From the Unité de Biochimie Physiologique,
Université Catholique de Louvain, Place Croix du Sud 2-20, B-1348
Louvain-la-Neuve, Belgium, the § Department of Physiology,
Emory University School of Medicine, Atlanta, Georgia 30322, and the
¶ Department of Chemical Pathology, University of Cape Town
Medical School, Observatory 7925, Cape Town, South Africa
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ABSTRACT |
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The Saccharomyces
cerevisiae genome encodes 15 full-size ATP
binding cassette transporters (ABC), of which
PDR5, SNQ2, and YOR1 are known to
be regulated by the transcription factors Pdr1p and Pdr3p
(pleiotropic drug resistance). We
have identified two new ABC transporter-encoding genes,
PDR10 and PDR15, which were up-regulated by the
PDR1-3 mutation. These genes, as well as four other ABC
transporter-encoding genes, were deleted in order to study the
properties of Yor1p. The PDR1-3 gain-of-function mutant was then used to overproduce Yor1p up to 10% of the total plasma membrane proteins. Overexpressed Yor1p was photolabeled by
[-32P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP
(K0.5 = 45 µM) and inhibited by
ATP (KD = 0.3 mM) in plasma membranes.
Solubilization and partial purification on sucrose gradient allowed to
detect significant Yor1p ATP hydrolysis activity (~100 nmol of
Pi·min
1·mg
1). This activity
was phospholipid-dependent and sensitive to low concentrations of vanadate (I50 = 0.3 µM) and
oligomycin (I50 = 8.5 µg/ml).
In vivo, we observed a correlation between the amount of Yor1p in the plasma membrane and the level of resistance to oligomycin. We also demonstrated that Yor1p drives an energy-dependent, proton uncoupler-insensitive, cellular extrusion of rhodamine B. Furthermore, cells lacking both Yor1p and Pdr5p (but not Snq2p) showed increased accumulation of the fluorescent derivative of 1-myristoyl-2-[6-(NBD)aminocaproyl]phosphatidylethanolamine.
Despite their different topologies, both Yor1p and Pdr5p mediated the ATP-dependent translocation of similar drugs and phospholipids across the yeast cell membrane. Both ABC transporters exhibit ATP hydrolysis in vitro, but Pdr5p ATPase activity is about 15 times higher than that of Yor1p, which may indicate mechanistic or regulatory differences between the two enzymes.
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INTRODUCTION |
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The yeast
YOR11 gene confers
oligomycin resistance on overexpression in a 2-µm plasmid (1). Its
nucleotide sequence reveals an ORF of 1477 amino acids encoding an ABC
protein highly homologous to mammalian transporters such as the
multidrug resistance-conferring enzyme MRP (BLAST (see Ref. 2) sequence
homology score: p = e228), the
organic anion transporter cMOAT (p = e
216), the sulfonylurea receptor
(p = e
164), and the cystic
fibrosis transmembrane conductance regulator CFTR (p = e
132). Yor1p is a "full-size" ABC transporter with
the topology (TM-NBF)2 (3, 4). It consists of two
homologous halves, with each containing a putative ATP-binding domain
(NBF) and a transmembrane domain of six membrane spans (TM). Cui
et al. (5) showed that Yor1p confers resistance to a series
of drugs including reveromycin A and suggested that Yor1p may be
involved in the cellular efflux of organic anions including the
fluorescent dye rhodamine B. They also showed that incubation with
reveromycin A increases the YOR1 mRNA level. The
transcription of YOR1 is controlled by the homologous pair
of transcription factors Pdr1p/Pdr3p. The level of YOR1
transcription is decreased by the deletion of either PDR1 or
PDR3 and increased in the presence of the gain-of-function
PDR1 alleles (1).
In this paper, we have investigated the transport activity of Yor1p. Building on previous studies, which indicated that the (TM-NBF)2-type Yor1p, together with the (NBF-TM)2-type Pdr5p and Snq2p ABC transporters, are overexpressed in the PDR1-3 mutant plasma membrane (6-8), the PDR1-3 mutant has been used as a tool that enhances the Yor1p protein level. As another investigative tool, we constructed a set of isogenic strains, in the PDR1-3 mutant, with multiple deletions of homologous ABC genes since, in situations where two or more proteins located in the same subcellular compartment share a common substrate, a clear phenotype is only seen when all the corresponding genes are deleted, as illustrated by the work of Mahé et al. (9), who showed that Pdr5p and Snq2p have an overlapping transport capacity for steroids. We deleted the yeast ABC transporter-encoding genes known or suspected to be controlled by the transcription factors Pdr1p and Pdr3p. The YCF1 gene, which encodes a glutathione S-conjugate pump (10), was also deleted. The multiply deleted mutants have allowed the demonstration that Yor1p and Pdr5p share several substrates, which include fluorescent phosphatidylethanolamine, rhodamine B, and oligomycin, even though previous studies had concluded that Pdr5p was not involved in oligomycin resistance (11, 12). The pumping of phospholipids is in line with reports of "flippase" activity with several human and mouse ABC transporters (13-16). It is also in agreement with the defective phospholipid accumulation of two new mutant yeast alleles, PDR1-11 and pdr3-11 (17). Construction of a stronger Yor1p-overexpressing strain allowed us to detect vanadate- and oligomycin-sensitive ATPase activity associated with Yor1p while no UTPase activity was detectable. Despite similar nucleotide binding specificities and transport capacities for Yor1p and Pdr5p, the Yor1p enzyme showed 15 times lower levels of ATP hydrolysis rate than Pdr5p.
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EXPERIMENTAL PROCEDURES |
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Chemicals--
n-Dodecyl -D-maltoside
was purchased from Boehringer Mannheim; bovine serum albumin,
2-deoxy-D-glucose, and oligomycin were from Sigma;
o-nitrophenyl-
-D-galactopyranoside and
rhodamine B were from Merck; molecular weight markers (range
53,000-212,000 Da) and Taq polymerase were from Amersham
Pharmacia Biotech; yeast extract was purchased from both KAT and Difco.
M-C6-NBD-PE, dioleoylphosphatidylcholine, and
N-rhodamine-dioleoylphosphatidylethanolamine were from
Avanti Polar Lipids Inc. (Alabaster, AL). TNP-8-azido-ATP and the
-32P species were synthesized as described previously
(18, 19). All other reagents were of analytical grade.
Yeast Strains-- The Saccharomyces cerevisiae strains used in this study are listed in Table I. Multiple deletions were performed sequentially in the US50-18C PDR1-3 strain by repeated use of the hisG-URA3-hisG cassette followed by selection of the ura3 auxotrophic marker with 5-fluoroorotic acid (20). The plasmids for the deletion of PDR5 (12), SNQ2, and YOR1 (1) genes were kindly provided by W. S. Moye-Rowley (Department of Physiology and Biophysics, University of Iowa, Iowa City, IA). For the deletion of PDR10, PDR11, PDR15, PDR3, and YCF1 genes, we amplified fragments of the promoter and the ORF 3'-end of each gene (Table II). The gene promoters were cloned into the EcoRI/BamHI sites of pSK, the ORF ends were cloned into the BamHI/XbaI sites (except for the PDR11 gene; see Table II), and the BamHI/BglII hisG-URA3-hisG cassette was cloned into the BamHI site.
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Transcriptional Activity of the Yeast ABC
Genes--
Transcriptional activity of the yeast ABC genes was
measured after transformation of the FY1679-28C/EC
(pdr1) strain with 2 centromeric plasmids. The first
plasmid carries either no allele (pRS315), or the wild type
(pRS315::PDR1), or the mutant allele (pRS315::PDR1-3) of the PDR1 gene as
described previously (21). The second plasmid (pSEYC102) bears a
translational fusion of the ABC gene promoters and the
Escherichia coli lacZ gene. The PDR5-lacZ fusion
(12), the SNQ2-lacZ fusion (8), and the YOR1-lacZ
construct (1) have been described previously. For the construction of
the PDR11-lacZ fusion, 687 bp of the PDR11 gene,
including 633 bp of the promoter, were amplified by PCR using the
primers 5'-CGGGATCCCGATCAAAGGTGACTGAAGC and
5'-GGAATTCCACTTTGACGCCCCTTTATGC (restriction sites are
underlined). The PCR-amplified promoter was cloned into a
BamHI-EcoRI-cleaved pSEYC102 vector. The same strategy was used to clone 680 bp of the PDR10 promoter
using the primer sequences: 5'-GGAATTCCTGCCTGACTTACAGATAC
and 5'-CGGGATCCCACATCCTAACAACTATG and 952 bp of
the PDR15 gene, including 897 bp of the promoter, with the
primer sequences: 5'-GGAATTCCGCCCAGCCTTTTATACCT and
5'-CGGGATCCCTTGAGCTCGAGCTCCG.
-Galactosidase activity
was measured as described by Sambrook et al. (22) on cell
extracts from yeast grown in synthetic complete glucose medium (SDC:
0.7% yeast nitrogen base, 2% glucose, complete amino acid
supplements) lacking uracil and leucine.
Complementation of Deleted yor1 Strains by the YOR1 Gene on a Centromeric Plasmid-- The pEGH452 cosmid was cut by MluI. The 5.7-kb restriction fragment containing YOR1, 0.4 kb of the gene promoter including the Pdr1p/Pdr3p binding site, and 0.9 kb downstream of the STOP codon was blunted with Klenow, cloned into the SmaI site of pRS316 (pRS316::YOR1) and used to transform the AD13 and AD1234568 strains for testing complementation of oligomycin and rhodamine B resistance on plates.
Isolation of Plasma Membranes-- Plasma membranes were isolated from the particulate fraction pelleted at 15,000 × g for 40 min after selective precipitation of mitochondria at pH 5.2 (23).
Solubilization of Plasma Membrane Proteins and Centrifugation on
a Continuous Sucrose Gradient--
AD1234567 and SUPERYOR 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 (7) and the solubilized proteins (7 mg)
were separated on a 33.3-ml linear sucrose gradient as described
previously (8).
Nucleoside Triphosphatase Assays-- Nucleotide hydrolysis of plasma membrane-enriched fractions was measured by incubation at 35 °C in a final volume of 100 µl containing 6 mM NTP, 7 mM MgSO4, 10 mM NaN3, 50 mM MES, 50 mM MOPS, and 50 mM Tris, adjusted to the right pH with either HCl or NaOH. In the sucrose gradient fractions, the NTP hydrolysis was measured in the presence of 6 mM NTP, 7 mM MgCl2, 50 mM MES, 50 mM MOPS, 50 mM Tris (pH adjusted with either HCl or NaOH) and 150 µg/ml asolectin. Assays were carried out as described previously (7).
Photolabeling--
Plasma membrane preparations (0.075 or 0.15 mg/ml) in 25 mM MES/tetramethyl ammonium hydroxide, pH 6.0, 1 mM MgCl2, 5 mM EGTA, and 20%
(v/v) glycerol, containing the indicated amount of
[-32P]TNP-8-azido-ATP and ATP were photolabeled using
a xenon lamp and toluene filters as described previously (18). The
samples were processed, subjected to SDS-PAGE and autoradiography, and the radioactivity quantitated by imaging (19).
Drug Resistance Assays-- The strains were tested for drug resistance on solid medium containing 1% yeast extract (Difco) and either 2% glucose and rhodamine B, or 4% glycerol and oligomycin dissolved in ethanol. Drug resistance assays after yeast transformation with the YOR1 gene-containing plasmid were performed on solid synthetic media containing either 2% glucose or 4% glycerol plus 0.7% yeast nitrogen base supplemented with amino acids lacking uracil. A 32-well replicator was used for plating and drug resistance was scored after 3-4 days at 30 °C as described previously (24, 25).
Rhodamine B Fluorescence Measurements in Intact Cells-- Four-ml YD cultures were inoculated with ~50 × 106 cells from an overnight preculture and incubated for 3 h at 30 °C. Culture aliquots of 750 µl (~40 × 106 cells/ml) were washed three times with buffer A (50 mM Hepes-NaOH, pH 7.0), resuspended in 2 ml of buffer A containing 100 µg/ml rhodamine B and either 5 mM D-glucose or 5 mM 2-deoxy-D-glucose and incubated for 2 h at 28-30 °C. A 1.5-ml sample was pelleted and washed three times with buffer A. The cell pellet resuspended in 800 µl of water was maintained on ice until cell fluorescence was measured using an SLM Aminco 48000 S spectrofluorimeter. The excitation wavelength was 555 nm (slit of 4 nm), and the emission wavelength was 575 nm (slit of 4 nm).
In rhodamine B extrusion experiments, cells from 3.5 ml of YD culture (~40 × 106 cells/ml) were washed three times with buffer A, incubated in 2 ml of buffer A containing 100 µg/ml rhodamine B and 5 mM 2-deoxy-D-glucose for 2 h at 28-30 °C and then washed three times with buffer A and resuspended in 1.5 ml of buffer A. Rhodamine B extrusion was measured in response to either 10 mM D-glucose, 4% ethanol, or no carbon source (control). At the indicated times, the fluorescence of 300 µl of both the cell-free supernatant and the cell pellet were measured.Yeast Cell Labeling with NBD-phosphatidylethanolamine-- Lipid vesicles including 50 µM total lipids comprising M-C6-NBD-PE (40 mol%), dioleoylphosphatidylcholine (58 mol%), and N-rhodamine-dioleoylphosphatidylethanolamine (2 mol%) were prepared as described previously (17). Phospholipid concentrations were determined by the lipid phosphorus assay (26). For internalization of M-C6-NBD-PE, yeast cells were grown overnight in SDC at 30 °C, diluted, and allowed to grow to an A600 of 0.2-0.3. Donor vesicles containing the fluorescent lipids were added to the yeast cells and incubated for 30 min at 37 °C. Cells were washed three times with ice-cold SCNaN3 (SDC lacking glucose but containing 2% sorbitol and 20 mM sodium azide) prior to analysis by fluorescence microscopy and flow cytometry.
Fluorescence Microscopy-- Fluorescence microscopy was performed on a Zeiss Axiovert microscope equipped with barrier filters that allowed no detectable crossover of NBD and rhodamine fluorescence. The fluorescence image was enhanced with a VE1000-SIT image-intensifying camera (DAGE-MTI Inc., Michigan City, IN), digitized, and stored. Image manipulation and editing were performed with Metamorph software (Universal Imaging Corp., West Chester, PA).
Flow Cytometry-- Flow cytometric analysis of the M-C6-NBD-PE labeled cells was performed with a FACScan cytometer (Becton-Dickinson Immunocytochemistry, San Jose, CA) equipped with an argon laser operating at 488 nm. Ten µl of a 50 µg/ml stock solution of propidium iodide was added to approximately 4 × 105 cells in 200 µl of SCNaN3 immediately prior to dilution (~3 times) and flow cytometric analysis. Ten thousand cells were analyzed without gating during acquisition. Analysis was performed with Lysis II (Becton-Dickinson Immunocytochemistry Systems) software. A dot plot of forward scatter versus the red fluorescence channel (propidium iodide) was used to set a gate that excluded dead cells from the analysis. The remaining live cells were plotted on a histogram with the green fluorescence (M-C6-NBD-PE) plotted on a log scale, and the mean (Fcell) and standard deviation of the fluorescence intensity of the live cells calculated.
Other Methods--
The protein content was measured as described
by Lowry et al. (27) with bovine serum albumin as the
standard. The protein samples were electrophoresed on
SDS-polyacrylamide gel according to Laemmli (28) and stained with
either Coomassie Blue or silver. The yeast cells were transformed as
described by Kuo and Campbell (29), and bacteria (DH5 strain)
transformation was performed by electroporation using a Bio-Rad Gene
Pulser apparatus, following the manufacturer's instructions.
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RESULTS |
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Overexpression of Yor1p and Genetic Purification--
The low
level of expression of YOR1 in wild-type yeast (1) precludes
characterization of its properties. Traditional overexpression of
plasma membrane proteins using strong promoters and multicopy vectors
often causes mistargeting and stimulates accumulation of intracellular
membranes (30). These problems have been overcome in a new approach,
which has allowed dramatically enhanced overexpression of Pdr5p and
Snq2p ABC transporters in the yeast plasma membrane (6-8). The method
takes advantage of point mutations in the transcription factor-encoding
genes PDR1/PDR3, which activate the transcription of their
target genes. The target gene promoters contain typical binding
sequences called PDREs (for PDR response elements) which correspond to
the 5'-TCCG(C/T)GGA-3' consensus sequence (12, 31, 32) (Table
III). An inherent problem to this
approach is the simultaneous overexpression of several Pdr1p-regulated
proteins, including other ABC transporters. It was therefore necessary
to identify all potentially interfering proteins and eliminate them by
gene deletion. Systematic sequencing of the yeast genome has revealed
new ORFs, which encode a total of 15 full-size ABC transporters (3)
including the PDR10 (33), PDR15 (34), and
PDR11 (35) genes whose promoters display at least one
putative PDRE. Table III shows the transcription activity mediated by
the PDR5, YOR1, SNQ2,
PDR10, PDR15, and PDR11 promoters in the
presence of PDR1 wild type-, null, or mutated
PDR1-3 allele as measured by the -galactosidase activity
of fusion constructs. The PDR11 promoter-mediated transcription activity was weak and not significantly affected by
Pdr1p, possibly because the 5'-TCCGCAGA-3'
sequence in the promoter was insufficient for Pdr1p/Pdr3p recognition. The PDR1-3 mutation slightly increased the efficiency of
the PDR15 gene promoter despite the presence of a perfect
Pdr1p/Pdr3p-binding site. In contrast, the PDR1-3 allele
increased the efficiency of the PDR10 promoter 11-fold.
Note, however, that the putative PDREs of the PDR10,
PDR15 and PDR11 gene promoters have yet to be
verified experimentally. In the presence of the wild-type allele of
PDR3, the PDR1-3 mutation increased expression
of
-galactosidase 25 times for the YOR1 promoter, 17 times for the PDR5 promoter, and 5 times for the
SNQ2 promoter.
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Yor1p Binds TNP-8-azido-ATP and Shows ATPase Activity-- Using the AD234567 multiply deleted strain overexpressing Yor1p, we started to investigate the Yor1p potential NTP hydrolysis activity. Pdr5p has an NTPase activity, which was distinguished from that of the H+-pump Pma1p by its broader nucleotide specificity and pH dependence (7, 8). With similar protein levels in the plasma membrane, the UTPase activity of Pdr5p was up to 10-fold higher than that of Pma1p (Fig. 2), which is the major ATPase in the yeast plasma membrane. The situation with Yor1p-enriched plasma membrane (AD234567) was very different since no significant difference in the NTPase activity was detected compared with the Yor1p-depleted strain (AD1234567). The same results were obtained with the AD2345678/AD12345678 strains. However, in the SUPERYOR strain obtained by fusion of the PDR5 promoter to the YOR1 ORF, a survey of pH from 5.0 to 8.5 revealed that some very low ATPase activity could be associated with SuperYor1p above pH 7.0 (data not shown).
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Overexpressed Yor1p Confers Increased Resistance to Oligomycin in
Vivo--
Overexpressed Yor1p has been reported to confer resistance
to oligomycin (1). The analysis of the multiple deletions shows that
oligomycin resistance of the PDR1-3 mutant strain is also dependent on the presence of Pdr5p. Unlike AD1 (yor1) and
AD12 (yor1
snq2
) strains, the AD13
(yor1
pdr5
) strain does not grow on plates containing
0.25 µg/ml oligomycin (Fig. 5).
Previous studies showed unmodified oligomycin sensitivity in single
PDR5 deletants (11, 12) but Kolaczkowski et al.
(43) showed that oligomycin is a competitive inhibitor of
Pdr5p-mediated transport of rhodamine 6G. As also shown by Fig. 5,
Pdr1p more strongly influences oligomycin resistance than its homolog
Pdr3p (compare the FY1679/EC (pdr1
) and FY1679/TD
(pdr3
) strains). This is in agreement with previous
observations (12) even though no difference in oligomycin resistance
between pdr1
and pdr3
strains was observed
by Delaveau et al. (44). As also shown by Fig. 2, oligomycin
resistance was increased 40 times in the strain AD234567 which
overexpresses Yor1p and was further increased by a factor of more than
8 in the SUPERYOR strain. These data indicate that, at the cell level,
the drug transport properties of Yor1p are conserved during
overexpression.
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Overexpressed Yor1p Confers Resistance to Rhodamine B--
Cui
et al. (5) recently found that deletion of YOR1
increases the cellular content of rhodamine B (which is negatively charged, unlike the Pdr5p substrate rhodamine 6G; see Ref. 43). Analysis of the multiply deleted mutants revealed that rhodamine B
resistance is mediated by both Yor1p and Pdr5p. The deletion of
PDR5 did not allow cell growth in the presence of 500 µg/ml rhodamine B and deletion of both PDR5 and
YOR1 (AD13) increased drug sensitivity so that the cells
fail to grow in 250 µg/ml rhodamine B (Fig. 5). Finally, the combined
deletion of the YOR1, SNQ2, PDR5, PDR10, PDR11, PDR15, and
YCF1 genes (strains AD1234568 and AD12345678) further
reduced the resistance to rhodamine B since growth was diminished at 50 µg/ml rhodamine B and abolished at 100 µg/ml rhodamine B. Pdr1p
affects rhodamine B and oligomycin resistance more drastically than
Pdr3p (FY1679/EC (pdr1) compared with FY1679/TD
(pdr3
)). Thus, Pdr1p and Pdr3p mediate rhodamine B and
oligomycin resistance through both Pdr5p and Yor1p. Notice that Yor1p
and Pdr5p share other common substrates, including the fungicide
miconazole (data not shown).
The Efflux of Rhodamine B in Yeast Cells Overexpressing Yor1p Is
Energy-dependent--
We therefore used the
Yor1p-overexpressing strain AD2345678 (snq2, pdr5
,
pdr10
, pdr11
, ycf1
, pdr3
, pdr15
) and its isogenic control, deleted in YOR1, AD12345678 (yor1
,
snq2
, pdr5
, pdr10
, pdr11
, ycf1
, pdr3
, pdr15
)
to demonstrate the involvement of Yor1p in the rhodamine B cell
content. In the yor1
strain, rhodamine B accumulation was
slightly lowered (82%) by deoxyglucose compared with glucose (100%).
However, in the presence of overexpressed Yor1p, rhodamine B
accumulation was much higher (58%) in energy-starved cells
(deoxyglucose), while glucose caused a drastic reduction (to 8%) in
the cellular rhodamine B content (data not shown). Fig.
6A shows the Yor1p-mediated
energy-dependent extrusion of rhodamine B from pre-loaded
cells incubated with deoxyglucose, which depletes the intracellular ATP
(45). The addition of glucose caused rapid extrusion of rhodamine B
from the Yor1p-expressing cells, while no glucose-dependent
effect was observed in the yor1
cells. Fig. 6B
shows the difference in the supernatant fluorescence (Fglucose
Fcontrol) in
the same experiment and establishes the Yor1p
energy-dependent extrusion of rhodamine B out of the cell. Addition of 30 µM protonophore FCCP in the presence of
the respiratory substrate ethanol as the sole energy source completely
abolished rhodamine B transport. while, in the presence of glucose,
FCCP only slightly affected Yor1p-mediated rhodamine B transport (Fig. 6C). This may be explained either by the partial involvement
of oxidative phosphorylation in the ATP formation of glucose-grown cells and/or by a possibly higher use of cellular ATP by the Pma1p H+-ATPase under these conditions. In the absence of an
energy source, the application of a pH gradient of 2 units (pH 7.0 inside the cell and pH 5.0 outside the cell) did not cause rhodamine B
extrusion (Fig. 6C).
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Yor1p and Pdr5p Are Involved in M-C6-NBD-phosphatidylethanolamine Accumulation in Vivo-- Several mammalian ABC transporters translocate phospholipids (13-16). A "flip-flop" of hydrophobic drugs from the inner leaflet to the outer one has been proposed as part of the mechanism of drug transport (49). We have tested whether overexpressed Yor1p was involved in the transport of a fluorescent phospholipid analog, M-C6-NBD-PE. Cell fluorescence was measured by flow cytometry after the incubation of yeast cells with M-C6-NBD-PE (Fig. 7A) and analyzed by fluorescence microscopy (Fig. 7B). The average accumulation of M-C6-NBD-PE in the PDR1-3 mutant strain (US50-18C) was about 13% of the PDR1 parent strains (IL125-2B and 2229-5C). In similar experiments, another mutant, PDR1-11, accumulated 1-2% of its isogenic parent.2 A strain in which the PDR1 gene was deleted (D1-3/3) accumulated about 70% more M-C6-NBD-PE than the PDR1 parent strains (Fig. 7A). It therefore seems likely that Pdr1p activates the expression of genes encoding proteins that decrease the steady-state accumulation of M-C6-NBD-PE by either increasing its efflux or decreasing its influx.
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DISCUSSION |
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This study provides some new information on the control of YOR1 and other yeast ABC genes by the transcription factors Pdr1p and Pdr3p. The expression of lacZ gene fusions with PDR10, PDR15, and PDR11 gene promoters containing putative PDREs reveals that PDR10 may be a target of Pdr1p/Pdr3p transcription factors. However, its level of expression was very low compared with that of PDR5, SNQ2, and YOR1, whose transcription was greatly enhanced by the PDR1-3 mutation. Transcription of the PDR15 gene, despite the presence of one perfect Pdr1p/Pdr3p-binding site (PDRE) in its promoter, was only increased 2 fold by the PDR1-3 mutation. The transcription level of the PDR11 gene was not modified upon addition of either the wild type PDR1 or the mutated PDR1-3 allele, possibly because the PDRE sequence in its promoter is not sufficient for Pdr1p/Pdr3p binding (TCCGCAGA instead of TCCG(T/C)GGA).
In the PDR1-3 gain-of-function mutant, the PDR5
gene promoter gave the highest absolute -galactosidase activity
among the fusion products with five different Pdr1p-regulated gene
promoters. This is consistent with the SDS-PAGE analysis of plasma
membrane-enriched fractions of multiply deleted strains. The latter
analysis also confirmed that Pdr5p, Snq2p, and Yor1p ABC transporters
are the major components of the 160-kDa overexpressed protein band in the PDR1-3 mutant.
Characterization of Yor1p, a minor protein of the plasma membrane, required both the overexpression of the Yor1p protein and also the deletion of related ABC transporters, in particular those suspected to be activated by the PDR1-3 mutation. We were able to delete up to 7 full-size yeast ABC transporters without impairing growth.
Membrane-bound Yor1p was labeled with TNP-8-azido-ATP in vitro in a saturable manner and the labeling was inhibited by ATP. Interestingly, the K0.5 for TNP-8-azido-ATP that we found for Yor1p (about 45 µM) is very close to the value found by Liu and Sharom for TNP-ATP binding to human P-glycoprotein (43 µM) (50). The nucleotide binding properties of Pdr5p and Yor1p appeared very comparable.
Investigation of the drug transport properties of intact cells
overexpressing Yor1p showed that the Yor1p enzyme was active in
vivo and required energy. The oligomycin resistance was increased 40 times in the strain overexpressing Yor1p and further increased by
about 8-fold in the SUPERYOR strain, indicating that overexpression of
Yor1p does not change its oligomycin transport capability in vivo. Absence of Yor1p or Pdr5p caused increased sensitivity to both oligomycin and rhodamine B. These effects were additive when both
proteins were missing. A requirement for energy was demonstrated by the
strong efflux of rhodamine B from Yor1p-enriched energy-starved cells
on glucose addition. The energy required for this process appears to be
provided by ATP rather than pH. Finally, absence of either Yor1p or
Pdr5p resulted in increased accumulation of a fluorescent
phosphatidylethanolamine. Again, the effect was more pronounced when
both ABC transporters were deleted, indicating that the transporters
may act independently. Conversely, none of the 5 other ABC
transporters, including the overexpressed Snq2p transporter, exhibited
this activity.
In plasma membranes from the AD234567 Yor1p-expressing strain, it was
difficult to measure ATPase activity distinct from that of Pma1p.
Enhancing the level of Yor1p by fusing the YOR1 ORF to the
PDR5 promoter in the PDR1-3 mutant (SUPERYOR
strain) allowed us, however, to detect ATPase activity in solubilized
and partially purified SuperYor1p fractions. The SuperYor1p activity of
~100 nmol of Pi·min1·mg
1
was sensitive to both vanadate and oligomycin, establishing that we are
not dealing with either P-type or F-type ATPase contaminants. The use
of the PDR1-3 gain-of-function allele of PDR1
has already allowed characterization of the Pdr5p and Snq2p NTPase
activities (7, 8). Surprisingly, Pdr5p, Snq2p, and (Super)Yor1p NTPase activities show distinct characteristics; Pdr5p hydrolyzes all Mg-NTPs
over a broad pH range, whereas Snq2p and Yor1p hydrolyze ATP
preferentially. The pH profile of Yor1p ATPase activity is also very
broad (with an optimum at pH 7.5), while it is much sharper for Snq2p
(pH 6.3). Only Pdr5p and Yor1p ATPase activities are
oligomycin-sensitive, while vanadate was shown to inhibit all three
enzyme ATPase activities. Finally, taking into account the relative
amount of each transporter in the plasma membrane, one can estimate
that the Yor1p ATPase activity is more or less 15 times lower than that
of Pdr5p or Snq2p.
As the pumping capacity (and specificity) of the Yor1p and Pdr5p
transporters appears similar in vivo, the low ATPase
activity of Yor1p (~0.1 µmol of
Pi·min1·mg
1) compared with
that of Pdr5p (~1.5 µmol of
Pi·min
1·mg
1) in similar
conditions may indicate that the purified Yor1p transporter is a more
highly coupled pump than Pdr5p. The high ATPase activity and apparently
low pumping capacity of ABC drug transporters (51-54) has been a
puzzling feature. For instance, Pdr5p ATPase activity is very high in
the apparent absence of drugs or other substrates, slightly stimulated
by substrates, and shows a broad nucleotide specificity despite an
in vivo requirement for ATP for transport (43). The poor
NTPase activity of Yor1p might be due to the fact that an activation
factor required for ATPase activity is lost during the plasma membrane
preparation or that some phospholipids block the Yor1p ATPase activity
when tested in membranes. Another possibility is that Yor1p, like its
ortholog CFTR, has low ATPase activity (~0.05 µmol of
Pi·min
1·mg
1) that is
sufficient for the control of channel gating (55). Very recently, the
close human ortholog of Yor1p, MRP, was purified and shown to hydrolyze
ATP at a rate of ~0.3 µmol of
Pi·min
1·mg
1 in the presence
of 6 mM Mg-ATP (56). If we consider that, in our
preparations, the "purified" SuperYor1p amounts to 20% of the
total proteins, one can estimate that SuperYor1p may exhibit an ATP
hydrolysis rate of ~0.5 µmol of
Pi·min
1·mg SuperYor1p
1.
The mediation of phospholipid efflux by yeast Pdr5p and Yor1p is consistent with recent reports that identified other ABC transporters as phospholipid transporters or flippases. These include mouse mdr1 (16) and mdr2 (13, 14) and human MDR1 (16) and MDR3 (15, 16). Phospholipid transport by MDR1 and mdr1 is not head-group or glycerol backbone-specific, whereas the MDR3 P-glycoprotein transports phosphatidylcholine exclusively. The specificity of Yor1p and Pdr5p for phospholipids other than phosphatidylethanolamine remains to be seen, but it could be different for the two pumps. It seems possible that the high steady state of ATP hydrolysis by Pdr5p and the low ATPase activity of Yor1p may be related to activation of the former with certain yeast phospholipids, which may not be substrates or even inhibitors of the latter. Anyway, it is intriguing that Pdr5p and Yor1p share similar phospholipid translocation properties which were not observed for Snq2p.
Finally, we wish to point out that, taking advantage of the strong PDR5 promoter associated with the gain-of-function PDR1-3 mutation, we have developed an important new tool for overexpression of ABC transporters in the yeast plasma membrane. This system allows the overexpression of functional yeast Yor1p at a level that represents more than 10% of total plasma membrane proteins. For comparison, overexpression of the human MDR1 from an high copy number expression vector in yeast by Mao and Scarborough (57) yielded protein amounts to only 0.4% of the total yeast membrane proteins. We anticipate that the PDR system, which dramatically overexpresses the functional yeast Yor1p in the plasma membrane without associated intracellular trafficking problems, and the use of the PDR5 promoter in strains deleted in the majority of the full-size ABC transporter-encoding genes provides a prototype for high level expression of orthologous ABC transporters of medical interest.
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ACKNOWLEDGEMENTS |
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We thank W. S. Moye-Rowley for the plasmid gift and J. Nader for useful technical assistance. We acknowledge E. Balzi, B. van den Hazel, M. Kolaczkowski, M. A. do Valle-Matta, and A. Cybularz-Kolaczkowska for helpful comments. We are grateful to B. van den Hazel for sharing unpublished observations and W. S. Moye-Rowley for constant interest in our work. We acknowledge B. C. Monk for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the Service des Affaires Scientifiques, Techniques et Culturelles, Pôles d'Attraction Interuniversitaires, the Fonds National de la Recherche Scientifique, and the Foundation for Research Development of South Africa, and by NATO Collaborative Exchange Research Grant CRG940493 and National Institutes of Health Grant GM52410 (to J. W. N.) and a National Institutes of Health minority predoctoral fellowship (to A. M. G.).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.
To whom correspondence and reprint requests should be
addressed. Tel.: 32-10-473614; Fax: 32-10-473872; E-mail:
goffeau{at}fysa.ucl.ac.be.
1 The abbreviations used are: YOR1, yeast oligomycin resistance; ORF, open reading frame; ABC, ATP-binding cassette; PDR, pleiotropic drug resistance; Pma1p, H+-plasma membrane ATPase; PDRE, Pdr1p/Pdr3p response element; M-C6-NBD-PE, 1-myristoyl-2-[6-(NBD)aminocaproyl]phosphatidylethanolamine; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; SDC, synthetic complete glucose medium; YD, rich glucose medium; YG, rich glycerol medium; MES, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; TNP, trinitrophenyl; TNP-8-azido-ATP, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine triphosphate; MOPS, 3-(N-morpholino)propanesulfonic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); TM, transmembrane; NBF, nucleotide-binding fold; CFTR, cystic fibrosis transmembrane regulator.
2 A. M. Grant and J. W. Nichols, unpublished observations.
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REFERENCES |
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