From the Unité de Biochimie Physiologique,
Université Catholique de Louvain, Belgium and the
Institut
für Biochemie und Lebensmittelchemie, Technische
Universität, Graz, Austria
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ABSTRACT |
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The Saccharomyces cerevisiae open
reading frame YNL231C was recently found to be controlled
by the multiple drug resistance regulator Pdr1p. Here we characterize
YNL231C (PDR16) and its homologue
YNL264C (PDR17). Deletion of PDR16
resulted in hypersensitivity of yeast to azole inhibitors of ergosterol
biosynthesis. While no increase in drug sensitivity was found upon
deletion of PDR17 alone, a The yeast Saccharomyces cerevisiae has, like many other
organisms, the ability to acquire multiple drug resistance,
i.e. become less sensitive to a broad range of chemically
and functionally unrelated cytotoxic compounds (1, 2). In yeast this
phenomenon can be provoked by a regulatory disorder, namely a mutation
in the transcription factors Pdr1p or Pdr3p. Pdr1p and Pdr3p are homologous Zn2Cys6 DNA-binding proteins which
control the expression of drug efflux pump-encoding genes (3, 4).
Gain-of-function mutations in the PDR1 or PDR3
genes may result in increased production of these efflux pumps, leading
to drug resistance (5, 6). Neither Pdr1p or Pdr3p nor the drug efflux
pumps which they regulate are required for growth of yeast in the
absence of drugs. It is not known whether the true physiological
function of these drug resistance determinants is to protect the cell
from external toxic compounds or whether they may play other roles. In
order to get more insight into the physiological role of Pdr1p, we
recently screened for target genes regulated by this transcription
factor. This screening resulted in the identification of a broad range of novel Pdr1p target genes, one of which was the open reading frame
with the systematic name YNL231C (PDR16).
Expression of the PDR16 gene is five times higher in strains
carrying pdr1-3, a strong constitutive allele of
PDR1, as compared with isogenic wild-type strains or strains
deleted for
PDR1.1
The PDR16 gene encodes for a protein of 351 amino acids.
This protein is 49% identical and 75% similar to the product of the YNL264C (PDR17) gene of S. cerevisiae.
Neither PDR16 nor PDR17 has been functionally
characterized. The Pdr16p is also 23% identical and 54% similar to
the product of the S. cerevisiae SEC14 gene. This homology
is spread throughout the protein sequence. However, three sequence
blocks which are strongly conserved among the SEC14 proteins
from different yeasts (around amino acid positions 55-60, 205-210,
and 235-240) (7) are also the most conserved areas between these
proteins and PDR16 and PDR17.
Sec14p was initially identified as a
PtdIns2 transfer protein
(PITP) which can perform transfer of phospholipids between membranes in vitro (8, 9). Subsequently, it was shown that Sec14p/PITP is required for transport of proteins through the Golgi complex (10).
It has been proposed that in vivo Sec14p senses the levels of PtdIns and PtdCho in the Golgi complex and exerts negative feedback
on PtdCho synthesis through the Kennedy pathway (11). More recently, it
was suggested that Sec14p/PITP may also regulate formation of secretory
vesicles from the Golgi by stimulating the turnover of phospholipids
(12, 13). The involvement of PITP in signal transduction of higher
eukaryotes has been discussed (14-16).
In the present work, we analyze the role of the distant yeast
SEC14 homologues, PDR16 and PDR17, in
drug resistance and lipid biosynthesis/sorting in a wild-type and a
pdr1-3 background. We find that deletion of
PDR16 leads to a strongly increased sensitivity to azole
antifungals. Deletion of both PDR16 and PDR17
leads to reduced resistance to a broad range of drugs. We, furthermore, show that the mutations affect the phospholipid and sterol composition of the plasma membrane, and that they change the total yeast lipid composition. We propose that the azole sensitivity of the
Strains and Deletions of Genes--
Escherichia coli
strain DH5
A construct for the disruption of the YOR1 gene (pDK30) (21)
was kindly supplied by Dr. W. Scott Moye-Rowley. All deletions of the
PDR16 gene and the deletions of the PDR17 gene in
US50-18c and its derivatives were constructed using the procedure
described by Alani et al. (22). A DNA fragment corresponding
to the 5'-flanking region upstream of the gene was generated by PCR and
cloned into pSK+ (Stratagene). For the PDR16 gene, the
primers for the PCR were 5'-GCACGAATTCTCAAAGACGGCGGATTCA-3' and
5'-AACCGGATCCCCTGGGTCTTCTGGAGCCC-3', while for the PDR17
gene, the primers were 5'-ACCGAATTCTGATTGAAGAGATCAAAGA-3' and
5'-TAAGGATCCGGCAGGAGGGTCCAA-3'. Subsequently, a 3'-flanking fragment, encompassing the 3' end of the gene, was generated by PCR. For the PDR16 gene, the PCR primers were
5'-TTTGGATCCTTGGTTAGCATGG-3' and 5'-TTGGAGCTCAGTGCATATAGACGCG-3', while
for PDR17, they were: 5'-ATGGGATCCTTGGAGGCATTGTCGGA-3' and
5'-CGGGAGCTCGATTGATTAGCTGGAAC-3'. This 3'-flanking PCR product was
subcloned into the plasmid that already contained the 5'-upstream
fragment. The resulting plasmids were termed pBVH742 for
PDR16 and pBVH1068 for PDR17. In a final subcloning step, a 3.8-kilobase BamHI-BglII
fragment containing a hisG-URA3-hisG cassette (22) was
inserted into the BamHI site between the 5'- and 3'-flanking
regions of the gene, resulting in the plasmids pBVH778
(PDR16) and pBVH1127 (PDR17). Each of the
resulting plasmids was treated with EcoRI and
SacI to generate a linear fragment consisting of the 5'- and
3'-flanking regions of the gene interrupted by
hisG-URA3-hisG. The fragment was transformed into yeast and
cells that had become Ura+ due to chromosomal replacement
of wild-type gene by the linear fragment were selected on SC plates
lacking uracil. Subsequently, the URA3 gene was
"looped-out" by growth on non-selective media, allowing
recombination between the hisG sequences. Cells in which a
loop-out event had occurred were selected by plating on media containing 5-fluoroorotic acid (22).
Deletions of PDR17 in strain FY1679-28C and its derivatives
were generated by one-step gene replacement as described by Winston et al. (23). A 1.15-kilobase BamHI fragment from
plasmid YDp-H, containing the HIS3 gene, was subcloned into
pBVH1068, resulting in plasmid pBVH1451. The pBVH1451 plasmid was
subsequently treated with EcoRI and SacI to
generate a linear fragment consisting of the 5'- and 3'-flanking
regions of the PDR17 gene interrupted by HIS3.
This fragment was transformed into yeast, and cells that had become
His+ due to chromosomal replacement of the wild-type
PDR17 gene by the linear fragment were selected on SC plates
lacking histidine.
All deletions were verified by PCR amplification of a chromosomal
region encompassing the deletion, followed by analysis of the PCR
product by agarose gel electrophoresis. Furthermore, the absence of an
mRNA signal from PDR16 in the US50-18c derivative deleted for this gene was verified by Northern blotting, using a
fragment of the gene as a probe.
Other Plasmid Constructions--
Centromeric plasmids containing
the PDR16 and PDR17 genes were obtained by "gap
repair." An EcoRI-SacI fragment consisting of
the 5'- and 3'-flanking regions of the PDR16 gene was
subcloned from pBVH742 into the URA3 centromere plasmid
pRS316 (24), resulting in plasmid pBVH1222. Similarly, a
ClaI-SacI fragment consisting of the 5'- and
3'-flanking regions of the PDR17 gene was subcloned from
pBVH1068 into pRS316, giving pBVH1378. The pBVH1222 and pBVH1378 plasmids were linearized using BamHI and transformed into
yeast strain US50-18c. Cells in which recombination between the
linearized plasmid and the chromosomal locus had generated a circular
centromeric plasmid containing the entire gene were selected on media
lacking uracil. Plasmids were isolated from yeast, transformed into
E. coli, and restriction analysis of plasmid preparations
was performed in order to verify the presence of the gene. A multicopy
plasmid containing PDR16 was generated by insertion of a
SalI-SacI fragment from the single-copy plasmid
containing the gene into pRS426 (25).
Growth Media and Drug Resistance Assays--
E. coliwas
grown in standard Luria broth medium (17). Yeast was grown on standard
rich glucose (YPD) or glycerol (YPG) media, or on SC medium lacking
appropriate amino acids for plasmid maintenance (26). For drug
resistance assays on solid media, drugs were added to the media
immediately prior to pouring. The drug concentrations tested were the
following: 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 1.5, 2, 4, and
8 µg of cycloheximide (Sigma, stock in ethanol) per ml of YPD; 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 200 µg of rhodamine-6-G (Merck,
stock in ethanol) per ml of YPG; 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2, and 5 µg of oligomycin (Sigma, stock in ethanol) per ml of YPG;
0.1, 0.25, 0.5, 1, 2, 3.5, 5, and 10 µg of 4-nitroquinoline oxide
(Sigma, stock in dimethyl sulfoxide) per ml of YPD; 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2, 5, 10, and 20 µg of miconazole (kindly
supplied by Janssen Pharmaceutica, stock in dimethyl sulfoxide) per ml
of YPD; 0.1, 0.25, 0.5, 1, 2, 5, and 10 µg of ethidium bromide
(Boehringer Mannheim, stock in water) per ml of YPG; 2, 5, 10, 15, 20, 25, 30, 40, 50, and 80 µg of nystatin per ml of YPD. 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, and 50 µg of ketoconazole (kindly supplied by Janssen Pharmaceutica, stock in dimethyl sulfoxide) per ml
of YPD; 0.5, 1, 2, 5, 10, 25, 50, and 100 µg of itraconazole (kindly
supplied by Janssen Pharmaceutica, stock in dimethyl sulfoxide) per ml
of YPD; 0.25, 0.5, 1, 2, 5, 10, and 20 µg of crystal violet per ml of
YPD; 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, and 1 µg of antimycin A
(Sigma, stock in dimethyl sulfoxide) per ml of YPG.
Growth tests on non-fermentable carbon sources, at high pH, osmolarity
and temperature were performed as described by Rank et al.
(27), i.e. on solid media containing 0.1% yeast extract, 0.5% bacto-peptone, and 100 mM potassium phosphate, pH
7.1, supplemented with 1% glucose (with or without 0.5 M
potassium chloride), glycerol or ethanol. Plates were incubated at 30 or 37 °C.
Rhodamine-6-G Uptake Assay--
Approximately 107
cells from an overnight culture were inoculated in 20 ml of YPD and
grown for 3 h at 30 °C. About 5.6 × 108 cells
were pelleted and washed three times with buffer A (50 mM
HEPES/NaOH, pH 7.0). The cells were subsequently resuspended in 4 ml of
de-energization buffer (1 µM antimycin A, 5 mM 2-deoxy-D-glucose in buffer A), incubated
for 2 h 30 min at 30 °C, and transferred to a water bath at
20 °C. A 200-µl aliquot of the de-energized cell suspension was
pelleted, washed once with 200 µl of cold buffer A, and resuspended
in 2 ml of the same cold buffer. Cell fluorescence background was
measured using an SLM Aminco 48000 S spectrofluorimeter. The excitation
wavelength was 529 nm (4 nm slit), and the emission wavelength was 553 nm (4 nm slit). Rhodamine-6-G was added to the remaining cell
suspension to a final concentration of 5 mM, and 200-µl
aliquots were taken every 5 min up to 1 h. The cells of each
aliquot were pelleted, washed, and resuspended as described above and
immediately subjected to fluorescence measurements. The cell
fluorescence background values after 0- and 1-h incubation were
averaged, and this average was subtracted from each fluorescence value.
Isolation of Yeast Subcellular Fractions--
Cells were grown
on YPD medium containing 2% glucose, 2% peptone (Oxoid), and 1%
yeast extract (Oxoid) under aerobic conditions at 30 °C. Growth of
yeast cells was monitored by measuring the OD (600 nm).
Highly purified plasma membrane was isolated as follows. The pellet of
crude plasma membrane (28) was suspended in 5 mM Mes, 0.2 mM EDTA, pH 6.0, with 10 strokes in a loose-fitting Dounce homogenizer and layered on top of a sucrose density gradient made of 10 ml of 38% (w/w), 10 ml of 43% (w/w), and 10 ml of 53% (w/w) sucrose
in 5 mM Mes, 0.2 mM EDTA, pH 6.0. Centrifugation was carried out at 100,000 × g for
2.5 h in an SW-28 rotor (Beckman). The highly purified plasma
membrane was withdrawn from the 43/53% sucrose interface, the
suspension was diluted 3-fold with 10 mM Tris-HCl, pH 7.4, and the plasma membrane was sedimented at 48,000 × g
for 20 min in an SS-34 rotor (Sorvall). The plasma membrane pellet was
suspended in 10 mM Tris-HCl, pH 7.4, using a loose-fitting Dounce homogenizer and stored at
Yeast spheroplasts and mitochondria were isolated by published
procedures (29). Microsomal fractions were prepared from the
post-mitochondrial supernatant that had been cleared of small mitochondria by centrifugation for 30 min at 20,000 × g in an SS-34 rotor (Sorvall). The resulting supernatant was
subjected to successive steps of differential centrifugation at 30,000, 40,000, and 100,000 × g (30). The 100,000 × g supernatant contains the cytosolic proteins. Protein
content and quality of the preparations as well as cross-contamination
with other organelle membranes were assessed as described previously
(30-33).
Lipid Analysis of Whole Cell Extracts and Subcellular
Fractions--
Cells were homogenized for 3 min with CO2
cooling in the presence of glass beads using a Merckenschlager
Homogenizer. Lipids of whole yeast cells and organelle preparations
were extracted by the procedure of Folch et al. (34).
Analysis of individual phospholipids and neutral lipids was carried out
by published procedures (35, 36). Alkaline hydrolysis of lipid extracts was carried out as described elsewhere (37). Individual sterols were
analyzed by gas-liquid chromatography (GLC) on an HP 5890 Series II
Plus GC equipped with electronic pressure control and an HP chemstation
software package. An HP 5972 mass selective detector and authentic
standards were used for identification of sterols. Injector and
interface were kept at 250 and 300 °C, respectively. GLC/MS analysis
was performed on a capillary column, HP-5MS 30 m × 0.25 mm × 0.25-µm film thickness, programmed from 150 °C to 320 °C at
20 °C/min after a 2-min hold at 150 °C. Finally, the column was
kept at 320 °C for 10 min. All analyses were carried out in the
constant flow mode. Helium was used as carrier gas with a linear
velocity of 34.1 cm/s. One-µl aliquots of the samples were injected
with an HP 7673 autosampler in splitless mode. Electron impact
ionization with 70 eV ionization energy was used for mass spectrometry.
Data were collected by scanning from 150 to 600 atomic mass units at
1.6 scans/s.
Alternatively, GLC was performed on an HP 5890 equipped with a flame
ionization detector (FID) operated at 320 °C using a capillary
column (HP5, 30 m × 0.32 mm × 0.25-µm film thickness). After a 1-min hold at 50 °C the temperature was increased to
310 °C at 10 °C/min. The final temperature was held for 10 min.
Nitrogen was used as carrier gas and 1-µl aliquots of samples were
injected cool on column. Relative retention times of sterols were
similar as described previously (38-40).
Determination of Lipid Transfer Activity--
Lipid transfer
activity of cytosolic fractions and peripheral organelle membrane
proteins was measured according to Ceolotto et al. (41).
Integral and peripheral membrane proteins were separated by treatment
of organelle membranes with 0.25 M KCl for 20 min on ice.
Insoluble membrane components were sedimented by centrifugation at
100,000 × g for 1 h, whereas solubilized proteins
were recovered in the supernatant.
The rate of protein-catalyzed transfer of fluorescently labeled
phospholipids from small unilamellar donor vesicles to unlabeled unilamellar acceptor membranes was measured as described previously (9,
40-43). The phospholipid transfer activity was measured using a
Shimadzu RF-5301 spectrofluorimeter. The excitation wavelength was set
at 342 nm (1.5 nm slit) and the emission wavelength was set at 380 nm
(3 nm slit). The assay was performed for 7 min with measurements taken
every 0.5 s. Fluorescently labeled PtdCho (44), PtdIns (45),
PtdSer (46), and N-trinitrophenyl phosphatidylethanolamine (47) were synthesized by published procedures.
Anisotropy Measurement--
Fluidity of the plasma membrane was
determined in vitro by measuring the fluorescence anisotropy
of TMA-DPH. Samples containing 100 µg of membrane protein were
incubated with 2.7 nmol of TMA-DPH for 30 min at 30 °C. Fluorescence
measurements were carried out using a Shimadzu RF 5301 spectrofluorimeter as described previously (48).
Drug Sensitivity of Strains Deleted for PDR16 and/or Its Homologue
PDR17--
We recently identified PDR16 (YNL231C) as one of
several novel genes controlled by the yeast multiple drug resistance
regulator Pdr1p.1 To investigate whether PDR16
like other Pdr1p targets is involved in multiple drug resistance, we
deleted the gene and studied the effects on drug sensitivity. In order
to maximize the possible effects, the deletion was made in a
pdr1-3 genetic background (strain US50-18c) which led to
overexpression of Pdr16p.
Deletion of PDR16 had no effect on yeast growth in the
absence of drugs. However, the PDR16-deleted strain
(
To verify whether the drug sensitivity phenotype was indeed due to loss
of PDR16 gene function, we introduced a single-copy plasmid
carrying the intact PDR16 gene in the
The PDR16 gene has a close homologue in S. cerevisiae termed PDR17 (YNL264C). To test whether
there is a functional relationship between these two genes, we
generated an isogenic strain deleted for PDR17, and a double
mutant deleted for both PDR16 and PDR17. The
single PDR17-deleted strain (
The PDR16- and PDR17-related phenotypes that we
described thus far were observed in the US50-18c genetic background.
US50-18c is highly drug resistant due to the pdr1-3
mutation which results in a strong overexpression of Pdr1p-regulated
drug efflux pumps such as Pdr5p, Snq2p, and Yor1p (5, 49). In order to
test whether the PDR16- and PDR17-related
phenotypes were specific for this particular genetic background, or
whether they also occurred in an otherwise wild-type context, we
deleted these two genes in the wild-type strain FY1679-28c and studied
drug sensitivity phenotypes.
As expected, due to lack of overexpression of the drug efflux pumps,
the FY1679-28c strain was generally much more sensitive to most drugs
than the US50-18c strain. As can be seen from Table II, however, the effects of deletion of
PDR16 and/or PDR17 in wild-type were roughly the
same as in US50-18c. The sensitivity to miconazole and ketoconazole
was increased about 10-20-fold upon deletion of PDR16, and
most drug sensitivities were increased 2-5-fold upon additional
deletion of PDR17. Thus, the
PDR16/PDR17-dependent drug sensitivity
phenotypes are not specific for US50-18c, but can also be observed in
a wild-type genetic background. The lower resistance of FY1679-28c
toward crystal violet and rhodamine-6-G allowed detection of a slightly
increased sensitivity to these drugs in the Deletions of PDR16 and PDR17 Rather Affect Drug Uptake Than Drug
Efflux--
Two yeast drug efflux pumps known to mediate resistance to
azoles are Pdr5p and Yor1p (50). In order to investigate whether the
effects of PDR16 on azole resistance were due to reduced
Pdr5p and Yor1p function, we constructed strains deleted for
PDR16 as well as for PDR5 and/or YOR1,
and investigated their drug sensitivities. As can be seen in Table
III, a triple mutant
To test whether a difference in passive drug transport was the reason
for the reduced drug resistance of the PDR16- or
PDR17-deleted strains, we investigated drug uptake into
cells in which active transport was blocked by energy depletion. As a
probe for drug uptake we used rhodamine-6-G, a toxic pink colored
fluorescent dye to which
Exponentially growing cells of wild-type, Plasma Membrane Lipid Composition of Strains Deleted for PDR16
and/or PDR17--
The fact that
Tables IV and V show that deletion of PDR16 and
PDR17 in the FY1679-28c and US50-18c background caused
several alterations of the plasma membrane lipid composition. Whereas
the amount of total phospholipids was similar in all strains tested and
the pdr1-3 mutation did not have marked effects, the
pattern of individual phospholipids was significantly changed in the
plasma membranes of
The total sterol content of the plasma membrane (Table
V) was significantly reduced by the
Despite the marked changes of the plasma membrane lipid composition
caused by the Total Lipid Composition of Strains Deleted for PDR16 and/or
PDR17--
To elucidate the possible role of PDR16 and
PDR17 in maintaining a distinct lipid composition of yeast
plasma membrane by either regulating biosynthesis or sorting of various
lipids we compared the lipid composition of plasma membrane
preparations to that of total membranes.
Both in wild-type and in the pdr1-3 background the
phospholipid composition of total cellular membranes was changed
significantly upon deleting PDR16 and PDR17
(Table VI). While the PtdCho content was
increased, the PtdEtn content was decreased in
Deletion of PDR16 and PDR17 also changed the
sterol pattern of total cells in the pdr1-3 and wild-type
background (Table VII). Especially the
accumulation of 4,4-dimethylzymosterol and lanosterol in the double
mutants can be correlated with the appearance of these two ergosterol
precursors in the plasma membrane of Lipid Transfer Activities of PDR16 and PDR17 Gene Products in
Vitro--
Since the PDR16 and PDR17 genes are
distant homologues of the SEC14 gene which encodes the yeast
PITP, it was tempting to speculate that Sec14p and the PDR16
and PDR17 gene products may have similar functions.
Therefore we investigated whether the levels of PtdIns, PtdCho, and
PtdSer transfer activities in isolated subcellular fractions differed
between the various strains used in this study. Cytosolic Sec14p/PITP
(9) and membrane-bound lipid transfer proteins (41) prefer PtdIns as a
substrate (Fig. 2). In the US50-18c
background the lipid transfer activity of the cytosol was only affected
to a minor extent by mutations of the PDR16 and
PDR17 genes. In contrast, the effect of these mutations on
the membrane-bound lipid transfer activity was pronounced. Especially
in microsomal fractions (Fig. 2) of Pdr1p has been known for several years as a transcriptional
regulator controlling yeast multiple drug resistance (for review, see
Ref. 2). Pdr1p together with its homologue Pdr3p regulates the
expression of the drug efflux pumps Pdr5p, Snq2p, Yor1p, Pdr10p, and
Pdr15p (18, 53). Recently, it was found that Pdr1p and Pdr3p control
transport of phospholipids across the plasma membrane via Pdr5p and
Yor1p (18, 54), raising the possibility that such transport is a
physiological function of the network of PDR proteins. Furthermore,
Pdr1p and Pdr3p regulate the expression of two hexose
transporter-encoding genes (6). Thus, Pdr1p controls plasma membrane
function by regulating the level of various active transport systems.
The present work suggests that Pdr1p may also affect structure and
function of the plasma membrane and drug resistance of the yeast
through another mechanism, namely by controlling the expression of the
PDR16 gene.
The PDR16 gene is specifically required for resistance of
yeast cells to miconazole and ketoconazole (see Tables I and II). These
two drugs have a similar mode-of-action: they affect ergosterol biosynthesis at the level of the ERG11 gene product, the
cytochrome P450-dependent lanosterol 14 Drug resistance phenotypes due to deletion of PDR17 were
only seen in the absence of PDR16. The double-deleted strain
Increase in passive uptake of drugs into the cell might be explained by
the changed lipid composition of the plasma membrane of the mutants.
Both PDR16 and PDR17 appear to affect the lipid composition of the plasma membrane, although in a different manner. Whereas deletion of the PDR16 gene mostly affects the sterol
composition (Table V) deletion of PDR17 rather alters the
phospholipid composition of the plasma membrane (Table IV). Most
strikingly, the ratio of the negatively charged phospholipids, PtdIns
and PtdSer, to the uncharged phospholipids, PtdCho and PtdEtn, is
dramatically increased in the In rich medium the changes of the lipid composition in the plasma
membrane in the double mutant do not cause a severe growth defect.
Local replacement of certain lipids appears to compensate for
deficiencies caused by the Both the PDR16 and PDR17 genes exhibit homology
to SEC14. In contrast to the sec14 mutation,
however, deletions of PDR16 and PDR17 are not
lethal. Furthermore, the cellular level of Sec14p in wild-type and
pdr1-3 background is the same independent of the intactness
of PDR16 and/or PDR173 indicating
that the expression of Sec14p is not regulated at the same level as
that of the PDR16 gene product. Sec14p is a regulator of
PtdCho synthesis through the Kennedy pathway (11, 55) or PtdCho
turnover (12, 13). The imbalance of the PtdCho level in the Golgi
caused by SEC14 dysfunction was shown to negatively influence the formation of Golgi-to-plasma membrane secretory vesicles.
Further investigation will be needed to demonstrate whether Pdr16p and
Pdr17p have functions similar to Sec14p, i.e. modulation of
lipid levels in subcellular compartments.
pdr16,
pdr17
double mutant was hypersensitive to a broad range of drugs. Both
mutations caused significant changes of the lipid composition of plasma
membrane and total cell extracts. Deletion of PDR16 had
pronounced effects on the sterol composition, whereas PDR17
deletion mainly affected the phospholipid composition. Thus, Pdr16p and
Pdr17p may regulate yeast lipid synthesis like their distant homologue,
Sec14p. The azole sensitivity of the PDR16-deleted strain
may be the result of imbalanced ergosterol synthesis. Impaired
plasma membrane barrier function resulting from a change in the lipid
composition appears to cause the increased drug sensitivity of the
double mutant strain
pdr16,
pdr17. The uptake rate of
rhodamine-6-G into de-energized cells was shown to be almost 2-fold
increased in a
pdr16,
pdr17 strain as compared with
wild-type and
pdr5 strains. Collectively, our results
indicate that PDR16 and PDR17 control levels of
various lipids in various compartments of the cell and thereby provide
a mechanism for multidrug resistance unrecognized so far.
INTRODUCTION
Top
Abstract
Introduction
References
pdr16 single mutant is mainly due to impaired sterol
synthesis and that the broad increase in drug sensitivity of the double
mutant is a result of a more general change in plasma membrane
composition. The possible roles of Pdr16p and Pdr17p in lipid
biosynthesis and sorting are discussed.
MATERIALS AND METHODS
(17) was used for plasmid propagation. S. cerevisiae strain US50-18c (MAT
pdr1-3 ura3 his1) (3), its derivative AD3 (MAT
pdr1-3 ura3 his1
pdr5::hisG) (18), and strain FY1679-28C
(MATa ura3-52 leu2
1 his3
200 trp
63
GAL2+) (derivative of FY1679) (19) were used for the construction
of strains deleted for the PDR16, PDR17, and
YOR1 genes, and as reference strains in drug sensitivity
assays. The US50-18c-derivatives AD1 (MAT
pdr1-3 ura3 his1
yor1::hisG) (18) and AD13 (MAT
pdr1-3 ura3
his1 pdr5::hisG yor1::hisG) (19) were used as
reference strains in drug sensitivity assays. PDR5-deleted strain FYMK-1/1 (20) was used for drug uptake experiments.
70 °C.
RESULTS
pdr16) exhibited a strongly increased sensitivity to
miconazole and ketoconazole as compared with the parental strain
US50-18c (Table I). For miconazole, the
sensitivity of
pdr16 was increased approximately 20-fold
over the control: while the minimal inhibitory concentration was 2 µg/ml for the parental strain, it was only 0.1 µg/ml for the
pdr16 strain. Sensitivity to ketoconazole increased about 10 times. Similar results were obtained with itraconazole (data not
shown). The
pdr16 strain was also slightly more sensitive to nystatin than the parental strain. No significant changes in sensitivity to any of the other drugs tested were observed (Table I).
Drug sensitivity of yeast strain US50-18c and various derivatives
after 3 days of growth at 30 °C
pdr16
mutant. The resulting transformant had a level of miconazole resistance
identical to that of the US50-18c parental strain, indicating that the
mutant phenotype was indeed due to loss of PDR16 function
(data not shown).
pdr17) did not
exhibit a growth defect as compared with US50-18c. Moreover, the
pdr17 strain did not show increased drug sensitivity,
except for a minor increase in sensitivity to
4-nitroquinoline-N-oxide (Table I). The growth rate of the
double mutant strain
pdr16,
pdr17, on the other hand, was slightly decreased on rich media as compared with the parental and
the single mutant strains (data not shown). Growth of the various
strains was also tested on non-fermentable carbon sources, at high pH,
osmolarity, and temperature. While most of these adverse growth
conditions did not differentially affect the growth of the parental and
mutant strains, growth of the double-deleted strain
pdr16,
pdr17 was severely reduced as compared with the parental and the single-deleted
pdr16 and
pdr17 strains at 37 °C on potassium phosphate-buffered
pH 7 plates containing 0.5 M potassium chloride (data not
shown). Furthermore, the
pdr16,
pdr17 strain was even
more sensitive to the azole antifungals miconazole and ketoconazole
than the
pdr16 strain (Table I). Moreover, the double
mutant also displayed increased sensitivities to cycloheximide, rhodamine-6-G, oligomycin, 4-nitroquinoline-N-oxide,
antimycin A, and crystal violet (Table I). The increase in sensitivity as compared with the
pdr16 single-deleted strain was
about 2-4-fold for most drugs; only sensitivities for ethidium bromide
and nystatin were not increased. The increased drug sensitivity
phenotypes of the
pdr16,
pdr17 strain were indeed due
to loss of PDR17 function, because introducing a single-copy
plasmid carrying PDR17 restored the miconazole and
cycloheximide resistance of the
pdr16,
pdr17 strain to
the levels of the single PDR16-deleted strain.
pdr16 strain
as compared with the parental strain (Table II).
Drug sensitivity of yeast strain FY1679-28c and various derivatives
after 3 days of growth at 30 °C
pdr16,
pdr5,
yor1 is more sensitive against some drugs,
e.g. ketoconazole and miconazole, than a
pdr5,
yor1 strain, indicating that at least part of the effect of the PDR16 gene on drug resistance is independent
of Pdr5p and Yor1p function. Furthermore, Table III shows a comparison of the drug sensitivities of strains deleted for PDR5 and/or
YOR1 to those of the
pdr16,
pdr17 strain.
The
pdr16,
pdr17 strain is more resistant to
cycloheximide and rhodamine-6-G, two typical substrates for Pdr5p, than
a
pdr5 strain, and more resistant to oligomycin, a
typical Yor1p substrate, than a
yor1 strain. This
strongly suggests that Pdr5p and Yor1p are at least partially active in
the
pdr16,
pdr17 strain, and that deletion of
PDR16 and PDR17 does not lead to loss of function
of these drug efflux pumps.
Drug sensitivity of derivatives of yeast strain US50-18c after 3 days
of growth at 30 °C
pdr16,
pdr17 strain is more
sensitive than the wild-type or PDR16 or PDR17
single-deleted strains (Table II), but less sensitive than
pdr5 (Table III).
pdr5, and
pdr16,
pdr17 strains were depleted for energy by
incubation with 2-deoxy-D-glucose and antimycin A. After
2.5 h at 30 °C, rhodamine-6-G was added and its cellular uptake
was followed (Fig. 1). The wild-type and the
pdr5 strain showed similar rates of rhodamine-6-G
uptake indicating that Pdr5p, a strong rhodamine-6-G pump, was not
active under these conditions and energy depletion was complete. Under the same conditions, the
pdr16,
pdr17 strain showed an
almost 2-fold higher rate of rhodamine-6-G uptake. These data indicate that the increased rhodamine-6-G sensitivity of the
pdr16,
pdr17 is at least partially due to an increased
passive drug uptake into these cells.
View larger version (20K):
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Fig. 1.
Rhodamine-6-G uptake in de-energized
cells. Cells were depleted for energy as indicated under
"Materials and Methods." The cellular uptake of rhodamine-6-G was
subsequently followed by determination of the cell-associated
fluorescence. The strains used for this experiment were FY1679-28c
(wild type) ( ), FY
pdr5 (
), and FY
pdr16,
pdr17 (
). Each time point represents the average of several
independent experiments (three experiments for the
pdr5
strain, five for the wild-type, and six for the
pdr16,
pdr17 strain). The variation indicated for each time point is
the standard error of the mean. There was no difference in survival of
the strains.
pdr16,
pdr17 mutations
appear to affect the uptake of drugs into yeast led us to investigate
some properties of the plasma membrane of the respective mutants.
Homologies of the PDR16 and PDR17 gene products
to Sec14p also suggested that perhaps lipid synthesis and/or sorting
might be controlled by these genes.
pdr16,
pdr17, and the double
mutant (Table IV). In plasma membrane
preparations of
pdr16,
pdr17 strains concentrations of
PtdCho and PtdIns were markedly increased as compared with the control
strains FY1679-28c and US50-18c, whereas the level of PtdEtn was
dramatically reduced. Furthermore, the amount of PtdSer was increased
in the plasma membrane of the double mutant in wild-type, but remained
constant in the pdr1-3 background. Changes in the PtdCho
and PtdIns levels appear to be a cumulative effect of both
pdr16 and
pdr17, whereas alterations in
PtdEtn and PtdSer seem to be more clearly expressed in the
pdr17 strain. It is noteworthy that the
pdr17 deletion causes a major increase in the amount of
negatively charged phospholipids, PtdSer and PtdIns, in the plasma
membrane. This fact may influence surface properties and/or function of
membrane bound proteins.
Phospholipid composition of purified plasma membrane
pdr16,
pdr17 mutations in the background of the
FY1679-28c strain. This effect was not seen in US50-18c which bears a
pdr1-3 mutation. In both backgrounds, however, ergosterol
precursors were observed in the plasma membrane of the double mutant.
Especially the unusual presence of 4,4-dimethylzymosterol and
lanosterol in the plasma membrane deserves our attention. It is well
known that Erg11p, the cytochrome P450-dependent lanosterol 14
-demethylase which uses lanosterol as a substrate, is a most sensitive target to azole inhibitors (for a review, see Ref. 51). A
possible effect on enzymes of ergosterol biosynthesis of the
pdr16,
pdr17 mutations might cause increased
sensitivity to azoles as shown in this study (see Tables I-III). As an
alternative, the PDR16 and PDR17 deletions might
cause mistargeting of the sterol precursor which is normally found at
significant amounts only in microsomal membranes and, in the form of
fatty acyl esters, in lipid particles (52).
Sterol composition of purified plasma membrane
pdr16,
pdr17 mutations the bulk fluidity of the plasma membrane appears to be largely preserved. Measurement of
anisotropy using the fluorescent marker TMA-DPH as a probe revealed
that membrane fluidity was not changed (data not shown). Thus, the
mutant cells obviously compensate in that respect almost perfectly for
the above mentioned alterations.
pdr16,
pdr17 compared with the control strains. The
level of PtdSer in total membranes was hardly affected by deleting the
two genes, and only a minor decrease of PtdIns was observed. Thus,
apart from PtdIns, the changes in phospholipid composition of total
membranes corresponded very well to the alterations of plasma membrane
phospholipid composition (see Tables IV and VI).
Phospholipid composition of total cellular membranes
pdr16,
pdr17 strains (see Tables V and VII). The increased total sterol content in
US50-18c
pdr16,
pdr17 relative to US50-18c may be
attributed to the slower growth of the former strain that is
accompanied by accumulation of steryl esters.
Sterol composition of total cellular membranes determined by GLC
pdr16,
pdr17 strains the PtdIns transfer activities were largely reduced as compared
with the control strain US50-18c. Reduced transfer activities were
also observed for microsomal fractions of both single mutants. Effects
of the
pdr16 and
pdr17 single mutations on
PtdIns transfer activity were not additive, which might be an
indication that the products of PDR16 and PDR17
somehow interact and deletion of one gene reduces the transfer activity
conferred by the other. However, absolute rates of phospholipid
transfer activities can only be measured with a mean standard deviation
of ±15-30% and should be therefore interpreted with caution. The
levels of transfer activity in mitochondrial and plasma membrane
fractions were low, and no significant difference was observed between
any of the strains (data not shown). In subcellular fractions of
FY1679-28c, the level of PtdIns transfer activity was similar to the
pdr16,
pdr17 double mutant in the pdr1-3
background, and deletion of PDR16 and PDR17 did
not further reduce the transfer activities (data not shown). In
summary, these data suggest that the increase of the in
vitro transfer activities due to the pdr1-3 mutation is the result of enhanced activities of the PDR16 and
PDR17 gene products. PtdCho and PtdSer transfer activities,
however, were generally much lower than PtdIns transfer activity in all
fractions tested. Furthermore, PtdCho and PtdSer transfer activities
were independent of the intactness of PDR16 and/or
PDR17.
View larger version (36K):
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Fig. 2.
Phosphatidylinositol transfer activity in
cytosol and 0.25 M KCl extracts of cellular membrane
fractions of yeast strains US50-18c and derivatives thereof.
CYT, cytosol; M20: 20,000 × g
microsomes; M30, 30,000 × g microsomes;
M40, 40,000 × g microsomes;
M100, 100,000 × g microsomes. Values of at
least three independent experiments with a standard deviation of ±15%
are shown.
DISCUSSION
-demethylase. One
possible explanation for the increased azole sensitivity of the
pdr16 strain is that the intracellular levels of azoles
are increased due to a change in structure and/or function of the
plasma membrane of the mutant strain. However, although plasma membrane
composition was found to be altered in the mutant, deletion of
PDR16 alone had little effect on resistance of yeast cells
to other drugs. Thus, it is not likely that the permeability of the
plasma membrane has changed severely in the single mutant. The
accumulation of precursor sterols in the plasma membrane of the
pdr16 mutant in the absence of azoles rather suggests
that the activity of enzymes that play a role in ergosterol
biosynthesis is affected in this strain, making it more sensitive to
inhibition by azoles. The finding that the level of precursor sterols
is not only elevated in the plasma membrane, but also in total cell
extracts of the double mutant
pdr16,
pdr17 supports
that hypothesis.
pdr16,
pdr17 exhibits a broad drug sensitivity
spectrum, although the most dramatic effects were observed with
miconazole and ketoconazole. The increased sensitivity to a broad range
of drugs, including mutagens, inhibitors of protein synthesis and
mitochondrial energy production, rather points toward a general change
in intracellular drug concentrations due to the mutations than to an
impairment of all the individual drug target functions. Most drugs for
which increased sensitivity was found are hydrophobic and are believed to enter the cell by passive diffusion. However, the yeast plasma membrane contains a number of protein pumps which can extrude drugs
from the cell in an ATP-dependent manner. Thus, a change in
intracellular drug concentrations could be due to increased passive
uptake of the drugs through the plasma membrane or to reduced active,
protein-mediated drug efflux out of the yeast. The
pdr16,
pdr17 strain was more resistant to cycloheximide
and rhodamine-6-G than a
pdr5 strain, and more resistant
to oligomycin than a
yor1 strain, indicating that Pdr5p
and Yor1p are active in the
pdr16,
pdr17 strain.
Moreover, deletion of PDR16 further reduced drug resistance
of a
pdr5,
yor1 strain, indicating that at least part
of the reduced resistance is independent of Pdr5p and Yor1p. Thus,
there is no indication that active protein-mediated drug efflux is
reduced in strains deleted for PDR16 and/or
PDR17. We cannot exclude that Pdr5p and Yor1p function is
partially affected, or that other yet unidentified drug efflux systems
are less active in the
pdr16,
pdr17 strain. The
observation, however, that energy-depleted
pdr16,
pdr17
cells exhibit an increased rate of rhodamine-6-G uptake as compared
with wild-type and
pdr5 cells suggests, that at least
part of the difference in drug sensitivity is due to a difference in
passive transport (see Fig. 1). Thus, the increased sensitivity of the
double mutant strain
pdr16,
pdr17 is at least partially, and perhaps entirely, due to an increased passive uptake of
drugs into the cell.
pdr17 deletion strain. To
distinguish between the influence of both mutations on lipid synthesis
and transport to the plasma membrane, the bulk membrane lipid
composition was compared with that of the plasma membrane. The changes
in plasma membrane lipid composition reflect to a large extent those of total membranes. Thus, mutation of PDR16 and
PDR17 rather appear to influence synthesis than transport of lipids.
pdr16,
pdr17 mutations.
Since the level of total membrane PtdCho in the
pdr16,
pdr17 strain is also increased it is most likely
that the mutations cause alterations in the biosynthesis of PtdCho,
probably by regulating the pathway in an as yet unknown way. The effect
of PDR16 on sterol biosynthesis could be direct by probing
local sterol concentrations and influencing the activity of ergosterol
synthesizing enzymes, or by modulating the local availability of sterol
precursors. Alternatively, the effect of PDR16 could be
indirect by changing phospholipid levels in the endoplasmic reticulum
in such a way that enzymes involved in sterol synthesis function less
well. This view is supported by the finding that the levels of
ergosterol precursors are much higher in the endoplasmic reticulum of
the double mutant strain
pdr16,
pdr17 as compared with
the corresponding wild-type strain FY1679-28c.3
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ACKNOWLEDGEMENTS |
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We thank Elisabetta Balzi, Marcin Kolaczkowski, and Patrick Marichal for helpful discussions, Anabelle Decottignies for supply of yeast strains deleted for efflux pumps, and Herbert Stütz for synthesis of pyrene-labeled phospholipids.
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FOOTNOTES |
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* This work was supported in part by grants from the Interuniversity Poles of Attraction Program-Belgian State Prime Minister's Office-Federal Office for Scientific, Technical and Cultural Affairs (to A. G.) and CAPES (Fundaçao Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior) and FIOCRUZ (Fundaçao Oswaldo Cruz), Brazil (to M. A. d. V. M.).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.
§ Contributed equally to the results of this work and should be considered co-first authors.
¶ Supported by the European Molecular Biology Organization. Present address: Janssen Research Foundation, Anti-infectives Research Departments, Beerse, Belgium.
** Supported by the Eurofan Project BIO4-CT95-0080, Austrian Ministry of Science and Transportation Project 950080, and Fonds zur Förderung der wissenschaftlichen Forschung in Österreich Project 12076. To whom correspondence should be addressed: Institut für Biochemie und Lebensmittelchemie, Technische Universität, Graz, Petersgasse 12/2, A-8010 Graz, Austria. Tel.: 43-316-873-6462; Fax: 43-316-873-6952; E-mail: f548daum{at}mbox.tu-graz.ac.at.
The abbreviations used are: PtdIns, phosphatidylinositol; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdSer, phosphatidylserine; YPD medium, yeast extract/peptone/dextrose medium; YPG medium, yeast/peptone/glycerol medium; GLC/MS, gas-liquid chromatography/mass spectroscopy; TMA-DPH, trimethylammonium diphenylhexatriene; Mes, 4-morpholineethanesulfonic acid.
1 H. B. van den Hazel and A. Goffeau, unpublished observation.
3 H. Pichler, unpublished observation.
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REFERENCES |
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