(Received for publication, April 17, 1995)
From the
The SNQ2 gene of Saccharomyces cerevisiae,
which encodes an ATP binding cassette protein responsible for
resistance to the mutagen 4-nitroquinoline oxide, is regulated by the
DNA-binding proteins PDR1 and PDR3. In a plasma membrane-enriched
fraction from a pdr1 mutant, the SNQ2 protein is found in the
160-kDa overexpressed band, together with PDR5. The SNQ2 protein was
solubilized with n-dodecyl Several genes sharing homology with the MDR genes,
which confer multidrug resistance in man and mouse (recently reviewed
by Gottesman and Pastan(1993)), have been identified in Saccharomyces cerevisiae. The predicted topography of the
products of these genes is typical of the ABC The MDR gene product, called P-glycoprotein, has been shown
to hydrolyze ATP in vitro and to mediate ATP-dependent
vanadate-sensitive drug efflux when reconstituted in plasma membrane
vesicles (Horio et al., 1988). PDR5 was the first yeast ABC
transporter shown to hydrolyze ATP in vitro (Decottignies et al., 1994), but no biochemical information is presently
available for SNQ2 or any other yeast multidrug resistance protein. In S. cerevisiae, a complex genetic network is responsible
for the regulation of the PDR phenotype. At least two transcription
factors, called PDR1 (Balzi et al., 1987) and PDR3 (Delaveau et al., 1992, 1994), have been shown to interact with the PDR5 promoter (Katzmann et al., 1994; Delahodde et al., 1995). Quite remarkably, point mutations in PDR1 lead to pleiotropic drug resistance correlated with overexpression
of PDR5 mRNA (Meyers et al., 1992). Impressive
amounts of PDR5 protein were found in the plasma membrane-enriched
fraction from the pdr1-3 mutant (Balzi et al.,
1994; Decottignies et al., 1994). On the other hand,
disruption of PDR1 and/or PDR3 leads to
hypersensitivity to drugs and decreases the level of PDR5 mRNA
(Delaveau et al., 1994; Katzmann et al., 1994).
Mutations in the PDR7 and PDR9 genes also influence
the level of PDR5 transcript (Dexter et al., 1994). In this study, we show that the level of SNQ2 transcript is
controlled by PDR1 and PDR3 and that disruption of SNQ2 affects cell growth. We have located the SNQ2 protein in the
plasma membrane-enriched fraction together with PDR5. By selectively
deleting or disrupting either the PDR5 or the SNQ2 gene in a pdr1-3 mutant, we were able to
distinguish, at the plasma membrane level, the nucleotide
triphosphatase activities of the two corresponding proteins
independently overexpressed. In a double PDR5,SNQ2 disruptant,
the two new ABC transporters PDR11 and YOR1 were detected in the
overexpressed 160-kDa protein band of the pdr1-3 mutant.
Our set of genetically purified strains was used to solubilize the
SNQ2, PDR11, and YOR1 proteins and to separate them from the plasma
membrane H
For plasma membrane preparations and growth
curves, the cells were grown in 5.8% glucose and 2% yeast extract (KAT)
adjusted to pH 4.5 with HCl.
The amino-terminal
sequences of YOR1 and PDR11 were determined as follows. After
pre-electrophoresis for 5 h at 50 mA/gel in separating gel buffer
containing 0.1 mM thioglycolate, 150 µg of plasma membrane
proteins from the US50-D25 strain with deletions in both the PDR5 and SNQ2 genes were separated on an SDS-7% polyacrylamide
gel as previously described (Laemmli, 1970), except that polyacrylamide
was replaced with 1% agarose in the stacking gel as described by Moos et al. (1988). After electrophoresis at 50 mA, the proteins
were blotted onto a polyvinylidene difluoride membrane (Problott,
Applied Biosystems) overnight at 40 V. The 160-kDa Serva blue-stained
band was excised with a razor blade and sequenced.
Figure 1:
Northern
blot analysis of PDR5 and SNQ2 transcripts in
different strains. 30 µg of total RNA was run in each well of a 1%
agarose-formaldehyde gel, transferred to a Hybond-N
Transcription of both PDR5 and SNQ2 is greatly enhanced in the pdr1-3 (Fig. 1, lane3) and pdr1-2 (Fig. 1, lane5) mutant strains, as in the pdr1-6 and pdr1-7 mutants (data not shown). When the gene
bearing the point mutation is replaced with a partially deleted PDR1 allele (pdr1- The regulation of SNQ2 transcription by PDR1 and PDR3 was confirmed by measuring the
Taken together,
these results clearly show not only that the pdr1 mutations
increase transcription of SNQ2 (and PDR5) but also
that the wild-type PDR1 and PDR3 proteins jointly control transcription
level of both SNQ2 and PDR5.
Figure 2:
Protein levels of PDR5, SNQ2, YOR1, and
PDR11 in the plasma membrane. Coomassie Blue-stained SDS-PAGE of plasma
membrane-enriched fractions is shown. Lanes1 and 10, molecular mass markers; lane2,
IL125-2B (PDR1, PDR5, SNQ2); lane3,
2229-5C (PDR1, PDR5, SNQ2); lane 4,
US50-18C (pdr1-3, PDR5, SNQ2); lane 5,
D1-3/3 (pdr1-
Figure 3:
Effect of PDR5 and/or SNQ2 disruption on growth in rich medium. The culture medium was
inoculated at 0.5
Figure 4:
In vivo resistance to
cycloheximide and 4-nitroquinoline oxide. Cells were spotted onto YPD
agar plates containing either 4-NQO (0.5 and 1.0 µg/ml) or
cycloheximide (0.1 µg/ml) and incubated for 3 days at 30 °C. A: 1, IL125-2B (wild type); 2,
2229-5C (wild type); 3, US50-18C (pdr1-3); 4, D1-3/3 (pdr1-
Figure 5:
Effect of pH on plasma membrane-bound
PDR5- and SNQ2-related ATPase activities. ATPase activity was measured
at different pH values, in the presence of 6 mM Mg-ATP and 59
mM MES (pH values from 5.2 to 7.0) or Tris (pH values from 7.3
to 9.0), in plasma membrane-enriched fractions from strains
US50-18C, US50-D5, US50-D2, and US50-D25. The pH was adjusted
with NaOH or HCl. PDR5- and SNQ2-related activities were calculated as
described in the text for three independent sets of four strains
derived from the same wild type. Data are expressed as means with the
S.D. (
Further NTPase activity
measurements were carried out at pH 6.3 for both SNQ2 and PDR5. Fig. 6shows that both PDR5 and SNQ2 hydrolyze magnesium
nucleotides at pH 6.3 in the plasma membrane but that the specificity
of PDR5 is broader. SNQ2 exhibits a marked ``preference'' for
ATP.
Figure 6:
Nucleoside phosphatase activities of
plasma membrane-bound PDR5 and SNQ2. Plasma membrane-enriched fractions
from strains US50-18C, US50-D5, US50-D2, and US50-D25 were
assayed at 35 °C in buffers containing 6 mM Mg-NTP and 59
mM MES-NaOH, pH 6.3. PDR5 and SNQ2 NTPase activities were
estimated as described in Fig. 5. The data are expressed as
means with the S.D.
Taken together, these results show that despite their high
sequence homology, the PDR5 and SNQ2 enzymes have different activity versus pH profiles and different substrate specificities.
Figure 7:
Effect of inhibitors on plasma
membrane-bound UTPase activities. The effects of oligomycin (A), erythrosine B (B), vanadate (C), and
Triton X-100 (D) on UTPase activity at pH 6.3 were tested on
samples of the plasma membrane-enriched fractions from strains US50-D5,
US50-D2, and US50-D25. The UTPase activities from the strain US50-D25
measured as described in Fig. 6were subtracted from those
measured in strains US50-D5 and US50-D2 to yield estimates of SNQ2 and
PDR5 inhibition, respectively.
After solubilization with n-dodecyl
Figure 8:
Partial purification of PDR5 and SNQ2.
Solubilized plasma membrane proteins (7 mg) from strains US50-D2 (lane1), US50-D5 (lane 2), and US50-D25 (lane 3) were separated by centrifugation through a continuous
sucrose gradient as described under ``Experimental
Procedures.'' 22 fractions (50 µl), corresponding to a sucrose
concentration of 12% (w/v), were analyzed by Coomassie Blue staining of
SDS-polyacrylamide gels.
SNQ2 has been isolated as a gene conferring
hyper-resistance to the mutagens 4-nitroquinoline-N-oxide and
triaziquone when introduced on a multi-copy plasmid (Haase et
al., 1992). It also confers resistance to sulfomethuron methyl and
phenanthroline (Servos et al., 1993) as well as to
staurosporine and fluphenazine (Hirata et al., 1994). Apart
from the observations that it encodes a putative ABC protein (Servos et al., 1993) whose amino acid sequence is 40% identical to
that of PDR5 (Balzi et al., 1994; Hirata et al.,
1994) and that its transcription is enhanced by heat shock or treatment
with a drug such as cycloheximide, 4-nitroquinoline-N-oxide or
fluphenazine (Hirata et al., 1994), nothing is known about the SNQ2 gene nor has the SNQ2 protein been biochemically
characterized. Our study establishes three important new facts about
SNQ2. First, transcription of the SNQ2 gene is controlled by
PDR1 and PDR3 as shown by Northern blot analysis and by expression of
an SNQ2 promoter-lacZ fusion gene in a wild-type
strain compared to otherwise isogenic pdr1 or pdr3 point or null mutants. This tallies with our observation that
mutations in PDR1 enable the mutated strain to grow in the
presence of higher concentrations of 4-NQO, while deletion of PDR1 and/or PDR3 renders the strain more sensitive. Second,
disruption of the SNQ2 gene in a PDR5/SNQ2-overexpressing pdr1-3 strain markedly lengthens the adaptation phase,
during which stationary cells undergo metabolic and physiological
changes before starting exponential growth. Yet, the exponential growth
rate itself is not affected. In contrast, deletion of PDR5 slightly enhances cell growth as compared to strains
overexpressing PDR5. This is in agreement with the suggestion made by
Romanos et al.(1992) that overexpressed membrane proteins
generally disturb yeast functions. It may also explain why wild-type PDR1 strains grow slightly better than the overexpressing pdr1-3 mutant (data not shown). Disruption of both PDR5 and SNQ2 genes reduces the exponential cell
growth rate, showing that the presence of either PDR5 or SNQ2 is
important for normal growth. As suggested by Hirata et al. (1994), these transporters may extrude intracellular cytotoxic
metabolites accumulated during growth. Third, the SNQ2 gene
product exhibits nucleoside triphosphatase activity. This activity,
like that of PDR5, is located in the plasma membrane. The study of the
SNQ2 activity was made possible by the development of three key
procedures: 1) the use of mutants affected in the transcription factor PDR1, which overexpress SNQ2; 2) the use of mutants where PDR5 and/or SNQ2 is/are disrupted, making it possible
to compare enzyme activities in plasma membranes lacking either PDR5,
SNQ2, or both; and 3) the use of magnesium nucleotides other than
Mg-ATP to measure activities not supported by the
H We were also able to
physically separate SNQ2 or PDR5 from PMA1 by solubilizing the plasma
membranes with n-dodecyl If, as predicted, PDR5 and SNQ2
are ``drug efflux pumps,'' one might expect a certain
substrate specificity for each pump. Hirata et al.(1994) have
indeed shown that multiple copies of the SNQ2 gene confer
resistance to 4-nitroquinoline-N-oxide, for instance, but not
to cycloheximide, while the opposite is true for the PDR5 gene
under the same conditions. Direct proof of this prediction and the
study of the mechanism involved require the development of new
procedures for reconstituting transport activities either in sealed
plasma membrane vesicles or in proteoliposomes containing the purified
proteins. It is noteworthy, in this respect, that Pawagi et
al.(1994) suggest that the transmembrane distribution of aromatic
amino acids in P-glycoprotein may play a functional role in drug
specificity. Amino acid sequence analysis of PDR5, SNQ2, and CDR1 (the
homolog of PDR5 in Candida albicans, which also confers
resistance to cycloheximide (Prasad et al., 1995)) reveals
that aromatic residues in transmembrane segments are more highly
conserved between PDR5 and CDR1 (75%) than between PDR5 and SNQ2 (50%).
Whether this difference in aromatic residue distribution is related to
the difference in substrate specificity is a matter of speculation. The
putative ATP binding sites of PDR5 and SNQ2 are also interesting;
although the GK(S/T) motif is found in the Walker A motifs of all ABC
transporters (reviewed by Thomas and Pedersen(1993)) and although the
middle lysine is said to be strictly required for ATPase activity (Sung et al., 1988; Reinstein et al., 1990; Tian et
al., 1990), this lysine is replaced by a cysteine in the
amino-terminal Walker A motif of both PDR5 and SNQ2, both of which
nevertheless actively hydrolyze ATP. After disruption of both PDR5 and SNQ2 in the pdr1-3 mutant, at
least two additional proteins, identified as PDR11 and YOR1, were found
in the 160-kDa overexpressed band. Both are ABC transporters; PDR11 is
homologous to SNQ2
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-D-maltoside from the
plasma membranes of a PDR5-deleted strain and separated from
the PMA1 H
-ATPase by sucrose gradient centrifugation.
The enzyme shows a nucleoside triphosphatase activity that differs
biochemically from that of PDR5 (Decottignies, A., Kolaczkowski, M.,
Balzi, E., and Goffeau, A.(1994) J. Biol. Chem. 269,
12797-12803) and is sensitive to vanadate, erythrosine B, and
Triton X-100 but not to oligomycin, which inhibits the PDR5 activity
only. Disruption of both PDR5 and SNQ2 in a pdr1 mutant decreases the cell growth rate and reveals the presence of
at least two other ATP binding cassette proteins in the 160-kDa
overexpressed band that have been identified by amino-terminal
microsequencing.
(
)superfamily, consisting of two hydrophobic domains
of six transmembrane segments each and two cytoplasmic
nucleotide-binding folds (reviewed by Balzi and Goffeau (1994)). Yeast
ABC proteins include the a mating factor transporter STE6
(McGrath and Varshavsky, 1989; Kuchler et al., 1989), the
multidrug transporters PDR5 (Balzi et al., 1994; Bissinger and
Kuchler, 1994; Hirata et al., 1994) and SNQ2 (Servos et
al., 1993), the yeast cadmium factor YCF1 (Szczypka et
al., 1994), the oligomycin resistance factor YOR1,
(
)and several newly discovered genes of totally
unknown function like PDR10
(
)and PDR11
(accession no. Z38113, NCBI gi:558389``) (reviewed by Balzi and
Goffeau(1994)). When overexpressed, the PDR5 gene confers
resistance to several unrelated drugs, such as the protein synthesis
inhibitor cycloheximide and the acetolactate synthase inhibitor
sulfomethuron methyl. Overexpression of SNQ2 confers
resistance to the mutagen 4-NQO and to sulfomethuron methyl (reviewed
by Balzi and Goffeau (1994)). With the exception of STE6, the
physiological substrates of the yeast ABC transporters remain unknown.
-ATPase PMA1 by centrifugation on sucrose
gradients.
Chemicals
n-Dodecyl
-D-maltoside and Triton X-100 were purchased from
Boehringer; ATP, UTP, GTP, CTP, ITP, bovine serum albumin,
lysolecithin, o-nitrophenyl
-D-galactopyranoside, cycloheximide, and 4-NQO were from
Sigma; erythrosine B and oligomycin were from Janssen Chemica;
molecular weight markers (range, 53,000-212,000 Da) were from
Pharmacia Biotech Inc.; nylon membranes (Hybond N
)
were purchased from Amersham Corp. Yeast extract was either from KAT
or from DIFCO. Asolectin was from Associated Concentrates (Woodside,
Long Island, NY). All other reagents were of analytical grade.
Yeast Strains and Growth Media
The S.
cerevisiae strains used in this study are listed in Table 1.
To disrupt PDR5, a 4.7-kb PvuII fragment of plasmid
pDR3.3 containing the PDR5 gene (Leppert et al.,
1990) was cut by double digestion with SmaI/SalI to
eliminate 1,243 bp of the promoter and 1,390 bp of the PDR5 open reading frame. The remainder was blunt ended by Klenow
polymerase treatment, ligated overnight to the URA3 gene
isolated by BamHI digestion of the YDp-U vector (Berben et
al., 1991), and blunt ended by Klenow treatment. Escherichia
coli strain C600 was transformed as in Hanahan(1983). The
extracted plasmid, designated as ppdr5::URA3, was
cleaved with BstEII, BglI, and EcoRI to
generate the 2,852-bp fragment containing the first 368 bp of the PDR5 promoter, the complete 1,108-bp URA3 gene, and
the 1,373-bp SalI-BstEII fragment of the PDR5 open reading frame. The haploid yeast strain US50-18C was
then transformed as in Kuo and Campbell(1983). Transformants (US50-D5)
appeared after 3 days at 30 °C on a selective uracil-free dropout
medium. The chromosomal structure of the disrupted pdr5
allele was checked by Southern blotting analysis. The PDR5 null allele obtained was designated as pdr5-
3::URA3 (two other PDR5 null alleles were previously constructed by Katzmann et
al.(1994) and Hirata et al.(1994). The ura3 auxotrophic marker was recovered by selection on 5-fluoroorotic
acid, and disruption of the SNQ2 gene was carried out as
follows. The linearized snq2::Tn10-LUK fragment
obtained by XbaI-SalI cleavage of pBS2218 (Servos et al., 1993), kindly provided by M. Brendel and J. Servos
(Goethe Universitat, Frankfurt, Germany), was used to transform the
yeast strains US50-18C and US50-D5. Disruption of the SNQ2 gene in the strains US50-D2 (SNQ2 disruption in
US50-18C strain) and US50-D25 (SNQ2 disruption in
US50-D5 strain) was checked by Southern blotting analysis. The strains,
grown on standard medium as previously described (Ulawzewski et
al., 1987), were tested for drug resistance on solid complex
medium containing 1% yeast extract (Difco), 2% glucose, and either
cycloheximide dissolved in ethanol or 4-NQO dissolved in acetone. A
32-well replicator was used for replica plating, and drug resistance
was scored after 3-4 days at 30 °C as described by Balzi et al.(1987).
Northern Blot Analysis
Total RNA was
extracted by the ``hot phenol'' procedure of Schmitt et
al.(1990). Standard techniques were used for agarose-formaldehyde
electrophoresis (Sambrook et al., 1989) and for transfer to
nylon membranes. A 4.7-kb PvuII fragment of plasmid pDR3.3, a
1.0-kb EcoRI fragment of plasmid pBS2218, and a 1.0-kb BamHI-HindIII fragment of plasmid pACT (kind gift of
J. Verdi re, Gif-sur-Yvette, France) were used as probes, specific for PDR5, SNQ2, and actin,
respectively, after labeling by nick translation with
[-
P]dCTP. Hybridizations were carried out
for 15 h at 42 °C in 40% formamide, 5
Denhardt's,
0.5% SDS, 5
SSPE, and 20 µg/ml denatured salmon sperm DNA.
The membranes were washed at 55 °C in 1
SSC, 0.2% SDS and
autoradiographed (KODAK x-ray film). For rehybridization, the membranes
were immersed once or twice in boiling 0.5% SDS and allowed to cool at
room temperature.
Isolation of Plasma Membranes
Plasma
membranes were isolated from the particulate fraction pelleted at
15,000 g for 40 min by selective precipitation at pH
5.2 as described by Goffeau and Dufour(1988).
Protein Microsequencing
The broad 160-kDa
band obtained from the plasma membranes of the PDR5-deleted
strain US50-D5:pdr1-3,pdr5-3::URA3 was excised from an SDS-PAGE gel and electroeluted overnight in 25
mM Tris, 192 mM glycine, 0.2% (w/v) SDS.
Electroeluted proteins were precipitated with 10% (w/v) trichloroacetic
acid and centrifuged for 15 min at 15,000
g at 4
°C. SDS was removed by acetone precipitation as described by le
Maire et al.(1993). After lyophilization, the sample was
dissolved in 0.1 M NH
HCO
, 10% (v/v)
acetonitrile, pH 8.0, and incubated overnight at 37 °C in the
presence of 1 µg of trypsin. The digestion product was lyophilized,
dissolved in 100 µl of 0.1% (w/v) trifluoroacetic acid, and
fractionated by reverse phase high pressure liquid chromatography
(C
; buffer A, 0.06% (w/v) trifluoroacetic acid; buffer B,
0.052% (w/v) trifluoroacetic acid, 80% acetonitrile; gradient:
0-10 min, 5% B, flow rate 0.3 ml/min; 10-10.01 min, 5% B,
flow rate 0.3 ml/min; 10.01-90 min, 75% B, flow rate 0.15
ml/min). The peaks, collected manually, were monitored for their OD at
210 mm. A peptide selected from the fractionation was sequenced with an
Applied Biosystems sequenator (model 477A) equipped with an on-line
amino acid derivative analyzer (model 120A).
Solubilization of PDR5 and SNQ2
US50-D5,
US50-D2, and US50-D25 plasma membrane proteins (5 mg/ml) were
solubilized with 0.2% (w/v) n-dodecyl
-D-maltoside in the presence of 8 mM Tris-HCl,
pH 7.5, as described by Decottignies et al.(1994).
Centrifugation on a Continuous Sucrose
Gradient
The solubilized proteins (7 mg) were separated on
a 33.3-ml linear sucrose gradient containing 6-30% (w/v) sucrose,
10 mM Tris, 1 mM EDTA, 1 mM ATP, pH 7.5
(AcOH). After centrifugation for 16 h at 76,000 g in a
Beckman type SW28 rotor, the sucrose gradient was divided into 30 1-ml
fractions.
Nucleoside Triphosphatase
Assays
Nucleotide hydrolysis was measured by incubation of
the sample at 30 °C in a final volume of 100 µl containing
either 6 mM NTP, 7.6 mM MgSO, 10 mM NaN
(to inhibit mitochondrial ATPase), and 59 mM MES-NaOH (from pH 5.0 to 7.0) or 6 mM NTP, 7 mM
MgSO
, 10 mM NaN
, and 59 mM Tris-HCl (from pH 7.3 to 9.0). Assays were carried out as
described by Decottignies et al.(1994).
Other Methods
The protein content was
measured as described by Lowry et al.(1951), with bovine serum
albumin as the standard. Protein samples were electrophoresed on SDS-7%
polyacrylamide gels according to Laemmli(1970) and stained with
Coomassie Blue. -Galactosidase activity was measured as described
by Sambrook et al.(1989) after transformation of the yeast
with a plasmid bearing an SNQ2 promoter-lacZ gene
fusion constructed as follows. 726 bp upstream of the SNQ2 ATG
were amplified by polymerase chain reaction, and an in-frame BamHI site was placed immediately downstream of this ATG.
Primer sequences were as follows: 5`-CGGGATATCCACGGCAAGGAAGTGGCGCG and
3`-GCGGGATCCATTGAATTCTCTTTACGTA. After sequencing, the SNQ2 promoter fragment was inserted as an EcoRV-BamHI
fragment into SmaI-BamHI-cleaved lacZ fusion
vector pSEYC102. The yeast cells were transformed as in Kuo and
Campbell (1983). When necessary, the ura3 auxotrophic marker
was obtained by selection on 5-fluoroorotic acid. Each value given is
the mean ± S.D. of at least 10 trials.
Transcriptional Control of the PDR5 and SNQ2 Genes
by the PDR1 and PDR3 Gene Products
The transcriptional
control of the PDR5 gene by the PDR1 gene product has
been previously demonstrated (Meyers et al., 1992; Balzi et al., 1994; Dexter et al., 1994; Katzmann et
al., 1994; Delahodde et al., 1995). The PDR3 gene encodes a transcription factor highly homologous but not
identical to PDR1 and also controls the expression of the PDR5 gene by binding to the same nucleotide sequence as PDR1 (Delaveau et al., 1994; Katzmann et al., 1994). SNQ2 is highly
homologous to PDR5; the two protein sequences display 40% identity over
their entire length. Several PDR1/PDR3 consensus binding sites are
present in the SNQ2 promoter. To test whether transcription of SNQ2 is affected by PDR1 and/or PDR3, Northern blot
hybridizations were carried out on total RNA from strains bearing
either mutant PDR1 alleles or null PDR1 and/or PDR3 alleles and from the corresponding parental strains. The PDR5 transcript level was also analyzed with a specific probe (Fig. 1).
membrane, and separately hybridized with probes specific for PDR5 (4.7-kb PvuII fragment from plasmid pDR3.3), SNQ2 (1.0-kb EcoRI fragment from plasmid pBS22-LB18),
and actin (1.0-kb BamHI-HindIII fragment
from pACT plasmid). After autoradiography, the membrane was stripped of
one probe and rehybridized with another probe. The actin gene was used
as a control for the mRNA levels. Lane2, D1 (pdr1-
1::URA3) strain obtained by
deletion of PDR1 in the wild-type strain 2229-5C (lane1); lane4, D1-3/3 (pdr1-
1::URA3) strain obtained by
deletion of pdr1 in the pdr1-3 strain
US50-18C (lane3); lane6,
D1-2 (pdr1-
1::URA3) strain
obtained by deletion of pdr1 in the pdr1-2 strain US54-17B (lane5); lane7, wild-type strain FY1679-28C; lane8, strain FY1679-28C/EC (pdr1-
2::TRP1); lane9, strain FY1679-28C/TD (pdr3-
1::HIS3); and lane10, strain FY1679-28C/TDEC (pdr1-
2::TRP1,
pdr3-
1::HIS3).
1), both PDR5 and SNQ2 transcript levels decrease drastically (Fig. 1, lane4versuslane3 and lane6versuslane5). As
compared to a parental wild type, total deletion of both PDR1 (pdr1-
2) and PDR3 (pdr3-
1) reduces the levels of both the PDR5 and SNQ2 transcripts, the effect being more
marked for PDR5 than for SNQ2 (Fig. 1, lanes10versus7). When only PDR1 or PDR3 is deleted, the PDR5 and SNQ2 transcript levels are but slightly affected (lane2versus1 and lanes7-9).
-galactosidase activity expressed from an SNQ2 promoter-lacZ fusion (Table 2). The results clearly
show the induction of lacZ expression in the pdr1-3 mutant compared to its parental strains as well as the decreased
-galactosidase activity when the mutated allele (pdr1-3 or pdr1-2) is deleted. As compared to a parental
wild type, a deletion mutant lacking either PDR1 or PDR3 expresses only slightly less
-galactosidase from the SNQ2 promoter-lacZ fusion gene, while deletion of both genes
reduces the
-galactosidase activity by 50%.
Analysis of PDR5 and SNQ2 in the Plasma
Membrane-enriched Fraction
We used SDS-PAGE to analyze the
proteins of the plasma-membrane-enriched fractions isolated from a set
of strains, all derived from the drug-resistant pdr1-3 mutant and isogenic but for the deletions/disruptions mentioned (Fig. 2, lanes4-8). The results are
also given for the drug-sensitive parental strains (Fig. 2, lanes2 and 3). Several components are
present in the overexpressed 160-kDa band from the drug-resistant pdr1-3 mutant US50-18C (Fig. 2, lane4). This band almost completely disappears after deletion
of pdr1 in the drug-hypersensitive strain D1-3/3 (Balzi et al., 1987; Decottignies et al., 1994).
Densitometric quantitation of the pdr1-3 plasma membrane
proteins separated by SDS-PAGE revealed that PDR5 and SNQ2 amount,
respectively, to 20 and 10% of the total membrane protein, while PMA1
H-ATPase totals 23%. As previously reported (Balzi et al., 1994; Decottignies et al., 1994), PDR5 is the
major component of the overexpressed 160-kDa band. The intensity of
this band is reduced by 66% after deletion of the PDR5 gene in
the US50-D5 strain (Fig. 2, lane6). We
detected no SNQ2 amino-terminal sequence in the remaining band, but
microsequencing of a tryptic product revealed the(1243)AIASR(1247)
sequence belonging to SNQ2. Analysis of the 160-kDa band from the SNQ2 disruptant showed that PDR5 occupies the lower part of
the band (Fig. 2, lane7) and that SNQ2
amounts to 27% of the total protein content of the band from the
parental strain US50-18C. When both PDR5 and SNQ2 were disrupted in strain US50-D25, two additional proteins were
detected in the band (lane8). These were shown by
microsequencing to contain the amino-terminal sequences SLSKYFNPIP and
SITVGDAVXE, respectively, matching those deduced from the PDR11 and YOR1 gene sequences without the initiating
methionine. A comparison of the pdr1-3 mutant (lane8) and the null mutant (lane5)
suggests that levels of both proteins are controlled by PDR1.
1::URA3, PDR5,
SNQ2); lane 6, US50-D5 (pdr1-3,
pdr5-
3::URA3, SNQ2); lane 7,
US50-D2 (pdr1-3, PDR5, snq2::Tn10-LUK); lane 8,
US50-D25 (pdr1-3, pdr5-
3::URA3,
snq2::Tn10-LUK). Each lane contained 150 µg of
protein. PDR5, SNQ2, YOR1, and PDR11 were found in the 160-kDa
overexpressed band.
Disruption of SNQ2 Alters Cell Growth
The
growth curves obtained with four of the above-mentioned strains in rich
medium (Fig. 3) show that a strain lacking the PDR5 gene reaches a higher cell density than a strain overexpressing
PDR5 due to a pdr1-3 mutation (the two strains being
isogenic apart from the features just mentioned). The PDR5 protein
would thus appear to become toxic when overexpressed, since wild-type PDR1 strains grow better than the pdr1-3 mutant
(data not shown). SNQ2 disruption, on the other hand, enhances
the lag phase without changing the exponential growth rate. This
suggests that the transition from the stationary phase to exponential
growth may require the SNQ2 transporter. Double PDR5-SNQ2 disruption alters both the lag and the log phase. Since
simultaneous loss of both genes results in slower growth and although
neither PDR5 nor SNQ2 is an essential gene, the
products of these genes do seem to perform, albeit redundantly, a
function or functions that affect growth.
10
cells/ml from stationary phase
precultures of US50-18C (pdr1-3), US50-D5 (pdr1-3, pdr5-
3), US50-D2 (pdr1-3,
snq2::Tn), and US50-D25 (pdr1-3, pdr5-
3,
snq2::Tn).
PDR5 and SNQ2 Confer Drug Resistance in
Vivo
PDR5 and SNQ2 are both involved in
multidrug resistance (Leppert et al., 1990; Meyers et
al., 1992; Servos et al., 1993; Balzi et al.,
1994; Hirata et al., 1994; Bissinger and Kuchler, 1994), but
the resistance spectra associated with these genes are different.
Resistance to cycloheximide is PDR5-specific; resistance to 4-NQO is
SNQ2-specific; resistance to staurosporine and fluphenazine is
attributable to both genes (Hirata et al., 1994). We therefore
compared the sensitivities to cycloheximide and 4-NQO of mutant and
parental strains (Fig. 4). The SNQ2 disruptants (Fig. 4C, 6 and 7) could not grow on
plates containing 4-NQO at 0.5 µg/ml, a concentration which failed
to inhibit growth of the PDR1 wild-type strain, the pdr1 mutant, or the deletion mutants lacking PDR1, PDR3, or both (Fig. 4C, 1-5 and 8-23). In contrast, deletion of PDR5 did not
affect growth on 4-NQO (Fig. 4C, 5). In the
presence of 1.0 µg of 4-NQO/ml, the pdr1 mutant strains
could still grow while the corresponding (and otherwise isogenic)
strains carrying the wild-type allele or lacking the PDR1 gene
grew more slowly or not at all (Fig. 4D, 3/4, 9/8, 10/11, 13/12, 15/14, 18/19). Deletion of PDR1 or PDR3 in a
wild-type strain enhanced the sensitivity of the strain to 4-NQO (Fig. 4D, 20-22). Deletion of both PDR1 and PDR3 was even more drastic in this respect (Fig. 4D, 23). These results show that
SNQ2-mediated resistance to 4-NQO is under the control of PDR1 and
PDR3, thus confirming the results obtained by Northern blot analysis.
When PDR5 was disrupted, the disruptant cells failed to grow
on plates containing 0.1 µg/ml cycloheximide (Fig. 4E, 5 and 7) while SNQ2 disruption had no effect (Fig. 4E, 5 and 7). The pdr1 deleted strains were more sensitive to
cycloheximide than the otherwise isogenic pdr1 mutant strains (Fig. 4E, 4/3, 11/10). Disruption of
both PDR1 and PDR3 in a wild-type strain caused cell
death on 0.1 µg of cycloheximide/ml (Fig. 4E, 23). These results fully confirm previous data showing that
PDR1 and PDR3 jointly control PDR5-mediated resistance to
cycloheximide. They also show that SNQ2 has no influence on
cycloheximide resistance but mediates resistance to 4-NQO through
transcriptional regulation involving both PDR1 and PDR3.
1); 5, US50-D5 (pdr1-3,
pdr5-
3); 6, US50-D2 (pdr1-3,
snq2::Tn); 7, US50-D25 (pdr1-3,
pdr5-
3, snq2::Tn); 8, FM11 (wild type); 9, 2-20 (pdr1-2); 10,
US54-17B (pdr1-2); 11, D1-2 (pdr1-
1); 12, IL125-2B (wild
type); 13, DRI9-T8 (pdr1-3); 14,
D286-2A (wild type); 15, BOR2-XI (pdr1-6); 16, JG200 (wild type); 17,
JG204 (pdr1-7); 18, 2229-5C (wild type); 19, D1 (pdr1-
1); 20,
FY1679-28C (wild type); 21, FY1679-28C/EC (pdr1-
2); 22, FY1679-28C/TD (pdr3-
1); 23, FY1679-28C/TDEC (pdr1-
2, pdr3-
1). Strains with
isogenic backgrounds are grouped.
SNQ2 Hydrolyzes Nucleoside Triphosphate in
Vitro
To investigate the ATPase activity of SNQ2, we
measured this activity at different pH values in plasma membrane
preparations from four strains (isogenic but for the features
mentioned): the parental pdr1-3 mutant (strain
US50-18C), the PDR5-deleted pdr1-3 strain
US50-D5 (pdr5-3::URA3), the SNQ2-disruptant pdr1-3 strain US50-D2 (snq2::Tn10-LUK), and the double PDR5-SNQ2 knockout pdr1-3 strain US50-D25 (pdr5-
3::URA3,
snq2::Tn10-LUK). The ATPase activities of PDR5 and SNQ2
were estimated by subtracting the ATPase activity of the doubly deleted
strain US50-D25 from the activities measured for the single disruptants
US50-D5 or US50-D2 or by calculating the loss of activity observed in
strains US50-D5 and US50-D2 as compared to their parental strain
US50-18C. This enabled us to eliminate the contribution of the
plasma membrane H
-ATPase PMA1 and possibly of other
ATPases (Fig. 5). The SNQ2 ATPase activity plotted against the
pH for strain US50-D5 or US50-18C shows a pH optimum at
6.0-6.5; the activity profile is shifted toward slightly more
alkaline pH values than that of the PMA1 H
-ATPase
(Borst-Pauwels and Peters, 1977; Dufour and Goffeau, 1980). The peak is
rather sharp, contrasting with the much broader profile obtained for
PDR5, in strain US50-D2 or US50-18C. The ATP-hydrolyzing ability
of PDR5 indeed remains almost constant from pH 6.0 to 9.0, as already
shown by Decottignies et al.(1994). Specific ATPase activities
in the plasma membrane-enriched fraction amount to 2.5 µmol
P
min
mg
for PDR5 at pH 6.3 and 1.7 µmol
P
min
mg
for SNQ2 at pH 6.0 (Fig. 5).
, PDR5;
, SNQ2).
Inhibition of PDR5 and SNQ2 UTPase
Activity
We have previously shown that oligomycin and
erythrosine B inhibit only partially the UTPase activity exhibited by pdr1-3 plasma membranes. In contrast, inhibition by
vanadate or Triton X-100 is almost total (Decottignies et al.,
1994). We show here that oligomycin strongly (I =
0.07 µg/ml) and almost totally inhibits the PDR5 UTPase activity at
pH 6.3, as reported for P-glycoprotein (Sarkadi et al., 1992;
Al-Shawi and Senior, 1993); it has very little effect, on the other
hand, on SNQ2 UTPase activity (Fig. 7A). As for
erythrosine B, it reduces the PDR5 UTPase activity by only 20% (Fig. 7B), whereas erythrosine B causes 65% inhibition
of SNQ2 UTPase activity at 100 µM and 50% inhibition at 35
µM. Vanadate strongly inhibits, to approximately the same
extent, both PDR5- and SNQ2-related UTPase activity, with I
values around 3-5 µM. Triton X-100 at 1
mM concentration totally inhibits both the SNQ2 and the PDR5
activities, as shown in Fig. 7D. Both enzymes are
half-inhibited at 40 µM Triton X-100. This result shows
that the H
-ATPase activity does not interfere with the
UTPase measurements, since at these concentrations Triton X-100
considerably enhances the H
-ATPase activity instead of
inhibiting it (Goffeau and Dufour, 1988).
Solubilization and Partial Purification of PDR5 and
SNQ2
At a detergent-to-protein ratio of 0.4 (w/v), n-dodecyl -D-maltoside solubilized the
components of the overexpressed 160-kDa protein band obtained from
plasma membranes of the pdr1-3 mutant. Solubilization by
this detergent led to the loss of 62% of the PDR5 UTPase activity
measured at pH 6.3 and 54% of the SNQ2 UTPase activity. The remaining
activity was about 20% stimulated by addition of 0.25 mg/ml asolectin
to the assay medium.
-D-maltoside, the plasma membrane proteins from the pdr1-3 strains US50-D5, US50-D2, and US50-D25 were
separated on continuous 6-30% sucrose gradients. Fractions
20-23 of each sucrose gradient were found to be enriched in the
overexpressed proteins of the 160-kDa band and devoid of the 100-kDa
major subunit of PMA1, detected in fractions where the sucrose density
exceeded that of fraction 19. Fig. 8shows the protein content
of fraction 22 from each gradient. In the US50-D5 strain gradient
fractions, at least two protein bands can be distinguished in the
150-160 kDa range (Fig. 8, lane 2). One of them
is SNQ2, since it disappears in the corresponding fractions of the
US50-D25 strain (Fig. 8, lane 3). Of the fractions
``enriched'' in solubilized SNQ2, fraction 21 showed the
highest ATPase activity: 0.4 µmol of
P
min
mg
.
Much higher ATPase activity was found in the PDR5-enriched fractions
20-24: 1.7 µmol of
P
min
mg
.
ATPase activities of about 4 µmol of
P
min
mg
were found at pH 6.3 in the PMA1-enriched fractions from all
strains.
-ATPase PMA1, previously reported to be very specific
for Mg-ATP (Borst-Pauwels and Peters, 1977).
-D-maltoside and
centrifuging overnight on sucrose gradients. This procedure, however,
reduced the SNQ2 ATPase activity from 1.7 µmol of
P
min
mg
in
the plasma membrane to 0.4 and the PDR5 ATPase activity from 2.4 to 1.7
µmol of
P
min
mg
.
The purification enabled us, nevertheless, to show that SNQ2 is much
more ATP-specific than PDR5; moreover, erythrosine B specifically
inhibits the SNQ2-related nucleoside triphosphatase activity and
oligomycin the PDR5-related activity. Vanadate and Triton X-100 inhibit
both enzymes. Also, the SNQ2 ATPase activity profile peaks sharply at
pH 6.0, while PDR5 can hydrolyze ATP from pH 6.0 to 9.0 with
approximately constant efficiency.
(
)and yor1 mutants
are sensitive to oligomycin.
These proteins seem to be in
relatively low amount in the plasma membrane, as judged by the
intensity of Coomassie Blue staining and by the low residual nucleoside
triphosphatase activity in plasma membranes isolated from the double
deletion mutant lacking both PDR5 and SNQ2.
Nevertheless, a further reconstitution study should use preparations
genetically purified of PDR11 and YOR1.
We gratefully thank M. Brendel and J. Servos for the
gift of the snq2::Tn10-LUK plasmid and G. R. Fink for
the gift of the FM11 strain. We also thank J. Nader for helpful
technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.