(Received for publication, September 29, 1995; and in revised form, November 20, 1995)
From the
Glutathione-dependent detoxification reactions are catalyzed by the enzyme glutathione S-transferase and are important in drug resistance in organisms ranging from bacteria to humans. The yeast Issatchenkia orientalis expresses a glutathione S-transferase (GST) protein that is induced when the GST substrate o-dinitrobenzene (o-DNB) is added to the culture. In this study, we show that overproduction of the I. orientalis GST in Saccharomyces cerevisiae leads to an increase in o-dinitrobenzene resistance in S. cerevisiae cells. To recover genes that influence o-DNB resistance in S. cerevisiae, a high copy plasmid library was screened for loci that elevate o-DNB tolerance. One gene was recovered and designated ROD1 (resistance to o-dinitrobenzene). This locus was found to encode a novel protein with no significant sequence similarity with proteins of known function in the data base. An epitope-tagged version of Rod1p was produced in S. cerevisiae and shown to function properly. Subcellular fractionation experiments indicated that this factor was found in the particulate fraction by differential centrifugation. Overproduction of Rod1p leads to resistance to not only o-DNB but also zinc and calcium. Strains that lack the ROD1 gene are hypersensitive to these same compounds. Rod1p represents a new type of molecule influencing drug tolerance in eukaryotes.
Cancer cells can often acquire a broad range resistance to cytotoxic drugs through alteration of certain genes. The most extensively described of these alterations is a change in expression of an ATP binding cassette transporter protein encoded by the MDR1 gene(1) . Increased production of this factor leads to a strong cross-resistant phenotype to a variety of chemotherapeutic agents and is a major problem in clinical treatment of human tumors.
Multidrug or pleiotropic drug resistance in Saccharomyces cerevisiae has served as a useful model for mammalian multidrug resistance. A large number of loci involved in the yeast pleiotropic drug resistance phenotype have now been identified due, in large part, to the ease of genetic manipulation in this organism. The identities of these gene products have been recently reviewed (2) and fall generally into two main classes: membrane transporter proteins and transcriptional regulatory proteins. Specific examples of these general classes of factors include the ATP binding cassette transporter Pdr5p (3, 4, 5) and the transcriptional regulatory protein yAP-1(6) . Pdr5p is believed to function as a drug efflux pump(7) , while yAP-1 activates the expression of a number of genes that directly act to confer drug resistance(8, 9, 10) .
One of the genes
activated by yAP-1 is the GSH1 locus which encodes the
-glutamylcysteine synthetase enzyme(8) . This protein
catalyzes the rate-limiting step in glutathione
biosynthesis(11) . Transcriptional control of GSH1 by
yAP-1 is essential for normal cadmium tolerance(8) . These
observations indicate that yAP-1 is important in regulating glutathione
production, which in turn is important in detoxification reactions in
yeast.
Glutathione has been found to be an important participant in
the inactivation of a large number of drugs in eukaryotic
cells(12) . The covalent attachment of glutathione to target
compounds is often catalyzed by the enzyme glutathione S-transferase (GST)()(13) . The involvement
of GSTs in glutathionedependent detoxification reactions has been well
documented in many eukaryotic cells, but no GST has yet been found in S. cerevisiae. A fungal GST-encoding gene has been isolated
from the yeast Issatchenkia orientalis(14) .
Expression of the I. orientalis GST locus is inducible by the
GST substrate o-dinitrobenzene (o-DNB). Although it
is attractive to speculate that production of the I. orientalis GST protein is involved in detoxification of o-DNB, there
is no direct evidence to support this belief.
We expressed the I. orientalis GST in S. cerevisiae and found that
this factor does elevate resistance to o-DNB. In addition, we
screened a high copy plasmid library of S. cerevisiae genomic
DNA for loci present in this organism that would elevate o-DNB
tolerance. A gene was recovered that was designated ROD1 (resistance to o-dinitrobenzene). This locus conferred
resistance to o-DNB, calcium, and zinc when carried on a high
copy plasmid. The ROD1 gene product (Rod1p) represents an
unusual class of resistance determinant and shares no significant
homology with proteins of known function. ROD1 is not
essential to the cell, but a rod1 mutant strain possesses
a hypersensitive phenotype to o-DNB, zinc, calcium, and
diamide.
The different protein fractions obtained were electrophoresed on sodium dodecyl sulfate (SDS)-8% polyacrylamide gels before being transferred to nitrocellulose(27) . Monoclonal antibody 9E10 (24) was used as the primary antibody to detect the c-Myc epitope. Monoclonal antibodies against carboxypeptidase Y (Molecular Probes), tubulin (YOL1/34, Seralabs), and Ras mAb Y13-259 were used as primary antibody for controls. Detection of the primary antibody was performed by using the chemiluminescence detection system (ECL, Amersham Corp.).
A plasmid (pSM84) expressing the I. orientalis
GST gene was able to tolerate 250 µMo-DNB,
while the expression vector alone was only able to support growth below
200 µMo-DNB (Fig. 1). While resistance to o-DNB was increased, S. cerevisiae transformants
carrying pSM84 did not show increased resistance to several other
inhibitors tested, including cycloheximide, cadmium, and
1,10-phenanthroline (data not shown). Furthermore, GST enzyme activity
present in S. cerevisiae transformants carrying pSM84 or a
high copy YAP1 plasmid was elevated relative to the vector
alone. GST activity produced by cells carrying pSM84 was 24.4
milliunits/mg, while cells bearing the expression vector only produced
2.8 milliunits/mg. Transformants bearing a 2-µm YAP1 elevated GST activity to 6.4 milliunits/mg. Also yap1 yeast strains were found to be hypersensitive to o-DNB
(see below). These data suggested that S. cerevisiae possesses
an o-DNB detoxification system that involves yAP-1 and might
also employ other glutathionedependent proteins (like GST). To further
explore the mechanisms of detoxification of o-DNB, we screened
a high copy plasmid library for loci that could elevate resistance to
this compound.
Figure 1: Function of I. orientalis glutathione S-transferase in S. cerevisiae transformants. S. cerevisiae strains containing (SEY6210) or lacking (SM10) the YAP1 gene were transformed with the indicated plasmids. Cells were transformed with plasmids overproducing the yAP-1 (pSEY18-R2.5) or I. orientalis glutathione S-transferase (pSM84) proteins. Transformants bearing the empty vector (pSEY18) were used as a control for the effect of overproduction. Appropriate transformants were placed on YPD medium with no drug (YPD) or the same medium containing 200 or 250 µMo-DNB. SM10 transformed with pSEY18-R2.5 was not tested.
The wild-type S. cerevisiae strain SEY6210
was transformed with DNA from a YEp24 S. cerevisiae genomic
library(18) . Approximately 10,000 Ura transformants were tested for o-DNB tolerance by replica
plating to YPD plates containing 400-µMo-DNB.
Plasmid DNAs from 10 o-DNB resistance colonies were recovered
and reintroduced back into the SEY6210 strain. Two different classes of
plasmid DNAs were able to reproducibly restore resistance to
400-µM o-DNB. We focused our attention on the function of
the plasmid containing the smallest insert of S. cerevisiae genomic DNA. This plasmid was designated 24-4-1. Restriction
mapping of this plasmid indicated that the insert was approximately 8.0
kb (Fig. 2). The gene conferring o-DNB resistance was
designated as ROD1 (resistance to o-dinitrobenzene).
Figure 2: Subcloning analysis of the ROD1 gene. The restriction map of the original ROD1 isolate (24-4-1) is shown on the top line. A bar indicating the relative scale in kilobase pairs is noted on the left-hand side of the figure. Restriction sites are abbreviated as: X, XhoI; B, BamHI, BII, BglII; E, EcoRI; H, HindIII. The cross-hatched bars indicate the amount of DNA present in the various subclones. The relative resistance phenotype of each plasmid when transformed into wild-type S. cerevisiae cells (SEY6210) is shown in the column labeled o-DNB Resistance Phenotype. + indicates that appropriate transformants were able to grow in the presence of 250 µMo-DNB, while - denotes the inability to tolerate this level of o-DNB. The location of the ROD1 open reading frame (ORF) is shown at the top of the figure.
DNA sequence analysis revealed a single large open reading frame of 837 amino acids with a calculated molecular mass of 92 kDa. Several TATA-like sequences are present upstream of ROD1 and a potential transcription termination sequence (TATATA) (29) is located from 2592 to 2597 in the 3`-noncoding region, 77 base pairs downstream of the translation termination codon. No good candidates for yAP-1 recognition elements were found in the ROD1 5`-flanking region.
Rod1p is rich in serine and asparagine residues containing 15 and 9%, respectively, of these amino acids. Computer search indicated that other than similarities based on the high content of these two amino acids, Rod1p did not show significant similarity to any proteins of known function in the data base. ROD1 has also been sequenced as a gene of unknown function present on chromosome XV as part of the effort to sequence the yeast genome (GenBank(TM) accession number: X87331). However, striking sequence similarity was found between Rod1p and a protein of unknown function identified on chromosome IV during the sequencing of this chromosome. This locus was designated YFR022W and shares 43% sequence identity with Rod1p throughout the length of both protein chains (Fig. 3). The most extensive sequence identity is in the amino-terminal segments of these two different proteins. A more distantly related protein encoded on chromosome II (YBL101c) was also found to exhibit significant sequence similarity to Rod1p (20%), but no functional information is available for this factor.
Figure 3: Sequence similarity between a predicted gene product from S. cerevisiae chromosome VI and Rod1p. A computer alignment generated through use of the Gap subroutine of the GCG software package (36) is shown. The Rod1p-like sequence is listed on the top lines while Rod1p is on the bottom. The one-letter code for amino acids is used throughout. A vertical bar indicates sequence identity, while one or two dots signify lesser or greater sequence similarity, respectively. The sequence information reported here was deposited into GenBank(TM) with the accession number U40561.
Computer
analysis of the Rod1p sequence indicated that this protein was highly
basic with a net charge of +10 at pH 7. Hydropathy analysis of
Rod1p indicated that the 350 amino acids at the COOH terminus are
predominantly hydrophilic, while the NH-terminal
three-fifths of the protein exhibited a more hydrophobic character.
Analysis of potential transmembrane segments using the Protean program
of the Lasergene sequence analysis package (DNAstar, Inc.) indicated
that one segment of Rod1p(246-263) might be able to serve as a
membrane spanning domain. Twelve potential N-linked
glycosylation sites (Asn-X-Ser/Thr) (30) are found in
the COOH-terminal region, although these may occur due to the high
asparagine and serine content of the factor. At least in the context of
the Rod1p-Myc fusion protein (see below), we were not able to detect
any endoglycosidase H-sensitive molecular mass forms of the protein
(data not shown).
Figure 4: Expression and function of epitope-tagged Rod1p. A, the Myc epitope was introduced onto the carboxyl terminus of Rod1p as described under ``Materials and Methods.'' A plasmid expressing this Rod1p-Myc fusion (pAW75) or the untagged parent plasmid (pAW49) were introduced into wild-type SEY6210 cells. Protein extracts were made and subjected to Western blotting analysis. The resulting blot was probed with the 9E10 anti-Myc antibody. Visualization of the antibody was accomplished through use of the ECL kit (Amersham). B, confirmation that the epitope-tagged Rod1p maintained normal function was achieved by comparing the o-DNB resistance of plasmids expressing wild-type Rod1p(24-4-1) or the Myc-tagged (pAW75) and untagged (pAW49) versions of this protein. A vector lacking ROD1 sequences was also tested as a control (pRS426). All plasmids were introduced into the wild-type strain SEY6210.
Whole cell protein extracts were prepared from wild-type cells carrying either pAW49 or pAW75. Western blot analysis of these extracts was carried out using the anti-Myc monoclonal antibody 9E10. A polypeptide with a molecular mass of approximately 100 kDa was detected in crude extracts from cells expressing the 2-µm Rod1p-Myc fusion construct but not from cells producing the untagged Rod1p (Fig. 4A). The predicted mass of the Myc-tagged Rod1p-Myc was 81 kDa. The cause of this discrepancy in the expected and observed molecular masses is not known but may be due to the unusual amino acid composition of this protein.
Figure 5:
Subcellular fractionation of Rod1p-Myc. A
crude protein extract was prepared from SEY6210 cells carrying a
plasmid expressing the Rod1p-Myc fusion protein. This crude extract was
resolved into particulate (P) or soluble (S)
fractions by differential centrifugation at 10,000 g (P10 and S10) or 100,000
g (P100 and S100). Prior to centrifugation, the
extracts were treated with lysis buffer, 0.1 M Na
CO
, or Triton X-100 (TX-100).
Aliquots of each sample were electrophoresed on SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose for Western blot
analysis. The filter was probed with antibodies directed against the
Myc epitope (Rod1p-Myc), Ras (Ras), or tubulin protein (TUB).
The location of molecular mass markers is shown on the left-hand
side of the figure.
This analysis indicated
that neither treatment with alkaline carbonate buffer (0.1 M NaCO
(pH 11)) nor the non-ionic detergent
Triton X-100 was able to release significant amounts of Rod1p. As a
control, the known plasma membrane-associated yeast proteins Ras1p and
Ras2p were examined. Treatment with alkaline carbonate buffer did not
release detectable amounts of Ras, whereas Triton X-100 efficiently
solubilized Ras from total membranes. To compare the behavior of Rod1p
with a known cytoskeleton protein, fractionation of yeast S.
cerevisiae protein was examined on the same blot using an
anti-tubulin antibody. The analysis indicated that the tubulin protein
stays in the supernatant fraction (S10 and S100) after 10,000
g and 100,000
g centrifugation.
To investigate the possibility that Rod1p might be a plasma membrane protein, differential subcellular fractions from cells carrying the pAW75 plasmid (Rod1p-Myc), enriched either in soluble portions, in mitochondria and ribosomal components, or in plasma membranes, were analyzed. Fig. 6shows that in both the crude membrane fraction and plasma membrane-enriched fraction, an approximately 100-kDa Rod1p-Myc fusion protein was detected. Ras, a known plasma membrane-associated protein, was found in the same fractions after immunoblotting, while the intracellular protein carboxypeptidase Y was detected in the soluble protein fraction. These data are consistent with the possibility that Rod1p is a plasma membrane-associated protein or associated in a large complex that cofractionates with the plasma membrane.
Figure 6:
Plasma membrane fractions are enriched for
Rod1p-Myc. SEY6210 cells were broken by glass bead lysis and separated
into particulate (P100) and soluble (S100) fractions
by centrifugation at 100,000 g. The P100 fraction was
then acid-extracted as described (3) to generate a
mitochondrial/ribosome-enriched (MIT-RIB) fraction and plasma
membrane-enriched (PL-MEM) fraction. Aliquots of each sample
were electrophoresed on SDS-polyacrylamide gel electrophoresis and
subjected to Western analysis. The antibodies used to visualize the
locations of the indicated proteins are described in the legend to Fig. 5with the exception of a mouse monoclonal directed against
carboxypeptidase Y (CPY). Molecular mass markers are indicated
on the left-hand side of the
figure.
There was no
detectable difference in growth between wild-type and rod1 cells in the absence of a toxic agent in the medium. This finding
suggests that ROD1 is not an essential gene. However, several
differences in growth between the wild-type and
rod1 mutant strains were observed on YPD plates containing o-DNB, zinc, diamide, or calcium (Fig. 7). The
rod1 mutant strain was unable to grow on plates
containing 200 µMo-DNB or 10 mM ZnCl2,
while the wild-type strain was still able to grow at these
concentrations. In addition, the
rod1 strain grows slower
than wild-type in medium containing 1.2 mM diamide or 10
mM CaCl
. These findings indicated that ROD1 gene plays a physiological role in resistance to o-DNB,
zinc, diamide, and calcium. These data also indicate that the role of
Rod1p in drug resistance is not limited to o-DNB tolerance.
Figure 7:
Overlapping pleiotropic phenotypes of rod1 and yap1 mutants. Yeast strains were constructed
that lacked the ROD1 (YAW14) gene or both the ROD1 and YAP1 loci (YAW15). These strains were assayed by spot
test analysis (20) along with isogenic wild-type (SEY6210) and yap1 (SM10) cells. Strains were tested on YPD medium
containing the indicated concentrations of inhibitors and were
photographed according to the pattern shown on the top of the
figure. Bars indicate the two different inhibitor
concentrations tested for o-DNB, diamide, and
CaCl
.
Since both yap1 and
rod1 strains were
hypersensitive to o-DNB, zinc, and diamide, we wanted to
further examine the overlap in resistance between these two genes. To
accomplish this, a strain lacking both these loci (
rod1/
yap1) was constructed. This strain
was designated YAW15. The
yap1/
rod1 strain
(YAW15) was dramatically more sensitive to o-DNB and calcium
than either
yap1 or
rod1 cells. This
finding indicates that the products of these loci are likely to
physiologically contribute to o-DNB and calcium tolerance. The
yap1/
rod1 strain was slightly more
sensitive to diamide than cells lacking the YAP1 gene,
although YAP1 was clearly the major resistance determinant. In
contrast, a strain lacking the ROD1 gene was quite sensitive
to zinc, but this sensitivity was not detectably enhanced in the
yap1/
rod1 background. These data indicate
that the functions of YAP1 and ROD1 are additive and
contribute roughly equally in terms of resistance to some agents (o-DNB, calcium). However, YAP1 contributes more to
diamide tolerance, and ROD1 has the major effect on zinc
resistance.
Figure 8:
Phenotypic suppression of rod1 and yap1 phenotypes by amplification of ROD1 or YAP1. A, functional dependence of a high copy ROD1 plasmid on the presence of the YAP1 gene. Either
high copy pRS426 vector only (vector) or the same plasmid containing
the ROD1 gene (2-µm ROD1) was transformed into
wild-type S. cerevisiae cells (wt) or isogenic yap1 mutants (yap1). Resistance phenotypes were
then assayed as described in the legend to Fig. 7. B,
high copy YAP1 suppression of rod1 mutant phenotypes.
A 2-µm plasmid containing the YAP1 gene (2-µm YAP1) was introduced into isogenic wild-type (wt) or rod1 (
rod1) cells and assayed for drug
resistance by spot test assay.
High copy ROD1 was only able to elevate o-DNB and zinc tolerance normally in the presence of an intact copy of chromosomal YAP1. In opposition to this YAP1 dependence of the ROD1 effects on o-DNB and zinc, loss of YAP1 had no effect on high copy ROD1-mediated calcium resistance. The 2-µm YAP1 plasmid did not elevate o-DNB or calcium resistance in a strain lacking the ROD1 gene. However, high copy YAP1-mediated zinc tolerance was not dependent on the presence of ROD1. These data are consistent with the belief that Rod1p and yAP-1 each contribute separate functions to o-DNB resistance with both of these functions necessary for normal tolerance to result. However, the relationship of Rod1p and yAP-1 to zinc and calcium resistance phenotypes suggests a more complicated interaction of these two gene products. This analysis clearly shows that Rod1p and yAP-1 are both key participants in the cellular handling of several different toxic compounds. Additionally, assay of GST present in ROD1 high copy transformants indicated that the activity of this enzyme was not elevated relative to a vector control (data not shown). This observation strongly suggests that the effect of Rod1p on drug resistance does not come about through an increase in GST activity.
Previous studies on pleiotropic drug resistance of yeast have provided information about several different gene products involved in allowing cells to deal with cytotoxic agents in their environment (reviewed in (2) ). Most of these loci have been integral membrane transport proteins and transcription factors. In the work described here, we provide an example of a new type of resistance determinant that does not obviously fit into these two well known classes of gene products.
Numerous studies in several laboratories
have established that elevating the copy number of yAP-1 increases
resistance to cycloheximide, 1,10-phenanthroline, cadmium,
HO
, and numerous other
agents(20, 31, 32, 33) . Our
observation that high copy ROD1 does not elevate tolerance to
all the same drugs as yAP-1 suggests that Rod1p is not likely to act
through yAP-1. Furthermore, Northern blot and lacZ gene fusion
experiments have established that ROD1 is not under the
transcriptional control of yAP-1. (
)These data are
consistent with the belief that Rod1p acts to contribute separate
functions to several yAP-1-mediated resistances.
The details of how
Rod1p acts are still unknown, but this factor clearly provides a
limiting function for o-DNB, calcium, and zinc resistance.
Loss of ROD1 causes cells to become hypersensitive to these
compounds, while overproduction of Rod1p elevates resistance to each of
these agents. The hypersensitivity elicited by loss of ROD1 was exacerbated with concomitant removal of YAP1, with
the exception of zinc resistance. Disrupting YAP1 in a rod1 background does not further reduce zinc tolerance
while overproduction of yAP-1 completely suppresses the need for ROD1. These data suggest that Rod1p is necessary for
physiological zinc homeostasis, while high levels of yAP-1 can bypass
this requirement. This situation is reminiscent of the effect of yAP-1
on cycloheximide resistance. Overproduction of yAP-1 leads to high
level cycloheximide tolerance, but a
yap1 strain is no
more sensitive to cycloheximide than a wild-type strain (34) .
Both yAP-1 and Rod1p are required for o-DNB resistance irrespective of the level of either factor. However, while both YAP1 and ROD1 are necessary for calcium resistance when these genes are present in single copy, the presence of ROD1 on a high copy plasmid eliminates the need for yAP-1. This observation suggests that the mechanism of resistance to o-DNB and calcium is not likely to be the same, since there are different genetic requirements for yAP-1 and Rod1p to effect tolerance to these compounds.
The localization information provided here provides a framework for beginning to understand the action of Rod1p. Our data suggests that Rod1p is associated with the plasma membrane fraction of yeast cells, but further experimentation is necessary to confirm this suggestion. Attempts to localize the Rod1p-Myc fusion protein by immunofluorescence were not successful, possibly owing to the finding that this fusion protein was extremely sensitive to zymolyase treatment (data not shown). A similar sensitivity has been noted for the plasma membrane protein Ste6p(35) . This proteolytic sensitivity of Rod1p-Myc is consistent with the enhanced accessibility of the fusion protein that could occur if the factor was exposed to the outside of the cell. The possibility that Rod1p is exposed to the outside of the S. cerevisiae cell is being investigated.
This work establishes several new facts about drug resistance in S. cerevisiae. First, production in S. cerevisiae of a fungal GST protein from I. orientalis is capable of elevating resistance to at least one toxic agent (o-DNB). Second, S. cerevisiae possesses a locus designated ROD1 that is required, along with the transcription factor yAP-1, for normal o-DNB tolerance. Additionally, Rod1p enhances the ability of the cell to tolerate several compounds known to be under the phenotypic influence of yAP-1. However, Rod1p only affects a small subset of the large number of resistance phenotypes influenced by yAP-1, indicating a much narrower range of actions for Rod1p. Finally, the ROD1 gene product fractionates in the particulate component of S. cerevisiae, unlike the soluble yAP-1 protein (data not shown). Understanding the action of Rod1p will provide important new information about how this novel protein influences drug resistance in eukaryotes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U40561[GenBank].