(Received for publication, May 31, 1995; and in revised form, October 30, 1995)
From the
The yeast cadmium factor gene (YCF1) from Saccharomyces cerevisiae, which was isolated according to its ability to confer cadmium resistance, encodes a 1,515-amino acid ATP-binding cassette (ABC) protein with extensive sequence homology to the human multidrug resistance-associated protein (MRP1) (Szczypka, M., Wemmie, J. A., Moye-Rowley, W. S., and Thiele, D. J.(1994) J. Biol. Chem. 269, 22853-22857). Direct comparisons between S. cerevisiae strain DTY167, harboring a deletion of the YCF1 gene, and the isogenic wild type strain, DTY165, demonstrate that YCF1 is required for increased resistance to the toxic effects of the exogenous glutathione S-conjugate precursor, 1-chloro-2,4-dinitrobenzene, as well as cadmium. Whereas membrane vesicles isolated from DTY165 cells contain two major pathways for transport of the model compound S-(2,4-dinitrophenyl)glutathione (DNP-GS), an MgATP-dependent, uncoupler-insensitive pathway and an electrically driven pathway, the corresponding membrane fraction from DTY167 cells is more than 90% impaired for MgATP-dependent, uncoupler-insensitive DNP-GS transport. Of the two DNP-GS transport pathways identified, only the MgATP-dependent, uncoupler-insensitive pathway is subject to inhibition by glutathione disulfide, vanadate, verapamil, and vinblastine. The capacity for MgATP-dependent, uncoupler-insensitive conjugate transport in vitro strictly copurifies with the vacuolar membrane fraction. Intact DTY165 cells, but not DTY167 cells, mediate vacuolar accumulation of the fluorescent glutathione-conjugate, monochlorobimane-GS. Introduction of plasmid borne, epitope-tagged gene encoding functional YCF1 into DTY167 cells alleviates the 1-chloro-2,4-dinitrobenzene-hypersensitive phenotype concomitant with restoration of the capacity of vacuolar membrane vesicles isolated from these cells for MgATP-dependent, uncoupler-insensitive DNP-GS transport. On the basis of these findings, the YCF1 gene of S. cerevisiae is inferred to encode an MgATP-energized, uncoupler-insensitive vacuolar glutathione S-conjugate transporter. The energy requirements, kinetics, substrate specificity, and inhibitor profile of YCF1-mediated transport demonstrate that the vacuolar glutathione conjugate pump of yeast bears a strong mechanistic resemblance to the MRP1-encoded transporter of mammalian cells and the cognate, but as yet molecularly undefined, function of plant cells.
Animal and plant cells have the capacity to eliminate a broad
range of lipophilic toxins from the cytosol after their conjugation
with glutathione (Ishikawa, 1992; Martinoia et al., 1993; Li et al., 1995). This process, mediated by a GS-X pump, ()a novel MgATP-dependent transporter that catalyzes the
efflux of glutathione S-conjugates and glutathione disulfide
(GSSG) from the cytosol via the plasma membrane and/or endomembranes,
is thought to constitute a terminal phase of xenobiotic detoxification.
The metabolism and detoxification of xenobiotics comprises three main phases (Ishikawa, 1992). Phase I is a preparatory step in which toxins are oxidized, reduced, or hydrolyzed to introduce or expose functional groups of the appropriate reactivity. Cytochrome P450s and mixed function oxidases are examples of phase I enzymes. In phase II, the activated derivative is conjugated with GSH, glucuronic acid, or glucose. In the case of the GSH-dependent pathway, S-conjugates of GSH are formed by cytosolic glutathione S-transferases. In the final phase, phase III, of the GSH-dependent pathway, GS-conjugates are eliminated from the cytosol by the GS-X pump.
A remarkable feature of the last component
of the GSH-dependent pathway, the GS-X pump, is the
pharmacological and agronomic importance of the compounds it
recognizes. Established transport substrates for the mammalian GS-X pump include the arachidonic acid metabolites, leukotriene
C, and prostaglandin A
, GS-conjugates of the
anticancer drugs cisplatin, chlorambucil, and acrolein, GSSG, and the
model compounds S-(2,4-dinitrophenyl)glutathione (DNP-GS) and
monochlorobimane-GS (Ishikawa et al., 1995). In plants, the
GS-X pump is competent in the transport of GS-conjugates of
the chloroacetanilide and triazine herbicides, metolachlor and
simetryn, respectively, as well as GSSG, DNP-GS, and
monochlorobimane-GS (Martinoia et al., 1993; Li et
al., 1995).
In spite of its relevance for understanding how
cells combat chemotherapeutic agents and herbicides and the interest
shown in this class of transporter because of its exclusive use of
MgATP, rather than preformed transmembrane ion gradients, as a direct
energy source for organic solute transport, the molecular identity of
any GS-X pump has remained elusive. Critical, therefore, is
the recent finding that the 190-kDa membrane glycoprotein encoded by
the human multidrug resistance-associated protein gene (MRP1), implicated in the resistance of
small cell lung cancer cell lines to a number of chemotherapeutic drugs
(Cole et al., 1992), catalyzes the MgATP-dependent transport
of leukotriene C and related glutathione S-conjugates (Leier et al., 1994; Muller et
al., 1994; Zaman et al., 1995).
MRP1 is a member of
the ABC (ATP binding cassette) superfamily
of transporters. Distributed throughout the major taxa, ABC
transporters catalyze the MgATP-dependent transport of peptides,
sugars, ions, and lipophiles across membranes. All are constituted of
one or two copies each of two basic structural elements, a hydrophobic,
integral membrane sector, containing approximately 6 transmembrane
-helices, and a cytoplasmically oriented ATP-binding domain
(nucleotide binding fold, NBF) (Hyde et al., 1990; Higgins,
1995). The NBFs, a diagnostic feature of ABC transporters, are 30%
identical between family members over a span of about 200 amino acid
residues, and each encompasses a Walker A and Walker B box (Walker et al., 1982) and an ABC signature motif (Higgins, 1995). The
most thoroughly investigated ABC family members in eukaryotes include
the mammalian P-glycoproteins (MDRs), some of which are implicated in
drug resistance and others in lipid translocation (Ruetz and Gros,
1994); the pleiotropic drug resistance protein (PDR5) and STE6
peptide-mating pheromone transporter of yeast; the cystic fibrosis
transmembrane conductance regulator (CFTR) chloride channel, mutation
of which is associated with cystic fibrosis in man; the malarial, Plasmodium falciparum, chloroquine transporter (PFMDR1); and
the major histocompatibiliy complex transporters responsible for
peptide translocation and antigen presentation in T lymphocytes
(reviewed in Balzi and Goffeau(1995) and Higgins(1995)).
Sequence comparisons between MRP1 and other ABC transporters reveal two major subgroups (Cole et al., 1992; Szczypka et al., 1994). One consists of MRP1, the Saccharomyces cerevisiae cadmium factor (YCF1) gene, the Leishmania P-glycoprotein-related molecule (Lei/PgpA), and the CFTRs. The other consists of the MDRs, major histocompatibiliy complex transporters, and STE6. However, of all the ABC transporters showing structural similarity to MRP1, YCF1 bears the closest resemblance. Unlike the similarities between MRP1, Lei/PgpA, and CFTR, which center on the NBFs, those between MRP1 and YCF1 are found throughout the sequence. YCF1 is 42.6% identical (63.4% similar) to MRP1, possesses NBFs with an equivalent spacing of conserved residues, and is collinear with respect to the location, extent, and alternation of putative transmembrane and extramembrane domains (Szczypka et al., 1994). Two features of YCF1 and MRP1 that distinguish them from other ABC transporters are their possession of a truncated CFTR-like regulatory domain rich in charged amino acid residues and an approximately 200 amino acid residue N-terminal extension. Thus, a potentially strategically important conclusion to derive from these considerations is the possibility that yeast YCF1 and human MRP1 bear so close a resemblance that they catalyze the same, or at least overlapping, reactions.
Anecdotal evidence in support of an
MRP1-like function for YCF1 is 4-fold. First, there is a strong
association between cellular GSH levels and resistance to cadmium,
implicating this peptide as one of the first lines of defense against
this heavy metal (Singhal et al., 1987). If YCF1 confers
resistance to cadmium through the transport of GSCd complexes, or
derivatives thereof, it may also be competent in the transport of other
GS-conjugates. Second, vacuolar membrane vesicles from wild type S.
cerevisiae catalyze high rates of MgATP-dependent,
uncoupler-insensitive S-conjugate transport, and the kinetics
of the transporter involved are akin to those of the mammalian and
plant vacuolar GS-X pumps. (
)Third,
vacuole-deficient mutants of S. cerevisiae exhibit markedly
increased sensitivity to cadmium, (
)suggesting that one
requirement for efficient elimination or detoxification of this metal
is maintenance of a sizable vacuolar compartment. Fourth, S.
cerevisiae yAP-1 transcription factor transcriptionally activates
both the YCF1 gene and GSH1 gene (Wemmie et
al., 1994; Wu and Moye-Rowley, 1994). Since GSH1 encodes
-glutamylcysteine synthetase, an enzyme critical for GSH
synthesis, expression of the YCF1 gene and fabrication of one
of the precursors for transport by the GS-X pump are
coordinately regulated.
In this report we demonstrate, through investigations of transport of the model compounds, DNP-GS and monochlorobimane-GS, by isolated membrane vesicles and intact cells, respectively, that YCF1 is indeed a membrane protein responsible for catalyzing MgATP-dependent, uncoupler-insensitive uptake of glutathione S-conjugates into the vacuole of wild type S. cerevisiae. In so doing, we show that yeast YCF1 is not only a structural homolog but also a functional homolog of human MRP1.
Plasmid pYCF1-HA, encoding epitope-tagged YCF1, was constructed in
several steps. A 1.4-kb SalI-HindIII fragment,
encompassing the carboxyl-terminal segment of the open reading frame of YCF1, from pIBIYCF1 (Szczypka et al., 1994), was
subcloned into pBluescript KS. Single-stranded DNA was
prepared and used as template to insert DNA sequence encoding the human
influenza hemagglutinin 12CA5 epitope immediately before the
termination codon of the YCF1 gene by oligonucleotide-directed
mutagenesis. The sequence of the primer for this reaction, with the
coding sequence for the 12CA5 epitope underlined, was
5`-GTTTCACAGTTTAAGCGTAGTCTGGGACGTCGTATGGGTAATTTTCATTGACC-3`. After
confirming the boundaries and fidelity of the HA-tag coding region by
DNA sequencing, the 1.4-kb SalI-HindIII DNA fragment
was exchanged with the corresponding wild type segment of pJAW50
(Wemmie et al., 1994) to generate pYCF1-HA.
Figure 4:
Sucrose density gradient fractionation of
vacuolar membrane-enriched vesicles prepared from DTY165 cells. One ml
(1.1 mg protein) of partially purified vacuolar membrane vesicles
derived from vacuoles prepared by the Ficoll flotation technique were
applied to a linear sucrose density gradient (10-40%, w/v) and
analyzed for protein (A), -mannosidase activity (B), V- ATPase activity (C), and MgATP-dependent,
uncoupler-insensitive [
H]DNP-GS uptake (D). [
H]DNP-GS uptake and enzyme
activity were assayed as described in Table 4and under
``Materials and Methods''.
Figure 5:
A, Effect of transformation with pYCF1-HA
or pRS424 on MgATP-dependent, uncoupler-insensitive
[H]DNP-GS uptake by vacuolar membranes purified
from DTY165 and DTY167 cells. Uptake was measured in standard uptake
medium containing 66.2 µM [
H]DNP-GS
and 5 µM gramicidin D. B, immunoreaction of
vacuolar membrane proteins prepared from pYCF1-HA-transformed and
pRS424-transformed DTY165 and DTY167 cells with mouse monoclonal
antibody raised against the 12CA5 epitope of human influenza
hemagglutinin. All lanes were loaded with 25 µg of delipidated
membrane protein and subjected to SDS-polyacrylamide gel
electrophoresis and Western analysis as described under
``Materials and Methods.'' The M
of
YCF1-HA (boldface type) and the positions of the M
standards are
indicated.
Microsomes and purified vacuolar membranes that were to be
employed for SDS-polyacrylamide gel electrophoresis and immunoblotting
were washed free of bovine serum albumin by three rounds of suspension
in suspension medium minus bovine serum albumin and
centrifugation at 100,000 g for 35 min. The final
membrane preparations were either used immediately or frozen in liquid
nitrogen and stored at -85 °C.
GSH and CDNB were purchased from Fluka; AMP-PNP,
aprotinin, ATP, creatine kinase (type I from rabbit muscle,
150-250 units/mg of protein), creatine phosphate, FCCP, GSSG,
gramicidin D, leupeptin, PMSF, verapamil, and vinblastine were from
Sigma; monochlorobimane was from Molecular Probes; cellulose nitrate
membranes (0.45-µm pore size, HA filters) were from Millipore;
[H]glutathione
[(glycine-2-
H]-L-Glu-Cys-Gly;
44 Ci/mmol) was from DuPont NEN; and metolachlor was a gift from
CIBA-Geigy, Greensboro, NC. All other reagents were of analytical grade
and purchased from Fisher, Fluka, or Sigma.
Figure 1:
Differential sensitivities of DTY165
(wild type) (A) and DTY167 (ycf1 mutant) cells (B) to growth inhibition by CDNB. Cells were grown at 30
°C for 24 h to an OD
of approximately 1.4 in YPD
medium before inoculation of aliquots into 15 ml volumes of the same
medium containing 0-60 µM CDNB. OD
was measured at the times indicated.
The isogenic wild type strain DTY165 and the ycf1 mutant strain, DTY167, were indistinguishable during growth in YPD
medium lacking CDNB: both strains grew at the same rate after a brief
lag. However, the addition of CDNB to the culture medium caused a
greater retardation of the growth of DTY167 cells (Fig. 1B) than DTY165 cells (Fig. 1A).
Inhibitory concentrations of CDNB resulted in a slower, more linear,
growth rate for at least 24 h for both strains, but DTY167 underwent
growth retardation at lower concentrations than did DTY165. The optical
densities of the DTY167 cultures were diminished by 65, 82, 85, and 91%
by 40, 50, 60, and 70 µM CDNB, respectively, after 24 h of
incubation (Fig. 1B), whereas the corresponding
diminutions for the DTY165 cultures were 14, 31, 59, and 92% (Fig. 1A). The increase in sensitivity to CDNB
conferred by deletion of the YCF1 gene was similar to that seen with
cadmium (data not shown).
Figure 2:
Time course of
[H]DNP-GS uptake by vacuolar membrane vesicles
purified from DTY165 and DTY167 cells. Uptake was measured in the
absence (-MgATP) or presence of 3 mM MgATP
(+MgATP) in reaction media containing 66.2 µM [
H]DNP-GS, 10 mM creatine
phosphate, 16 units/ml creatine kinase, 50 mM KCl, 0.1% (w/v)
bovine serum albumin, 400 mM sorbitol, and 25 mM Tris-Mes (pH 8.0) at 25 °C. Values shown are means ±
S.E. (n = 3).
Agents that dissipate both the pH (pH) and
electrical (
) components of the
t;ex2html_html_special_mark_amp;mgr;
Of these two pathways, the -dependent pathway
predominated in membranes from DTY167 cells (Table 1). FCCP,
gramicidin D, and bafilomycin A
diminished net DNP-GS
uptake by DTY167 vacuolar membranes from 15.4 ± 0.4 nmol/mg/10
min to between 4.3 ± 0.3 and 6.4 ± 0.3 nmol/mg/10 min.
Moreover, although the effects of FCCP or gramicidin D and V-ATPase
inhibitors in combination were slightly greater than those seen when
these agents were added individually, the transport remaining was only
about 10% of that seen with wild type membranes and only 2-4-fold
stimulated by MgATP. In conjunction with the negligible inhibitions
seen with NH
Cl, alone, indicating that
, not
pH, is the principal driving force for the transport activity
remaining in their vacuolar membranes, DTY167 cells are inferred to be
preferentially impaired in MgATP-energized,
t;ex2html_html_special_mark_amp;mgr;
The nonhydrolyzable ATP analog, AMP-PNP, did not
promote DNP-GS uptake by vacuolar membrane vesicles from either DTY165
or DTY167 cells (Table 1), indicating a requirement for
hydrolysis of the -phosphate of ATP regardless of whether uptake
was via the YCF1- or
-dependent pathway.
Figure 3:
Kinetics of uncoupler-insensitive
[H]DNP-GS uptake by vacuolar membrane vesicles
purified from DTY165 and DTY167 cells. A, MgATP
concentration-dependence of uncoupler-insensitive uptake. B,
DNP-GS concentration-dependence of MgATP-dependent,
uncoupler-insensitive uptake. The MgATP concentration-dependence of
uptake was measured with 66.2 µM [
H]DNP-GS. The DNP-GS
concentration-dependence of uptake was measured with 3 mM MgATP. Uptake was allowed to proceed for 10 min in standard uptake
medium containing 5 µM gramicidin D. The kinetic
parameters for vacuolar membrane vesicles purified from DTY165 cells
were K
86.5 ±
29.5 µM, K
14.1 ± 7.4 µM, V
38.4 ± 5.6 nmol/mg/10 min, V
51.0 ± 6.3 nmol/mg/10 min. The lines of best fit and
kinetic parameters were computed by nonlinear least squares analysis
(Marquardt, 1963). Values shown are means ± S.E. (n = 3).
Direct participation of the
plasmid-borne YCF1-HA gene product in DNP-GS transport and
CDNB detoxification was verified by the finding that vacuolar membrane
vesicles purified from pYCF1-HA-transformed DTY167 cells exhibited a
6-fold enhancement of MgATP-dependent, uncoupler-insensitive
[H]DNP-GS uptake (Fig. 5A) which
was accompanied by a decrease in the susceptibility of such
transformants to growth retardation by exogenous CDNB (Fig. 6).
Whereas pYCF1-HA-transformed DTY167 cells exhibited a similar
resistance to growth retardation by CDNB as untransformed DTY165 cells
(compare Fig. 6B with Fig. 1A), the
same mutant strain showed neither increased vacuolar DNP-GS transport in vitro nor decreased susceptibility to CDNB in vivo after transformation with parental plasmid pRS424, lacking the
YCF1-HA insert (Fig. 6B).
Figure 6:
Effect of transformation with pYCF1-HA (A) or pRS424 (B) on sensitivity of DTY167 cells to
growth retardation by CDNB. Cells were grown at 30 °C for 24 h to
an OD of approximately 1.4 in AHC medium before
inoculation of aliquots into 15-ml volumes of the same medium
containing 0-60 µM CDNB. OD
was
measured at the times indicated.
Fluorescence microscopy of DTY165 and DTY167 cells after incubation in growth medium containing monochlorobimane provided direct evidence that YCF1 contributes to the vacuolar accumulation of its glutathione S-conjugate by intact cells (Fig. 7). DTY165 cells exhibited an intense punctate fluorescence, corresponding to the vacuole as determined by Nomarski microscopy, after 6 h of incubation with monochlorobimane (Fig. 7, A and C). The fluorescence associated with vacuolar monochlorobimane-GS was by comparison severely attenuated in most, and completely absent from many, DTY167 cells (Fig. 7, B and D).
Figure 7: Photomicrographs of DTY165 (A and C) and DTY167 cells (B and D) after incubation with monochlorobimane. Cells were grown in YPD medium for 24 h at 30 °C and 100-µl aliquots of the suspensions were transferred into 15-ml volumes of fresh YPD medium containing 100 µM monochlorobimane. After incubation for 6 h, the cells were washed and examined in fluorescence (C and D) or Nomarski mode (A and B) as described under ``Materials and Methods.''
The experiments described demonstrate that uptake of the
model glutathione S-conjugate, DNP-GS, by vacuolar membrane
vesicles isolated from S. cerevisiae proceeds by two parallel
pathways: one that is directly energized by MgATP and another that is
driven by the inside-positive established by the V-ATPase (Fig. 8). Of these two pathways, only the former is catalyzed by
YCF1. Vacuolar membrane vesicles purified from the wild type strain,
DTY165, are competent in both MgATP-dependent, uncoupler-insensitive
uptake and uncoupler-sensitive uptake but membranes from the ycf1
strain, DTY167, are more than 90% deficient in
MgATP-dependent, uncoupler-insensitive uptake.
Figure 8:
Two parallel pathways for the transport of
GS-conjugates (GS-X) across the vacuolar membrane of S.
cerevisiae. YCF1-mediated, MgATP-energized GS-X uptake (GS-X Pump) is insensitive to uncouplers and V-ATPase
inhibitors but inhibited by vanadate and the alkaloids, verapamil, and
vinblastine. YCF1-independent, -driven GS-X uptake (Anion Uniport) is inhibited by V-ATPase inhibitors
(bafilomycin A
) and electrogenic uncouplers (FCCP and
gramicidin-D), but insensitive to alkaloids, vanadate, and
electroneutral uncouplers (NH
Cl). The vacuolar membrane of
DTY165 cells contains both pathways; the vacuolar membrane of DTY167
cells is deficient in the YCF1-dependent
pathway.
Three primary
findings implicate MgATP, rather than a preformed electrochemical
gradient, as the immediate energy source for YCF1-mediated DNP-GS
transport. (i) Abrogation of the development of an inside-acid,
inside-positive t;ex2html_html_special_mark_amp;mgr;
YCF1-independent uptake, by contrast, has the characteristics of a
DNP-GS (anion) uniport driven by the inside-positive
established as a result of electrogenic
H
-translocation by the V-ATPase. Much of the DNP-GS
transport activity remaining in the vacuolar membrane fraction from ycf1
mutants is abolished by V-ATPase inhibitors and
electrogenic uncouplers (FCCP and gramicidin D) but unaffected by
vanadate, verapamil, vinblastine, and electroneutral uncouplers
(NH
Cl) (Fig. 8).
The functional resemblance
between the YCF1-encoded DNP-GS transporter of S.
cerevisiae and the GS-X pumps of animal and plant cells
is striking. Like YCF1-mediated transport, DNP-GS uptake by membrane
vesicles derived from rat liver canaliculus (Akerboom et al.,
1991; Kobayashi et al., 1990), MRP1-transfected human
cells (Leier et al., 1994; Muller et al., 1994) and
plant vacuoles (Martinoia et al., 1993; Li et al.,
1995) is sensitive to inhibition by vanadate and GSSG but not GSH.
Moreover, in strict correspondence with the functional characteristics
of MRP1 (Muller et al., 1994) and the plant vacuolar GS-X pump (Li et al., 1995), YCF1-catalyzed DNP-GS transport
is also subject to inhibition by S-conjugates of herbicides (e.g. metolachlor-GS) and the alkaloids, vinblastine and
verapamil. These similarities, in combination with the congruence of
their kinetic parameters, notably K and K
(see Table 4in Li et al., 1995), suggest that the
glutathione-conjugate transporters of animals, plants, and yeast are
catalytically equivalent. Two important corollaries therefore follow.
First, YCF1 is not only a structural homolog of MRP1 but also a
functional homolog. By implication, the yeast YCF1 gene and ycf1
mutants, respectively, fulfill the requirements of
probes for and null backgrounds against which the corresponding genes
from molecularly less well characterized systems, such as plants, may
be identified. Second, it is probable that MRP1 and YCF1 serve similar
roles in vivo. Since the glutathione S-conjugation reaction
mediated by cytosolic GSTs is known to be instrumental in the
detoxification of lipophilic electrophiles derived from exogenous or
endogenous sources, it has been proposed that through the concerted
actions of GSTs and the GS-X pump, mammalian and plant cells
can confer a common structural determinant on and increase the water
solubility of the toxins in question and thereby eliminate them from
the cytosol by MgATP-dependent transport (Ishikawa et al.,
1991; Martinoia et al., 1993; Li et al., 1995). The
existence of ostensibly the same transport function in the vacuolar
membrane of wild type cells, the increased sensitivity of ycf1
mutants to the toxic effects of CDNB and the
capacity of plasmid-encoded YCF1 to rescue the hypersensitive mutant
phenotype collectively indicate operation of an analogous
transport-based, detoxification mechanism in yeast.
The tight
association between YCF1 and the vacuolar membrane is notable.
YCF1-mediated DNP-GS transport copurifies with -mannosidase and
V-ATPase activity but not with any of the nonvacuolar membrane markers
examined (NADPH cytochrome c reductase, GDPase, F-ATPase,
P-ATPase), exposure of intact wild type cells to monochlorobimane
results in vacuolar localization of its fluorescent GS derivative, and
expression of functional plasmid-encoded YCF1-HA in ycf1
cells enhances vacuolar DNP-GS uptake concomitant with rescue of the
CDNB hypersensitive phenotype.
Localization of YCF1 to the vacuolar membrane may be instructive in the context of two other recent studies. The first of these is the demonstration that cisplatin-resistant human promyelocytic leukemia HL-60 (HL-60/R-CP) cells, displaying functional overexpression of the GS-X pump, mediate MgATP-dependent accumulation of monochlorobimane-GS into intracellular vesicles (Ishikawa et al., 1994). Given the capacity of HL-60/R-CP cells for the eventual excretion of monochlorobimane-GS, this finding is consistent with the initial accumulation of S-conjugates into intracellular vesicles by the GS-X pump and their subsequent elimination by exocytosis. The second of these is the finding that temperature-sensitive S. cerevisiae sec6-4 mutants, defective in the final step of the vesicular secretory pathway (fusion with the plasma membrane) accumulate secretory vesicles containing glutathione S-conjugate pump activity at the nonpermissive temperature (St-Pierre et al., 1994). Since the transporter concerned is kinetically similar to the GS-X pump of mammalian cells and the corresponding functions of plant and yeast vacuolar membrane vesicles but the rates and extents of transport by yeast secretory vesicles are some 30-40-fold lower, two alternative explanations may apply. Either the secretory vesicles prepared by St-Pierre et al.(1994) were 2-4% contaminated with vacuolar membrane vesicles or the presence of GS-X pump activity in this fraction reflects its redistribution in preparation for the fusion of S-conjugate-loaded vesicles with the plasma membrane. Further experiments are required to distinguish between these alternatives but the second interpretation would agree with the conclusions of Ishikawa et al.(1994) and implicate ultimate exocytosis of intracellularly compartmented glutathione S-conjugates in both animal and yeast cells. A fundamental distinction between animal, yeast and plant cells may therefore correspond to the necessity for ``storage excretion'' in plants (Martinoia et al., 1993). Mammalian and yeast cells have the option of excreting conjugates to the external medium, for eventual elimination by the kidneys in mammals, but terrestrial plants are primarily dependent on the sequestration of noxious compounds in their large central vacuoles. Thus, what might be an intermediate step in the elimination of xenobiotics from the cytosol of mammalian, and possibly yeast cells, intracellular compartmentation, probably constitutes the ultimate phase of detoxification in plants.
Given MRP's membership of a new subclass of ABC transporter and its involvement in resistance to chemotherapeutic drugs, disclosure of its functional and structural equivalent in an organism of such manipulability as yeast opens the way for its detailed molecular characterization. In addition, since it is now evident that the transport of herbicides in plants after their conjugation with GSH is catalyzed by a similar transporter, there is a strong possibility that studies of yeast YCF1 will also directly impinge on xenobiotic detoxification in plants and bioremediation in general.