(Received for publication, October 16, 1995; and in revised form, December 11, 1995)
From the
The multidrug resistance-associated protein (MRP) is a member of
the ATP binding cassette superfamily of transporters which includes the
mammalian P-glycoproteins (P-gp) family. In order to facilitate the
biochemical and genetic analyses of MRP, we have expressed human MRP in
the yeast Saccharomyces cerevisiae and have compared its
functional properties to those of the mouse Mdr3 P-gp isoform.
Expression of both MRP and Mdr3 in the anthracycline hypersensitive
mutant VASY2563 restored cellular resistance to Adriamycin in this
mutant. MRP and Mdr3 expression produced pleiotropic effects on drug
resistance in this mutant, as corresponding VASY2563 transformants also
acquired resistance to the anti-fungal agent FK506 and to the
K/H
ionophore valinomycin. The
appearance of increased cellular resistance to the toxic effect of
Adriamycin (ADM) in MRP and Mdr3 transformants was concomitant with a
reduced intracellular accumulation of [
C]ADM in
spheroplasts prepared from these cells. Moreover, MRP and Mdr3, but not
control spheroplasts, could mediate a time-dependent reduction in the
overall cell-associated [
C]ADM from preloaded
cells, suggesting the presence of an active ADM transport mechanism in
MRP and Mdr3 transformants. Finally, human MRP was found to complement
the biological activity of the yeast peptide pheromone transporter Ste6 and partially restored mating in a sterile ste6 null mutant. These findings suggest that despite their relatively
low level of structural homology, MRP and P-gp share similar functional
aspects, since both proteins can mediate transport of chemotherapeutic
drugs and the a mating peptide pheromone in yeast.
The overexpression of P-glycoprotein in cultured cells in
vitro and in certain types of human cancers has been shown to
confer cellular resistance to many types of chemotherapeutic drugs such
as anthracyclines, Vinca alkaloids, epipodophyllotoxins, and several
others, a phenomenon known as multidrug resistance or
MDR()(1) . P-glycoprotein (P-gp) is an integral
membrane protein of approximately 170 kDa which binds drugs, binds and
hydrolyzes ATP, and that functions as a drug transport system to reduce
the intracellular accumulation of structurally unrelated drugs in
resistant cells(2) . The emergence of MDR in the absence of
P-gp expression in a number of cell lines has been associated with the
overexpression of another membrane protein, the multidrug
resistance-associated protein or MRP(3) . MRP was initially
discovered in, and its cDNA cloned from, the H69AR small cell lung
carcinoma cell line selected for increasing levels of resistance to the
anthracycline Adriamycin (ADM)(3, 4) . The protein was
subsequently found to be overexpressed in several other
multidrug-resistant cell
lines(5, 6, 7, 8) . Transfection
experiments in drug-sensitive cells have clearly established a direct
causal relationship between MRP overexpression and pleiotropic
resistance to drugs, but have also shown that the MDR phenotype
conveyed by MRP, although similar, is distinct from that encoded by
P-gp(9, 10, 11) . Drug accumulation and
efflux studies in MRP-transfectants have identified an ATP-dependent
decrease in drug accumulation associated with a concomitant increase in
drug efflux from preloaded cells(10, 11) . Studies in
inside-out membrane vesicles have indicated that MRP can transport in
an ATP-dependent fashion leukotriene
C
(12, 13) , and several other types of
glutathione S-conjugates (12, 13, 14) . This transport can be
inhibited by a leukotriene D
receptor antagonist, and also
by relatively high concentrations of cyclosporin A and PSC833, the
latter two being known modulators of P-glycoprotein-mediated MDR. These
findings have led to the suggestion that MRP may be the multiple
organic anion transporter and may therefore transport drug molecules as
GSH conjugates(13) .
Predicted amino acid sequence analysis of MRP identifies an integral membrane protein (minimal size, 170 kDa), with two putative nucleotide binding domains and two large highly hydrophobic, probably membrane-associated regions(3, 7) . It has been confirmed biochemically that MRP is an integral membrane glycophosphoprotein capable of binding ATP(15) . These characteristics and its predicted primary amino acid sequence place it in the ATP binding cassette superfamily of membrane transporters, known as the ABC superfamily(16) . In prokaryotes, these transporters form the large group of bacterial periplasmic permeases or traffic ATPases, that import or export at the expense of ATP, a large number of structurally unrelated substrates(19) . In eukaryotes, the best characterized ABC transporters include the mating pheromone a transporter Ste6 of the yeast Saccharomyces cerevisiae(20) , the pfmdr1 gene of Plasmodium falciparum associated with chloroquine resistance in the malarial parasite(21) , the CFTR chloride channel in which mutations cause cystic fibrosis in humans(18) , the TAP1 and TAP2 peptide pumps participating in antigen presentation in lymphocytes(22) , and the family of mammalian P-gps(1, 2) . A number of eukaryotic proteins of this group, including P-gps (2) and CFTR(18) , appear to be formed by two relatively similar halves each encoding six putative transmembrane domains and one nucleotide binding site (17) .
We have previously used the yeast S.
cerevisiae as to characterize the mechanism of action of the three
murine P-gp isoforms (23, 24, 25, 26, 27) . The
Mdr1 and Mdr3 P-gp isoforms expressed in yeast bind drug analogs and
confer cellular resistance to known MDR drugs (e.g. the
anti-fungal macrolite peptide FK506) by reducing their intracellular
accumulation(23, 24) . The structural similarity
between P-gps and the endogenous yeast Ste6 peptide pheromone
transporter translates into functional homology, since the mouse Mdr3
P-gp isoform can complement the biological activity of Ste6 and partially restore mating in a sterile ste6 null
mutant(23) . Studies of P-gp expressed in the membrane of
secretory vesicles from the yeast mutant sec6-4 have shown
that P-gp uses direct ATP hydrolysis to transport drug molecules across
the membrane, this being independent of an intact membrane
electrochemical gradient (,
pH)(26) . Finally,
using the same expression system, we have been able to show that the
normal physiological role of the liver-specific P-gp isoform Mdr2 is an
ATP-dependent phosphatidylcholine flippase that translocates lipid
molecules from the inner to the outer leaflet of the membrane lipid
bilayer(27) .
The exact mechanism by which MRP confers multidrug resistance and the normal physiological function of this protein are not understood. To facilitate the functional dissection of this protein in a system that would permit biochemical and genetic analyses, we have expressed MRP in normal and mutant strains of yeast. We have compared in this system the phenotypic characteristics acquired by MRP transformants to those observed in the same hosts expressing the mouse Mdr3 P-gp isoform, with respect to cellular resistance to known cytotoxic drugs, accumulation, and release of radiolabeled ADM and complementation of a null mutation at the endogenous Ste6 gene.
The aim of this study was to use yeast as an experimental
system to express and functionally characterize the MRP. Yeast
expression plasmids pVT containing full-length cDNA clones for human MRP and mouse wild type mdr3 (used as control) were
transformed into S. cerevisiae strain JPY201(MATa,
ste6::his3). MRP protein expression was monitored by
immunofluorescence on permeabilized spheroplasts using a polyclonal
antiserum raised against a MRP peptide (15) . As shown in Fig. 1A (panel 1), a strong fluorescent signal
was detected in JPY201 cells transformed with the pVT-MRP plasmid, which was absent in control cells transformed with pVT (panel 2). Likewise, a strong fluorescent signal was produced
by the polyclonal anti-P-gp antiserum ES4 in JPY201 cells transformed
with the pVT-mdr3 plasmid (panel 3). While in Mdr3
cells, the fluorescent signal had a ring-like pattern, seemingly
associated with cell membrane expression of the protein, the anti-MRP
antibody generated a strong additional punctate fluorescence in the
cytoplasm, suggesting the presence of MRP protein associated with
intracellular organelles.
Figure 1:
Functional expression of MRP in JPY201
cells. A, the expression of the MRP protein in yeast cells was
tested by immunofluorescence using either a specific antiserum or by
expression of a naturally fluorescent MRP-GFP fusion protein. For
immunofluorescence staining, control cells (pVT) and MRP- and
Mdr3S-expressing JPY201 transformants were converted to spheroplasts,
fixed, and permeabilized as described(31) . MRP cells (panel 1), MRP-GFP (panel 5), and control (panel
2) cells were incubated for 2 h at room temperature with the
antiserum MRP-2 (15) at a 1:100 dilution, whereas Mdr3S cells (panel 3) were exposed to the anti-Pgp polyclonal antiserum
ES4 (32) at a 1:250 dilution. Rhodamine-conjugated goat
anti-rabbit IgG was used at a 1:2000 dilution in all experiments except
for panel 5, where a Texas red-conjugated second antibody was
used. To demonstrate full-length expression of MRP, a GFP tag was
introduced at the COOH terminus of the MRP cDNA as described under
``Experimental Procedures.'' Panel 4 depicts the
distribution of the fluorescent MRP-GFP fusion protein expressed in
JPY201 cells. B, time-dependent cell growth in the absence
(,
,
,
) or in the presence of 50 µg/ml
FK506 (
,
,
,
) was measured in Mdr3S (
,
)-, Mdr3F (
,
)-, MRP (
,
)-, and MRP-GFP-
(
,
) expressing JPY201 transformants using a growth
inhibition assay in liquid
cultures(24) .
Although expression of wild type and mutant Mdr3 proteins in yeast plasma membranes can be readily demonstrated by immunoblotting and immunoprecipitation(23, 24) , similar attempts using a number of well characterized anti-MRP monoclonal or polyclonal antibodies (15) failed to detect the presence of full-length MRP polypeptides (data not shown). These results suggest a high turnover and short half-life of mammalian MRP in yeast cells, resulting in low steady state levels of intact protein. To determine if full-length MRP was indeed synthesized in our yeast strain, we introduced a DNA fragment coding for the naturally fluorescent GFP (29) in-frame at the carboxyl terminus of MRP and determined if a full-length fluorescent MRP-GFP protein could be synthesized in yeast. As shown in panel 4, expression of this MRP-GFP construct in JPY201 cells resulted in the production of a naturally fluorescent polypeptide (ring-like signal), which was also recognized by the anti-MRP antiserum (panel 5). These results suggest that a full-length recombinant MRP-GFP protein was indeed synthesized in these yeast cells.
We then determined if MRP expression in yeast JPY201 cells could alter sensitivity to cytotoxic drugs. We tested the sensitivity of yeast cells transformed with pVT plasmids carrying full-length cDNAs for wild type mdr3, wild type MRP, or the MRP-GFP chimera to the anti-fungal macrolite FK506 (at 50 µg/ml), a known P-gp substrate(24) . In these experiments, we included as an additional control a mutant mdr3 cDNA carrying a single Ser to Phe substitution at position 939 (Mdr3F) which shows drastically reduced biological activity in mammalian and yeast cells(34) . Control experiments in Fig. 1B (left panel) indicate that growth of Mdr3F cells was completely inhibited by FK506, while growth of Mdr3S cells was unaffected, reaching cellular densities at 24 h similar to those of Mdr3S and Mdr3F cells grown in the absence of drug. Likewise, cells expressing either wild type MRP or the MRP-GFP fusion protein showed a significant degree of resistance to FK506, showing only a small growth retardation (lag phase), but reaching cell densities at 20 h similar to those observed in the absence of drug (Fig. 1B, right panel). These results strongly suggest that MRP overexpression in yeast is sufficient to confer cellular resistance to the anti-fungal drug and Mdr3 substrate FK506, suggesting functional MRP expression in these cells.
A major goal of our study was first,
to determine if MRP can directly confer resistance to ADM in yeast (a
drug to which MRP confers resistance in mammalian cells), and second,
to gain insight into the underlying mechanism of resistance.
Preliminary experiments in JPY201 and other commonly used S.
cerevisiae laboratory strains indicated that yeast cells are
intrinsically resistant to the cytotoxic effect of ADM (and other MDR
drugs), precluding the analysis of MRP or Mdr3 activity against ADM in
these strains (data not shown). To circumvent this limitation, we took
advantage of the unique phenotype of the mutant S. cerevisiae strain VASY2563 (MAT, his4-15, lys9, ura3-52,
erg6
, leu2::his G, rad52::LEU2). This strain carries a null
allele at the ERG6 locus with pleiotropic effects on membrane
permeability and fluidity(31) , including hypersensitivity to
anthracyclines such as ADM. In addition, this strain carries a mutation
at the rad52 locus, which is phenotypically expressed as a
defect in DNA repair. We tested in a growth inhibition assay in liquid
culture the capacity of MRP and wild type Mdr3 (Mdr3S) to increase
cellular resistance to ADM in this strain. Control, MRP-, Mdr3S-, and
Mdr3F-expressing VASY2563 cells at stationary phase were diluted to 5
10
cells/ml, further incubated 1 h at 30 °C,
and then supplemented with ADM (1, 2, and 3 µg/ml). After 48 h of
incubation at 30 °C in the dark (ADM is light-sensitive), the
extent of cell growth was measured by optical density (at A
) and was expressed as a percentage of growth
observed for the same clone in drug-free medium. In these experiments,
we also tested the sensitivity of the various transformants to FK506
and the K
/H
ionophore VAL (both
present at 50 µg/ml), two known Mdr3S substrates in yeast (24) . As shown in Fig. 2, ADM (at all concentrations
tested), FK506 and VAL completely inhibited growth of control pVT and
Mdr3F VASY2563 transformants. By contrast, Mdr3S- and MRP-expressing
VASY2563 cells were resistant to all concentrations of ADM, reaching
cell densities after 48 h similar to those observed for control
cultures in drug-free medium. Likewise, both Mdr3S and MRP VASY2563
transformants displayed significant resistance to growth inhibition by
FK506 and VAL (Fig. 2), in agreement with results obtained with
Mdr3 and MRP JPY201 transformants (Fig. 1B). To verify
that the results shown in Fig. 2truly reflected cellular
resistance of the MRP and Mdr3S VASY2563 transformants, culture
aliquots were sampled after 4 h of drug incubation and plated onto YPD
agar (free of drug) to determine the number of live yeast cells (Fig. 3). Upon quantitation, only a small fraction of the
control (8.5% ± 2.5%) and of Mdr3F-expressing cells (6% ±
1.5%) were found to survive 4 h of incubation with all ADM
concentrations tested. In contrast, MRP and Mdr3S transformants were
not affected by a 4-h drug exposure, even at the highest ADM
concentration. On the other hand, incubation of MRP but not Mdr3S
transformants in FK506 (70.5% ± 3.5% survival) or valinomycin
(43% ± 4% survival) resulted in a decreased fraction of
surviving cells (data not shown). Together, these results suggest that
MRP and Mdr3S are functionally expressed in VASY2563 cells and confer
resistance to at least three known MDR drugs. Since we have shown in
yeast spheroplasts that Mdr3S confers resistance to FK506 by reducing
its intracellular accumulation(24) , our results suggested that
MRP may confer drug resistance in yeast by a similar drug transport
process.
Figure 2: Growth inhibition of control and MRP- and Mdr-expressing VASY2563 cells by structurally unrelated cytotoxic drugs. Growth inhibition of VASY2563 cells transformed with either control plasmid pVT (open bars), pVT-MRP (filled bars), pVT-mdr3S (hatched bars), or pVT-mdr3F (stippled bars) by FK506, VAL, or ADM was determined in liquid cultures as described under ``Experimental Procedures.'' Cell growth after 48-h incubation in drug-containing medium was quantitated by optical density and is expressed as a percentage of growth observed in control wells free of drugs. The means and standard deviations of triplicate measurements of three independent experiments are shown.
Figure 3: Drug resistance characteristics of control and MRP- and Mdr-expressing VASY2563 cells. VASY2563 transformants were grown and diluted as described for the growth inhibition assay under ``Experimental Procedures.'' After a 4-h incubation with FK506, VAL, and ADM, 20-µl aliquots were spotted on YPD plates (free of drugs) and grown for 72 h at 30 °C. In addition, 100-µl aliquots (in triplicates) were plated on 100-mm YPD-plates to determine relative growth efficiencies.
We next used [C]ADM to test the
possibility that cellular resistance to this drug in MRP and Mdr3S
VASY2563 transformants may be due to a reduced intracellular
accumulation. The low specific activity of radiolabeled ADM, together
with the high nonspecific binding of this drug to intact yeast cells
and spheroplasts required to determine first optimal experimental
conditions for ADM accumulation in control yeast cells. Spheroplasts
from JPY201 and VASY2563 cells grown in either YPD or YCG medium were
found to accumulate [
C]ADM poorly, with total
cell-associated radioactivity reaching only a 2-3-fold increase
above background after a 2-h incubation period (data not shown). On the
other hand, JPY201 spheroplasts derived from cells grown in LA medium
(which requires robust mitochondrial respiration) were found to
accumulate at least 20 times more ADM (8.25 pmol/10
cells
prior compared with 247.5 pmol/10
cells after a 2-h
incubation at 30 °C) than spheroplasts prepared in either YPD
medium (9.25 pmol/10
cells prior compared with 57.5
pmol/10
cells after a 2-h incubation at 30 °C) or YCG
medium (11.5 pmol/10
cells prior compared with 62
pmol/10
cells after a 2-h incubation at 30 °C).
Therefore, we used such preparations of JPY201 spheroplasts to
monitor the effect of MRP and Mdr3S expression on cellular accumulation
of [C]ADM. Spheroplasts were diluted in LA
medium containing [
C]ADM, placed at 30 °C,
and at 5-min intervals the amount of cell-associated ADM was
determined. During the first 15 min of incubation, ADM accumulation in
control, MRP, and Mdr3S spheroplasts was rapid and followed identical
kinetics in the three groups (Fig. 4). ADM accumulation
continued at the same rate in control pVT spheroplasts to reach a
maximum of approximately 200 pmol/10
cells after 45 min of
incubation. By contrast, the rate of ADM accumulation in both MRP and
Mdr3S spheroplasts started to decrease significantly after the initial
15-min incubation period, resulting in a lower level of steady state
ADM accumulation in these cells compared with pVT controls at 45 min
(110-125 pmol/10
cells; Fig. 4). These results
suggest that Mdr3S and MRP are functionally expressed in yeast and may
mediate cellular resistance and reduced ADM accumulation through an
active drug transport process.
Figure 4:
ADM accumulation in spheroplasts prepared
from control and MRP- and Md3S-expressing JPY201 cells. Control
() and MRP (
)- and Mdr3S-expressing (
) cells were
grown in LA medium to midlogarithmic cultures and converted to
spheroplasts. Subsequently, the spheroplast suspensions were diluted
20-fold in cold LA medium supplemented with 1 µM ADM,
[
C]ADM and placed at 30 °C. At 5-min
intervals, aliquots were removed and cell-associated
[
C]ADM was determined by separating the
spheroplasts from the drug containing medium after centrifugation
through an oil cushion as described under ``Experimental
Procedures.'' Data represent means of six determinations of two
independent experiments.
We initiated experiments to determine
if MRP could actively decrease intracellular drug pools through an
active drug efflux mechanism(24) . In these experiments, we
loaded pVT control, MRP and Mdr3S spheroplasts with
[C]ADM for 25 min at 30 °C under normal
conditions, at which time we attempted to deplete the medium of free
drug molecules by adding excess amounts of BSA. This was done with the
intent of measuring potential drug efflux from ADM-preloaded cells, as
previously performed by others using DNA(11) . The analysis of
the fluorescence profile of a 1 µM solution of ADM in the
presence and absence of a 20 molar excess of BSA shown in Fig. 5A indeed demonstrated complete quenching of
fluorescence associated with the free drug molecules by BSA (Fig. 5A). Addition of BSA to pVT control spheroplasts
after the initial 25-min exposure to ADM caused a small decrease of the
rate of drug accumulation, with a 5-10% reduction in the steady
state level of ADM (compare Fig. 4and Fig. 5B).
The addition of BSA to MRP and Mdr3S spheroplasts caused a small
reduction in the rate of ADM accumulation in these cells, which was
followed 10 min later by an actual decrease in the total amount of
spheroplasts associated radioactivity. This decrease resulted in a
reduction of total ADM accumulation of 20-25% from peak levels
attained in the presence of BSA (Fig. 5B, compare 45-
and 90-min time points) and 30-35% from peak levels reached in
these cells in the absence of BSA (compare 90-min time points from Fig. 4and Fig. 5B). Such a decrease in cell
associated ADM from steady state levels was not seen in pVT control
transformants (Fig. 5B). These results are compatible
with the proposition that MRP and Mdr3S can reduce the intracellular
ADM concentration from preloaded cells, possibly through an active
transport (efflux) mechanism. In this system, the presence of
extracellular BSA probably contributes to 1) reduce the total amount of
free drug available for uptake, and 2) possibly forming complexes with
ADM molecules extruded from preloaded cells.
Figure 5:
ADM release from spheroplasts prepared
from control and MRP- and Md3S-expressing JPY201 cells. A,
emission spectra of ADM (1 µM) in LA medium in the absence
() and presence (
) of BSA (1 mg/ml). B, ADM
accumulation into spheroplasts from control (
) and MRP (
)-
and Mdr3S-expressing (
) cells was initiated and monitored as
described in the legend to Fig. 4. BSA (1 mg/ml) was added to
the reaction mixture (arrow) at 25 min. Data represent means
of six determinations of two independent
experiments.
The similarities
detected between MRP and Mdr3S yeast transformants in drug cytotoxicity
and drug transport experiments prompted us to test the possibility that
MRP shares another functional characteristic of Mdr3S in yeast, the
ability to complement the mating defect of a ste6 null mutant.
Ste6 is an ABC transporter which shares some structural similarity and
limited sequence homology with both Mdr3 and MRP and which transports
the a mating pheromone in S. cerevisiae. We have
previously shown that expression of wild type (Mdr3S), but not mutant,
forms of Mdr3 (e.g. Mdr3F) in a deletion ste6 JPY201
mutant partially restores the ability of these cells to transport a factor and mate to MAT cells(23) . Therefore, MRP and
Mdr3S transformants together with control pVT and pVT-Ste6
transformants in JPY201 cells were analyzed for their ability to form
diploids upon mating with a cell of the opposite mating type (DC17, MAT
) in a quantitative mating assay(23) . Results
shown in Fig. 6indicate that expression of MRP in otherwise
sterile JPY201 cells caused an 800-fold increase in the ability of
these cells to mate and form diploids on selective medium. Although the
mating frequencies measured in MRP transformants (1.9
10
) were only 13% of those detected in yeast cells
expressing wild type Ste6(23) , they were in the same range and
sometimes superior to those measured in cells expressing Mdr3S (1.9
10
) (Fig. 6). Similarly, the MRP-GFP
fusion protein also complemented ste6 with efficiency 80% of wild type
MRP (data not shown). These findings establish that MRP can, like
Mdr3S, mediate the export of the yeast mating pheromone a and
restore mating in a sterile mutant.
Figure 6:
Complementation of the ste6 mutation by MRP and Mdr3S. JPY201 cells transformed with either
control pVT plasmid, or with the same plasmid carrying full-length
cDNAs for mdr3S, Ste6, or MRP, were allowed
to mate with a haploid MAT- tester stock (DC17), and the
formation of diploid cells on selective medium was monitored as
described under ``Experimental Procedures.'' A depicts the growth of diploid cells on MM medium agar plates after
48-h incubation at 30 °C. B, data of three independent
experiments are expressed as the average relative mating frequencies,
calculated from the number of haploid cells introduced in the mating
reaction(23) .
MRP was originally identified in the P-gp-negative drug-resistant small-cell lung carcinoma cell line H69AR, in which the gene is both amplified and overexpressed(3, 4) . The specific drug resistance profile of this cell line together with that characteristic of MRP transfectants (10) indicate that MRP overexpression also confers cellular resistance to a large group of structurally distinct drugs such as vincristine, VP-16, daunorubicin, and colchicine, all of which are known MDR drugs and P-gp substrates. Also analogous to P-gp isoforms, MRP seems to be expressed at the cell surface where it functions as an active, ATP-dependent drug transporter(10, 11, 15) , although it may also be active in subcellular membranous compartments. Distinguishing features of MRP, however, are that it can confer resistance to heavy metal oxyanions (10) and can efficiently transport anionic amphiphiles such as cysteinyl leukotrienes and glutathione S-conjugates(12, 14) . This suggests that MRP may have a broader substrate specificity than P-gp, being able to recognize and transport anionic (leukotrienes) and possibly conjugates or metabolites of cationic amphiphiles (MDR drugs). Although attractive, this model is difficult to reconcile with the observations that 1) MRP overexpression does not confer cellular resistance to cis-platinum(9) , a drug detoxified after modification via glutathione conjugation, and 2) ADM, a drug to which MRP confers resistance to(9) , is metabolized to a C-13-OH, and is not known to be biotransformed to conjugates(35) . In the present study, we have used yeast as a heterologous expression system to initiate the characterization of the mechanism of MRP action.
Immunofluorescence was used to monitor MRP protein expression in
yeast. Anti-MRP antibodies produced in MRP transformants a fluorescent
signal which displayed a ring-like pattern typical of cell membrane
staining, but we also observed a strong cytoplasmic punctate staining
pattern, suggesting that a portion of the overexpressed protein may be
associated with intracellular organelles. In parallel analyses of
membrane-enriched fractions of MRP transformants by immunoblotting, we
were unable to identify a polypeptide of molecular weight compatible
with that predicted from MRP amino acid sequence analysis (in the range
of 170 kDa). Rather, specific 95-100-kDa polypeptides were always
detected by the anti-MRP antibodies in cell fractions (data not shown),
suggesting that the majority of the MRP protein produced in yeast was
either truncated or rapidly degraded during cell growth or in the
isolation procedure. Degradation due to incomplete synthesis or
aberrant processing of heterologous mammalian membrane proteins by the
yeast machinery may be linked to the absence of important signals
missing from the primary amino acid sequence of the protein (for
sorting, targeting) and/or to very active proteolysis in these cells.
Finally, the standard plasma membrane isolation protocol used here
includes as a first step, the conversion of intact cells to
spheroplasts by enzymatic digestion of the cell wall with Zymolyase.
This enzymatic step is critical and can be significant cause of
degradation, as not only exogenous but also endogenous yeast membrane
proteins (such as the PMA1 H-ATPase) may be very
sensitive to proteases frequently contaminating the commercial
preparations of this enzyme. To unequivocally demonstrate that MRP was
indeed produced in yeast as a full-length protein, we constructed a
chimeric MRP cDNA encoding at its 3` end the GFP
protein(29) . The staining pattern of the expressed MRP-GFP
fusion protein detected by fluorescence analysis suggested that the
MRP-GFP chimera was associated with the plasma membrane. In addition,
we observed that both MRP- and MRP-GFP-expressing yeast cells showed
cellular resistance toward the P-gp substrate and anti-fungal drug
FK506, but could also restore mating in an otherwise sterile ste6 mutant. These results strongly suggest that at least a fraction of
the MRP protein in yeast is intact, expressed properly folded at the
plasma membrane, and functional.
Since ADM is the drug that had been
used in the initial selection of the H69AR cell line, we focused on
this cytotoxic drug as a tool to further characterize MRP in yeast. The
use of ADM as a cytotoxic drug and a pharmacological transport
substrate turned out to be problematic in yeast, for various reasons.
First, most common laboratory strains of S. cerevisiae are
resistant to extremely high concentrations of this drug. Second, ADM is
photolabile and quite unstable at room temperature, as ADM solutions of
less than 5 µM are known to loose 95% of their activity in
less than 1 h at 20 °C(36) . Third, ADM binds
nonspecifically to plastic and other surfaces and can also form
aggregates with itself or other molecules at concentrations higher than
1 µM(36) . Despite these limitations, we found
that MRP expression in the mutant S. cerevisiae strain
VASY2563, which is hypersensitive to anthracyclines and other
lipophilic compounds, dramatically increased resistance to ADM. We
noticed that MRP expression in these cells also conferred cellular
resistance to two P-gp substrates FK506 and to the
K/H
ionophore VAL(24) .
Overall, MRP behaved in a manner very similar to P-gp and conferred
cellular resistance to structurally unrelated drugs.
Since we had
previously established that increased resistance to drugs such as FK506
in yeast Mdr3 transformants is linked to a decreased drug accumulation
and increased drug efflux(24) , and since studies in mammalian
MRP transfectants also indicate that overexpression of MRP is
associated with decreased drug accumulation and increased
efflux(10, 11) , we used [C]ADM
to monitor and compare the kinetics of drug accumulation in MRP and
Mdr3 transformants. Results in Fig. 4indeed showed that
spheroplasts from MRP and Mdr3 transformants accumulated significantly
less ADM (steady state level after 60 min) than spheroplasts from
control pVT yeast cells. We attempted to create ADM efflux conditions
from preloaded spheroplasts in order to determine if the reduced
accumulation seen in MRP and Mdr3 transformants was indeed associated
with increased drug release from these cells. This was done by adding
large excess of BSA extracellularly after a 25-min drug preloading
period. The rationale for using BSA in such a manner is that this
protein binds ADM nonspecifically, which quenches its natural
fluorescence (Fig. 5A), but, more importantly, reduces
the availability of the free form of the drug. We speculated that this
may allow monitoring drug release into the medium from preloaded cells,
as released drug molecules would bind nonspecifically BSA causing an
overall ``displacement'' of the drug chemical equilibrium
toward the outside (reduction of total cell-associated radioactivity).
Results shown in Fig. 5B indicated that actual drug
efflux may be ongoing in spheroplasts from MRP and Mdr3 transformants.
Indeed, while control spheroplasts continued to accumulate ADM
(although at a slower rate) after the addition of BSA, a gradual
reduction of ADM followed by an actual decrease in the amount of total
cell-associated radioactivity was detected in MRP and Mdr3 spheroplasts
(30-35% reduction in steady state accumulation). Taken together,
the results presented in Fig. 4and Fig. 5clearly
indicated that MRP can reduce intracellular accumulation of ADM, and
this reduction may be associated with an increase drug release from
these cells.
The fact that overexpression of MRP and P-gp (Mdr3) in yeast cells confers cellular resistance to three structurally unrelated drugs (ADM, FK506, and VAL), and is associated with reduced drug accumulation, suggests that both proteins probably act by very similar mechanisms. In addition, the fact that one of these three cytotoxic drugs is known to act at the level of the cell membrane without binding to an intracellular target clearly suggests that MRP and P-gp can recognize and act on intact drug molecules that are not otherwise modified. The data presented here, together with the previously demonstrated ability of MRP to transport leukotrienes and glutathione S-conjugates(12, 13, 14) , are in agreement with the proposal that MRP may be capable of acting on both anionic and cationic amphiphiles. Therefore, although both proteins may act according to a common mechanistic basis, their substrate specificity, although overlapping, is clearly distinct. We have demonstrated that MRP can complement a null mutation at the Ste6 locus and restore a mating factor transport and mating in an otherwise sterile yeast mutant (Fig. 6). These experiments suggest that structural and/or functional determinants required for the transport of this pheromone by MRP, Mdr3, and Ste6 proteins have been evolutionarily conserved in these three very distant members of the ABC superfamily. The nature of these common determinants is unknown and can only be speculated upon. The a mating pheromone is a dodecapeptide, which is modified on its terminal cysteine by methylation and by the addition of a fernosyl lipid moiety, and both modifications of the peptide backbone are absolutely required for export. Since we have previously established that one of the members of the mouse P-gp family (the liver-specific Mdr2 isoform) is a lipid flippase capable of actively translocating phosphatidylcholine molecules from the inner to the outer leaflet of the cell membrane(27) , it is tempting to speculate that some aspect of lipid transport by Mdr2 may be preserved and be important for the translocation of the lipidated a pheromone by MRP, Mdr3, and Ste6 in yeast. Although highly speculative, such a proposition can now be tested experimentally in yeast.
The yeast proved to be extremely useful as an experimental system for the study and characterization of eukaryotic ABC transporters such as MRP. Functional expression of this protein in yeast will permit detailed pharmacological and biochemical analyses of the mechanism of transport using well defined secretory vesicles from sec mutants(26) , where the parameters of transport can be experimentally modulated by the use of specific inhibitors or with the combined use of additional mutant yeast genetic background.