35 revealed that it is a
gain of function allele of PDR1, a transcription factor that
regulates genes involved in multidrug resistance. The mutated
amino acid in PDR1(R821H) is near the mutation in the gain
of function PDR1-3 allele, which has a phenylalanine to
serine substitution at position 815 (28). The mechanism of the
PDR1(R821H) and PDR1-3 gain of function
mutations is unclear, as the altered amino acids lie neither in the
inhibitory domain nor in the activation domain of Pdr1p (32). It has
been postulated that this region, when mutated, could affect binding of
the "major inhibitory effector."
We discovered that overexpression of PDR1-regulated genes
results in a decrease in cytosolic iron, as shown by reduced growth of
PDR1(R821H) expressing cells in low iron medium and
increased survival of PDR1(R821H) transformed
These results suggest that PDR1 targets, depending on iron
concentration, may affect both iron efflux and sequestration. The hypothesis that both efflux and sequestration occur is strengthened by
the identification of the genes that are required to produce the low
iron phenotype (see Table II). Some of the known genes encode ABC
transporters present on the vacuole (TPO1) or
plasma membrane (YOR1 and SNQ2). There is some
limited data on the role of multidrug resistance genes in transition
metal metabolism. A double deletion of SNQ2 and
PDR1 were shown to effect Mn2+ and
Ni2+ resistance (33), as wild type cells were shown to
export Mn2+ at a greater rate than
Expression of the PDR1(R821H) allele resulted in an increase
in transcription of at least 19 different genes. We took advantage of a
homozygous deletion collection to determine which of those genes were
involved in transition metal metabolism by transforming the
PDR1(R821H) allele into strains that have a deletion in a single gene and examining their phenotype on low iron and on
cycloheximide containing medium. We observed that deletion of a single
gene, PDR5, abrogated cycloheximide resistance but did not
eliminate the low iron phenotype. This result confirms previous studies that show that PDR5 is responsible for cycloheximide
resistance. In contrast, we discovered that at least six genes were
required for the low iron phenotype. That multiple genes are required
to fully express a multidrug resistance phenotype is not unprecedented (for review see Ref. 23). It is of interest that whereas both Snq2p and
Pdr5p were shown to be required for resistance to Mn2+ and
Li+, we observed that deletion of PDR5 did not
affect the low iron growth phenotype (33).
We further demonstrated that PDR1(R821H) provided resistance
to copper and manganese but rendered cells more sensitive to cobalt.
The increased cobalt sensitivity may result from the affect on
cytosolic iron on the allele of the PDR1(R821H). A
recent study demonstrated that reduced cytosolic iron levels lead to
increased cobalt sensitivity, whereas increased cytosolic iron
protected against cobalt toxicity (34). The finding that expression of PDR1(R821H) results in low cytosol iron is consistent with
the expectation that such cells might be cobalt-sensitive. Expression of PDR1(R821H) also led to increased resistance to copper
and manganese. Measurement of cellular copper indicates that resistance is because of lowered cellular levels. Interestingly, the genes required for resistance to copper with one exception (SNQ2)
are different from those required for the iron sensitivity. In both cases simultaneous expression of multiple genes are required to develop
the phenotype. Our biochemical experiments suggest that copper
resistance may be because of metal export. The high level of media
copper should result in the down-regulation of copper transporters,
suggesting that decreased cellular copper may be because of increased
copper efflux. Experiments are currently in progress to determine
whether the same genes that are required for copper resistance are also
required to rescue ccc1 cells, which were previously shown to have
increased sensitivity to high iron medium because of defective vacuolar
iron storage (Li, L., Chen, O. S., Ward, D. M., and Kaplan, J. (2001) J. Biol. Chem. 276, 29515-29519), conferred
resistance to high iron medium. Cells expressing
PDR1(R821H) also showed increased resistance to copper and
manganese because of increased metal export. These results suggest that
expression of PDR1-regulated genes affects both efflux and
storage of transition metals.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ccc1 strain was made as previously
described (7). A library of strains with deletions of each of the
nonessential genes in homozygous diploid (BY4743) backgrounds was
purchased from Research Genetics.
. The strain was supplemented with antibiotics as required (8). Yeast extract-peptone-dextrose (YPD) and yeast nitrogen-base synthetic complete media (CM) were used for the standard growth of
S. cerevisiae and were supplemented as needed (9).
Cycloheximide containing medium was prepared by adding 0.7 µg/ml
cycloheximide to standard growth media (10). Oligomycin containing
medium was prepared by adding 0.7 µg/ml oligomycin to yeast
extract-peptone-glycerol ethanol (YPGE) (11). Low iron growth medium
was made by adding 40 µM bathophenanthroline disulfonate
(BPS), an iron chelator, to standard growth medium and then adding back
varying amounts of FeCl3 (3). Synthetic low iron medium
(LIM), supplemented with varying amounts of FeCl3 and
CuSO4, was used to limit both iron and/or copper (12). The
various LIM and BPS media used in this work are referred to as LIM(x)
and BPS(x), where x equals the concentration in micromolar of added
FeCl3. High metal growth media was made by adding the
indicated amount of
Fe(NH4)2(SO4)2·6H2O, ZnSO4, MnCl2, CoCl2, or
CuSO4 to standard CM.
pdr1 strains were generated by double fusion
polymerase chain reaction (13). The primers were:
5'-GTCGTGACTGGGAAAACCCTGGCGCTTAGG-3', 5'-CATAGTAACGCCAAACGATCGC-3',
5'-TCCTGTGTGAAATTGTTATCCGCTTACAGTATCCTGTGGAGCGACGT-3', and
5'-GACTATCAGAGATTGTGGCGC-3'. The HIS3 gene was used as
the selectable marker. The PCR fusion products were transformed into DY150 and MS35 cells, and the deletion of the PDR1 gene was
confirmed by PCR.
-32P-labeled
PDR5 probe was generated using random primers and a PDR5 (YOR153W) open reading frame template from the
Research Genetics open reading frame collection. Radioactive
ICT1, FET3, and ACT1 probes were
generated in a similar manner using ICT1 (YLR099C), FET3 (YMR058W), and ACT1 (YFL039C) open reading frames.
20 °C. Vacuoles were prepared as previously described (7) and also
frozen at
20 °C. The frozen samples were then digested in 200 µl
of 5:2 nitric acid:perchloric acid at 80 °C for 1 h. After
digestion, the samples were diluted to 1.0 ml with sterile-deionized
water and then flamed in a Perkin-Elmer inductively coupled plasma
atomic absorption spectroscope (19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Mutant MS35 shows a
semidominant low iron medium growth defect, despite having a functional
high affinity iron transport system. A, wild type (DY150)
and MS35 cells were grown in either CM (uninduced) or in CMBPS(0)
medium (induced) for 8 h. Cells were washed and iron transport
activity was assayed using 0.5 µM
59FeCl3. B, serial dilutions of wild
type (DY150) and mutant (MS35 and MM19) cells were plated on low iron
(LIM (1)), low copper (0.125 µM CuSO4), and
low iron, copper-supplemented (500 µM CuSO4)
plates. As a control we included MM19, a yeast strain with a mutation
in the GEF1 gene, which leads to a nonfunctional iron
transport system that can be suppressed by the addition of copper.
C, serial dilutions of the wild type diploid (DY150XDY1457),
the heterozygous diploid (MS35XDY150), and the homozygous mutant
diploid (MS35XMS35), plated on low iron (CMBPS(1)) plates.
Up-regulated genes in the mutant MS35
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Fig. 2.
Mutant MS35 shows increased multidrug
resistance. A, increased expression of two genes in MS35
compared with wild type (DY150) was verified by Northern analysis.
Total RNA was isolated from mid-log phase cultures grown in CM or
CMBPS(0) (BPS). Northern blots were probed for PDR5,
ICT1, and ACT1. B, serial dilutions of
wild type cells and MS35 cells were plated on either low iron
(CMBPS(2)), cycloheximide (0.7 µg/ml), or oligomycin (0.7 µg/ml)
plates. C, same as B except the wild type diploid
(DY150XDY1457), the heterozygous diploid (MS35XDY150), and the
homozygous mutant diploid (MS35XMS35) were plated on low iron,
oligomycin, or cycloheximide.
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Fig. 3.
The MS35 phenotype of low iron growth
sensitivity and multidrug resistance is because of a mutation in
PDR1. A, wild type and MS35 cells were
transformed with either a high copy control vector (pTF63) or a high
copy vector containing PDR1 or PDR3. Serial
dilutions of transformed cells were plated on either low iron
(CMBPS(2)) or cycloheximide (0.7 µg/ml) plates. The presence of the
PDR1 or PDR3 plasmids attenuated the MS35
phenotype of low iron growth sensitivity and multidrug resistance.
B, PDR1 was deleted in wild type cells and in
MS35 cells, and serial dilutions were plated on either low iron
(CMBPS(2)), cycloheximide (0.7 µg/ml), or oligomycin (0.7 µg/ml)
plates. Deletion of PDR1 in MS35 led to loss of both the low
iron growth defect and multidrug resistance. C, wild type
cells were transformed with a single copy plasmid containing the
PDR1-3 allele or the PDR1(R821H) allele. Serial
dilutions of the transformed cells were plated on either low iron
(CMBPS(2)-uracil) or cycloheximide (0.7 µg/ml) plates. Both of the
PDR1up alleles resulted in low iron growth
sensitivity and multidrug resistance. D, wild type (DY150)
cells, transformed with either a control vector (pTF63) or
PDR1(R821H), and MS35 cells were grown in either CM
(uninduced) or CMBPS(0) (induced) for 6 h. Cells were washed and
iron transport activity was assayed using 0.5 µM
59FeCl3.
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Fig. 4.
Cells transformed with the
PDR1(R821H) allele show increased low iron growth
sensitivity and high iron resistance. A, wild type cells and
fet3 cells were transformed with either a control vector
(pTF63) or PDR1(R821H). Serial dilutions were plated on low
iron (CMBPS(2)-uracil for DY150, CMBPS(5)-uracil for
fet3) or cycloheximide (0.7 µg/ml) plates. The result
shows that the low iron growth defect of
PDR1(R821H) can occur in the absence of a
required component (FET3) of the high affinity iron
transport system. B, wild type or
ccc1 cells
were transformed with a control vector or with PDR1(R821H).
Serial dilutions were plated on high iron (4 mM
Fe2+) plates. CCC1 encodes a vacuolar iron
transporter; thus, a
ccc1 strain is sensitive to high
iron. Transformation with PDR1(R821H) rescues
ccc1 cells from high iron toxicity.
ccc1 cells
suppressed this high iron sensitivity (Fig. 4B), suggesting
that PDR1(R821H) affects iron efflux from the cell,
sequestration of iron into cellular compartments, or a combination of
these two effects.
ccc1 cells transformed with
PDR1(R821H) or a control vector. We observed that, when
incubated in high iron medium, either wild type cells (Fig.
5A) or
ccc1
cells (Fig. 5B) transformed with PDR1(R821H) had
higher levels of iron than cells transformed with only the vector.
Interestingly, we observed that in low iron medium the reverse was
true: wild type or
ccc1 cells transformed with
PDR1(R821H) had less iron than vector-transformed cells. As
mentioned previously, the sensitivity of
ccc1 cells to
high iron is thought to be because of cytosolic iron accumulation. If
PDR1(R821H)-transformed
ccc1 cells have more
total cellular iron but are less iron sensitive, then we might expect
that the increased iron is not in the cytosol. In fact, vacuoles
isolated from PDR1(R821H)
ccc1 cells were
found to have a higher iron content than vacuoles isolated from
vector-transformed
ccc1 cells (Fig. 5C). This
result suggests that the PDR1(R821H) allele leads to
increased iron storage.
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Fig. 5.
Iron content of
PDR1(R821H)-transformed ccc1 cells
grown in low or high iron. A, PDR1(R821H) or
control vector-transformed wild type (DY150) cells were incubated in CM
containing either 200 µM
Fe(NH4)2(SO4)2·6H2O
(high iron) or no additional iron (low iron). After 6 h of growth,
cells were thoroughly washed and iron content was determined by atomic
absorption spectroscopy. B, PDR1(R821H) or
control vector-transformed
ccc1 cells were incubated in
CM containing either 200 µM
Fe(NH4)2(SO4)2·6H2O
(high iron) or no additional iron (low iron). After 6 h of growth,
cells were thoroughly washed and iron content was determined by atomic
absorption spectroscopy. C, PDR1(R821H) or
control vector-transformed
ccc1 cells were incubated in
medium containing 200 µM
Fe(NH4)2(SO4)2·6H2O.
After 6 h of growth, cells were sphereoplasted, vacuoles were
isolated, and the iron content of the isolated vacuoles determined by
atomic absorption spectroscopy. Data are shown as iron content/mg of
vacuolar protein.
pdr5
strain transformed with PDR1(R821H) was
cycloheximide-sensitive but still showed decreased growth on low iron
medium (Fig. 6B). This result confirmed previous studies indicating that Pdr5p is responsible for cycloheximide resistance (31),
and showed that Pdr5p is not involved in the low iron phenotype
conferred by PDR1(R821H).
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Fig. 6.
Specific genetic deletions affect the
PDR1(R821H)-mediated low iron growth or multidrug
resistance phenotypes. Wild type diploid (BY4743) or diploid
strains homozygous for deletions in specific genes were transformed
with a control plasmid, PDR1-3, or PDR1(R821H)
on a high copy plasmid. Serial dilutions of the transformed cells were
plated on low iron and cycloheximide containing plates. A,
wild type diploid results are shown. B, deletion of
PDR5 leads to loss of multidrug resistance but has no affect
on the low iron growth phenotype. C, deletion of
YOR1 does not affect multidrug resistance but does result in
the loss of the low iron growth defect.
Genes required for low iron sensitivity and copper resistance
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Fig. 7.
The PDR1(R821H) allele
affects transition metal sensitivity and resistance. A, wild
type (DY150) cells were transformed with either a control vector
(pTF63) or PDR1(R821H). Serial dilutions were plated on the
following plates: 20 mM MnCl2
(manganese), 2.0 mM CuSO4
(copper), 5 mM ZnSO4
(zinc), and 1.5 mM CoCl2
(cobalt). B, PDR1(R821H) or control
vector-transformed wild type cells were incubated in media containing
50 µM CuSO4, or C, 200 µM MnCl2. After 6 h of growth, cells
were thoroughly washed and metal content was determined by atomic
absorption spectroscopy.
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Fig. 8.
PDR1(R821H) mediates increased
resistance to copper through activation of specific genes. Wild
type diploid cells (BY4743) and diploid cells homozygous for a specific
gene deletion were transformed with either PDR1(R821H) or a
control vector. The cells were spotted onto media containing 2.2 mM CuSO4 and incubated for 3 days prior to
being recorded.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ccc1 cells in high iron medium. Whereas there is a
decrease in the activity of the high affinity iron transport system in
PDR1(R821H) expressing cells, the decreased low iron growth
is not because of a defect in iron transport. This point is suggested
by the fact that PDR1(R821H) expressing cells that lack a
component of the high affinity iron transport system
(
fet3) are more sensitive to low iron than
fet3 cells transformed with a control vector. Whereas we
were not able to directly demonstrate increased iron efflux caused by
PDR1(R821H), we did find that expression of
PDR1(R821H) led to increased resistance to high iron medium
in
ccc1 cells. We observed that, whereas expression of
PDR1(R821H) led to decreased cellular iron when either wild
type or
ccc1 cells were grown in low iron medium, there
was an increase in cellular iron when the same cells were grown in high
iron medium. This observation suggests that there is a difference in
the mechanism(s) leading to low iron growth sensitivity and high iron
resistance conferred by PDR1(R821H). As high iron conditions
down-regulate most of the known iron transporters, PDR1(R821H) is unlikely to affect changes in iron uptake.
Rather, we observed that vacuoles from
PDR1(R821H)-transformed cells have higher levels of iron
than those from vector-transformed cells. As CCC1 encodes
the major, if not the only, vacuolar iron transporter, PDR1(R821H) expression must lead to an increase in vacuolar
iron accumulation.
snq2
pdr1 cells. It was not clear, however, whether the
loss of these genes directly affected Mn2+ efflux or
whether this effect was a downstream consequence of the gene deletions.
It is known that many transporters that recognize Mn2+ also
recognize Fe2+, suggesting that Snq2p could also modulate
iron export (7). However, the formal possibility remains that
expression of Snq2p somehow modulates plasma membrane permeability
rather than mediating active transport of metal ions.
ccc1 cells from high iron.
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FOOTNOTES |
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* This work was supported in part by NIDDK National Institutes of Health Grant DK-30534. Support for use of Core facilities was provided NCI National Institutes of Health NCI-CCSG P30CA 42014.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 in part by a grant from the Pfizer Co.
§ Supported by National Institutes of Health Genetics Predoctoral Training Grant GM 07464. Present address: Division of Life Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
¶ To whom correspondence should be addressed: Dept. of Pathology, School of Medicine, 50 N. Medical Dr., University of Utah, Salt Lake City, UT 84132. Tel.: 801-581-7427; Fax: 801-581-4517; E-mail: jerry.kaplan@path.utah.edu.
Published, JBC Papers in Press, October 30, 2002, DOI 10.1074/jbc.M209631200
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ABBREVIATIONS |
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The abbreviations used are: PDR, pleiotropic drug resistance; ABC transporters, ATP-binding cassette transporters; BPS, bathophenanthroline disulfonate; LIM, low iron medium; CM, complete media.
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