From the Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322
Received for publication, October 4, 2000, and in revised form, December 8, 2000
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ABSTRACT |
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The yeast Saccharomyces
cerevisiae readily accumulates short-chain, fluorescent
7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled phosphatidylcholine and phosphatidylethanolamine at the nuclear envelope/endoplasmic reticulum and mitochondria. The net intracellular accumulation reflects the sum of their inwardly and outwardly directed
transbilayer translocation across the plasma membrane (flip and flop,
respectively). The rate of flop is negligible in energy-depleted cells
as well as at low temperature (2 °C). Although flip is reduced at
2 °C, it can still be measured by flow cytometry, allowing the rate
of flip, independent of flop, to be characterized at this temperature.
Flip requires the energy of the plasma membrane proton electrochemical
gradient and is down-regulated as cells pass through the diauxic shift
and enter stationary phase. Furthermore, drug-resistant,
gain-of-function mutations in the transcription factors,
PDR1 and PDR3, result in a dramatic
down-regulation of flip in addition to their already established
up-regulation of flop. These results imply that down-regulation of the
NBD-phospholipid flip pathway is a physiological response to
environmental stress.
Selective, inwardly directed transport (flip) of the
aminophospholipids, phosphatidylserine
(PtdSer)1 and
phosphatidylethanolamine (PtdEtn), across the plasma membrane of
erythrocytes (1-4) has been proposed to establish and maintain their
asymmetric distribution between the two leaflets of the plasma membrane
(5, 6). Loss of this asymmetry is mediated by a
Ca2+-activated, nonselective phospholipid transporter (7,
8). The observation of similar aminophospholipid transport (9-16) and
nonselective phospholipid transport (17, 18) activities in a wide range
of nucleated cells has led to the current consensus that the general
features of the asymmetric bilayer distribution measured in
erythrocytes exist in most, if not all, eukaryotes (reviewed in Refs.
19 and 20). Maintenance of this asymmetry appears to be critical to
cell survival in higher eukaryotes, since the exposure of PtdSer on the
cell surface is a signal for macrophage clearance (21-23).
Furthermore, PtdSer exposure on the surface of platelets is essential
to initiate the coagulation cascade (24), and regulation of
phospholipid asymmetry across the plasma membrane may modulate a
variety of other processes including membrane budding (25) and
activation of PtdSer-dependent enzymes (26).
Although the activity of aminophospholipid transport has been well
characterized, the identity of the aminophospholipid translocase has
remained elusive. A 115-kDa P-type ATPase, purified and cloned from
bovine chromaffin granules, has been proposed to have aminophospholipid translocase activity based on inference from the absence of selective PtdSer uptake in yeast cells in which its yeast orthologue,
DRS2, was deleted (27). However, failure to confirm the
dependence of PtdSer uptake on the expression of DRS2 in two
independently constructed null strains (28, 29) calls into question the inference that the chromaffin granule P-type ATPase has
aminophospholipid translocase activity. Other membrane proteins have
been identified as aminophospholipid translocases (30-33); however, a
definitive demonstration of selective phospholipid translocase activity
is lacking in each case (20). As such, there is no consensus as to the
identity of this transporter.
Identification of the Ca2+-activated, nonselective
phospholipid transporter (scramblase) has been more successful. The
protein responsible for the loss of asymmetry in erythrocytes has been purified (34), and the cDNA has been cloned and sequenced (35). Although the expression of scramblase in human cancer cell lines is
directly correlated with Ca2+-activated exposure of PtdSer
in the exoplasmic leaflet (18), scramblase expression in these same
cell lines does not correlate with PtdSer exposure activated by
apoptosis (36). This discrepancy suggests a second unidentified pathway
for PtdSer translocation across the plasma membrane that is responsive
to the apoptotic cascade. This pioneering work illustrates the need for
further research on the molecular details of the regulation and
maintenance of phospholipid distribution in eukaryotic cells.
The use of phospholipids with the fluorescent label NBD covalently
attached to a short acyl chain provides a means by which phospholipid
transbilayer translocation can be addressed (37). In previous studies,
we and others have applied these NBD-labeled phospholipids to study the
mechanisms and regulation of phospholipid transbilayer transport across
the plasma membrane in the yeast Saccharomyces cerevisiae
(29, 38-40). NBD-labeled PtdCho (29, 40) and PtdEtn (39) are
internalized predominantly by inwardly directed transbilayer transport
(flip) across the plasma membrane. However, at ambient growth
temperatures, the net accumulation of both NBD-phospholipids reflects
the sum of inwardly and outwardly directed transbilayer translocation,
flip and flop, respectively (39, 40).
The transcription factors Pdr1p and Pdr3p regulate the net accumulation
of NBD-labeled PtdCho and PtdEtn (39). Pdr1p and Pdr3p are among the
primary regulators of pleiotropic drug resistance (PDR) in yeast, and
gain-of-function mutations in these genes confer the ability to grow in
the presence of structurally unrelated compounds that are normally
cytotoxic. This drug-resistant phenotype is thought to be mediated
primarily by transcriptional up-regulation of a variety of ATP binding
cassette (ABC) transporters as well as members of the major facilitator
superfamily, many of which have been convincingly shown to
efflux drugs (Refs. 41-43; reviewed in Ref. 44). Therefore, it was
previously assumed that the NBD-lipid accumulation defect seen in some
PDR1 and PDR3 gain-of- function mutants was due
solely to up-regulated NBD-phospholipid flop. The fact that deletion of
the ABC transporters PDR5 and YOR1, individually
or in combination, partially rescues the NBD-PtdEtn accumulation defect
of a PDR1 gain-of-function mutant (45) confirms the role of
efflux in modulating lipid and drug accumulation within the cell.
To date, few, if any, studies have examined the role of drug
resistance networks in regulating the unidirectional uptake of drugs or
lipid analogs. By monitoring the flip of NBD-phospholipids at low
temperature when flop is inhibited, we have made the novel observation
that in addition to their established role in up-regulating flop,
PDR1 and PDR3 also dramatically down-regulate
NBD-phospholipid flip. Furthermore, we show here that flip is
down-regulated when cells experience nutrient starvation, suggesting
that down-regulation of the expression and/or activity of the
transporter responsible for NBD-phospholipid flip is a physiological
response to environmental stress.
Materials--
Yeast medium was from Difco, and NBD-PE and
NBD-PC were purchased from Avanti Polar Lipids (Alabaster, AL).
Unless otherwise noted, all other
materials were purchased from Sigma.
Yeast Strains and Culture--
The S. cerevisiae
strains used are listed in Table I. Early
log phase cultures (A600 = 0.1-0.2)
were grown from overnight cultures in synthetic complete medium
(SDC; 0.67% yeast nitrogen base 2% glucose, complete amino acid
supplements) at 30 °C as described in (46). For diauxic shift and
stationary phase experiments, cells grown to varying culture densities
were harvested and resuspended at an A600 = 0.1-0.2 in nutrient-depleted medium obtained from the original
culture. SDC lacking glucose but containing 2% sorbitol and 20 mM sodium azide (SC-azide) with or without 10 mM sodium fluoride was used as indicated.
Internalization of Phospholipids into Yeast Cells--
For low
temperature internalization assays, early log phase cultures were
chilled in an ice bath for ~10 min. 10 µl of 0.5 mM
NBD-phospholipid in Me2SO was added per ml of cell
culture while vigorously vortex mixing. Cultures were returned to the ice bath, and aliquots of labeled cells were removed at the indicated times and washed three times with two volumes of ice-cold SC-azide. Samples were analyzed by fluorescence microscopy or flow cytometry. For
room temperature labeling experiments, 20 µl of a vesicle stock
containing 1 mM total lipid (40 mol % of NBD-phospholipid) were added to 1 ml of yeast cells and incubated at 30 °C for 30 min
with shaking as described in Ref. 38. After labeling, cells were washed
three times with two volumes of ice-cold SC-azide and analyzed by
fluorescence microscopy or flow cytometry.
Efflux of Phospholipids from Yeast Cells--
Cells were labeled
at low temperature as described above to achieve similar levels of
fluorescence in the strains to be compared. After labeling, the cells
were washed three times with two volumes of ice-cold SDC. Cells were
resuspended and subsequently incubated in either SDC or SC-azide at 2 or 30 °C. At the given time points, cells were rapidly cooled in an
ice bath, washed three times with two volumes of ice-cold SC-azide, and
analyzed by flow cytometry.
Flow Cytometry--
Flow cytometric analysis of the
NBD-lipid-labeled cells was performed with a FACScalibur or FACScan
cytometer (Becton-Dickinson Immunocytochemistry, San Jose, CA) as
described previously (45). Briefly, 10 µl of a 50 µg/ml stock
solution of propidium iodide were added to ~4 × 105
cells in 1 ml of SC-azide immediately prior to dilution (~3 times) and flow cytometric analysis on a cytometer equipped with an argon laser operating at 488 nm. Analysis was performed with CellQuest (Becton-Dickinson Immunocytochemistry Systems) software, using the
propidium iodide staining to exclude dead cells when quantifying NBD fluorescence.
Fluorescence Microscopy--
Fluorescence microscopy was
performed on a Carl Zeiss microscope, Axiovert 135 model (Carl Zeiss,
Inc., Thornwood, NY). Fluorescence images were enhanced with an
image-intensifying camera (model VE1000-SIT; DAGE-MTI, Inc., Michigan
City, IN), digitized, and stored. Contrast enhancement was performed
with Metamorph software (Universal Imaging Corp., West Chester PA).
NBD-Phospholipid Flop Is Inhibited at Low Temperature--
Our
laboratory has previously observed that when yeast cells are labeled
with NBD-PE, washed, and incubated at 30 °C, the intracellular
fluorescence gradually decreases with time (39). Based on data obtained
by fluorescence microscopy, fluorometry, and chemical analysis, we
concluded that this decrease results from the transport of intact
NBD-PE to the outer leaflet of the plasma membrane, where it is
degraded by periplasmic phospholipases to water-soluble products with
low quantum yields (39). Since the rate of NBD-PE exposure to the
exoplasmic surface is not inhibited in a strain defective in a late
stage of vesicle secretion (sec6-4), we concluded that
NBD-PE is not secreted, but rather flopped to the surface (39). In
addition, we observed that the rate of appearance of NBD-PE at the
exoplasmic surface was essentially the same whether it was measured by
degradation or transfer to excess liposomes in the solution. Thus, we
concluded that the rate of fluorescence decrease was limited by the
rate of flop, not degradation (39), and therefore provided an accurate
measurement of the rate of flop.
In Fig. 1, we demonstrate that flow
cytometry can be used to measure the time-dependent loss of
fluorescence in a wild-type yeast strain. Flow cytometry has the
advantage over fluorometry of allowing the exclusion of dead cells,
which readily absorb NBD-PE, from the analysis of the mean
fluorescence. Briefly, a culture of yeast in early log phase was
labeled with Me2SO-solubilized NBD-PE at low temperature
and washed with cold medium. Aliquots of labeled cells were then
resuspended in the indicated medium at either 30 or 2 °C, and
changes in fluorescence as a function of time were measured by flow
cytometry. As expected (39), the fluorescence of labeled cells
resuspended in standard medium at 30 °C gradually decreased
over time. However, cells incubated at low temperature (2 °C) or
following energy depletion with sodium azide experienced no significant
change in cellular fluorescence during a 90-min incubation. These data
show that NBD-PE flop is an energy-requiring process that is
essentially blocked at low temperature. Similar results were obtained
for NBD-PC (data not shown).
NBD-Phospholipids Are Internalized by Flip at 2 °C--
At
ambient growth temperatures (~30 °C), yeast cells grown to early
log phase readily accumulate both NBD-PC (29, 38, 40) and NBD-PE (39,
40) by transbilayer transport across the plasma membrane. At this
temperature, since the rate of flop is substantial, it is not possible
to determine whether regulation of the extent of net accumulation
results from regulation of flip, flop, or a combination of both.
However, since the rate of flop is essentially nil at 2 °C (Fig. 1)
and since endocytosis and vesicle fusion and budding (40, 47-49) are
blocked at this temperature, measurement of NBD-phospholipid
accumulation at 2 °C reflects the rate of flip independent of flop.
Flow cytometry was used in Fig. 2 to
demonstrate that the cell-associated NBD-PC and NBD-PE fluorescence
gradually increases with time at 2 °C. Since no fluorescence is
detectable in the plasma membrane by fluorescence microscopy following
labeling with either NBD-PC or NBD-PE at 2 °C (40), nor is it
transported to and degraded in the vacuole under these conditions (40),
the cell-associated fluorescence originates solely from internalized,
membrane-associated NBD-PC or NBD-PE. Thus, measurement of low
temperature accumulation by flow cytometry provides a measure of the
average rate of flip, independent of flop, for a large population of
live cells and provides a means to investigate its activity and
regulation. These data (Fig. 2) illustrate that the flip of NBD-PC is
slightly faster than NBD-PE and that neither flip rate begins to
saturate during the first 2 h.
NBD-Phospholipid Flip Is Dependent on the Plasma Membrane Proton
Electrochemical Potential--
We have shown previously that the low
temperature flip of NBD-PC and NBD-PE is inhibited by energy depletion
with sodium azide and fluoride. Here we show that the flip of both
NBD-phospholipids is also inhibited by the protonophore CCCP (Fig. 2).
Because maintenance of the proton gradient by the proton pump Pma1p
requires ATP, it is likely that the inhibition of flip by azide and
fluoride is a secondary effect of ATP depletion and that the energy
required for flip is provided primarily by the proton electrochemical
gradient. Consistent with this model, we have observed that PMA1
mutants with decreased membrane potential show a concomitant reduction in low temperature internalization of NBD-phospholipids (data not shown).
Since a large fraction of NBD-PC and NBD-PE fluorescence is localized
in the mitochondria following low temperature labeling, it is possible
that depolarization of the mitochondrial proton-electrochemical gradient by CCCP inhibits the uptake of NBD-PC and NBD-PE by inhibiting their accumulation in the mitochondria in addition to inhibiting flip
across the plasma membrane. To rule out this possibility, we studied
petite strains obtained by treatment of the CRY2 strain with ethidium
bromide (50). The resulting rho
This result was confirmed by examining the intracellular localization
of NBD-PC and NBD-PE in the petite strain by fluorescence microscopy
(Fig. 3B). As in wild-type strains (40), both
NBD-phospholipids gave a punctate staining pattern that often
colocalized with DAPI staining of the mitochondrial DNA (Fig.
3B). Thus, the mitochondrial membrane potential is not
required for NBD-phospholipid flip across the plasma membrane; nor is
it required for mitochondrial localization. Furthermore, disruption of
the mitochondrial membrane potential by CCCP does not affect the
maintenance of mitochondrial NBD-PE localization. After labeling with
NBD-PE and DAPI, wild-type cells were thoroughly washed with cold SDC
to remove donor lipid, resuspended in 30 °C SDC, and treated with
and without 50 µM CCCP for 15 min. Cells were then
harvested, washed with cold SC-azide to prevent any additional lipid
efflux, and visualized by fluorescence microscopy. The intracellular
distribution of NBD-PE and its colocalization with DAPI-stained
mitochondrial DNA were not significantly altered by CCCP (Fig.
3C). Thus, neither NBD-PE flip across the plasma membrane
nor its distribution to or maintenance in the mitochondria are
dependent upon the mitochondrial membrane potential.
NBD-Phospholipid Flip Is Inhibited as Cells Progress through the
Diauxic Shift--
To gain a better appreciation of the physiological
regulation of flip, we chose to monitor this process as yeast
experience one of their most common stresses, nutrient starvation. As
seen in Fig. 4A, when cells
progress through the diauxic shift and enter stationary phase, their
net uptake of NBD phospholipids at 30 °C tends to decrease. To rule
out the possibility that this decreased uptake was due to
starvation-associated alterations in the cell wall, we labeled cells
with a passively absorbed, short-chain NBD-ceramide and observed no
correlation between its cellular accumulation and culture density (data
not shown). Thus, changes in the permeability of the cell wall do not
account for the observed decrease in NBD-phospholipid at increased
culture density over the range studied. The reduced net accumulation
reflects some combination of decreased flip and increased flop across
the plasma membrane.
To determine the relative roles of flip and flop in decreasing
NBD-phospholipid accumulation in response to growth
phase-dependent nutrient starvation, we measured NBD-PE
flip and flop independently as described above. By comparing the low
temperature uptake of cells at various growth phases, we determined
that flip is gradually down-regulated as cells enter stationary phase
(Fig. 4, A and B). On the other hand, nutrient
starvation has only a modest effect, if any, on NBD-phospholipid flop
at 30 °C (Fig. 4C). Because nutrient starvation has no
effect on the flop rate at 30 °C, the observed decrease in net
NBD-PE accumulation at this temperature is due to a decrease in the
rate of flip. Given that the dependence of net NBD-PE accumulation on
culture density is similar for measurements made at 2 and 30 °C
(Fig. 4A), it is reasonable to infer that the same flip
pathway is inhibited at both temperatures. Inhibition may result from
down-regulation of the expression or activity of the gene product
responsible for NBD-PE flip or a reduction of the proton
electrochemical potential driving force. The observation that
internalization of the potential-dependent dye, rhodamine 123, actually increases as cells pass through the diauxic
shift2 argues against the
latter possibility. These data support the conclusion that the
expression and/or activity of an inwardly directed NBD-PE transporter
is regulated by the nutrient status of these cells.
PDR1 and PDR3 Up-regulate Flop and Down-regulate
Flip--
Drug-resistant, gain-of-function mutations in
PDR1 and PDR3 have been shown to inhibit the net
accumulation of NBD-PE and NBD-PC at normal growth temperatures
(39).2 Because these transcription factors are known to
up-regulate expression of a variety of drug pumps, the accumulation
defects of these mutant strains were thought to be primarily the result of up-regulated NBD-phospholipid flop. As seen in Fig.
5, the pdr3-11
gain-of-function mutant is clearly capable of up-regulating NBD-PE flop
compared with its isogenic parent, and this increased flop can be
inhibited by azide and low temperature. Similar results are seen in
other accumulation-defective PDR1 and PDR3
gain-of-function mutants (data not shown). However, by monitoring the
low temperature uptake of NBD-phospholipids, we have determined that
these mutants are also dramatically down-regulated in flip (Fig.
6, A and B). Furthermore, disruption of PDR1 and PDR3 results
in a modest yet significant increase in NBD-phospholipid accumulation
(Fig. 6, C and D), indicating that the wild-type
alleles also down-regulate flip.
A recent report has shown that PDR16 and PDR17,
two downstream targets of PDR1, can alter the passive uptake
of drugs by affecting lipid biosynthesis (52). Deletion of
PDR16 and PDR17 results in hypersensitivity to a
broad range of drugs and increased uptake of rhodamine-6-G in cells
deenergized with antimycin A and 2-deoxy-D-glucose (52). To
rule out the possibility that the observed differences in
NBD-phospholipid accumulation were a reflection of alterations in the
passive permeability of the plasma membrane, we measured the low
temperature uptake of NBD-PE in cells treated with the ATP-depleting
agents azide and fluoride or alternatively with CCCP. As seen in Fig.
7, any strain-specific differences in the passive uptake of NBD-phospholipids do not account for the
down-regulated rates of flip. In fact, the strains down-regulated in
flip, pdr3-11 and PDR1-11, may exhibit a
slightly increased passive uptake of NBD-phospholipids under these
conditions. Thus, we conclude that PDR1 and PDR3
regulate the expression and/or activity of transporters capable of
translocating NBD-phospholipids in both directions across the plasma
membrane.
We have shown here and previously that under normal growth
conditions the net accumulation of NBD-PC and NBD-PE in yeast cells is
determined by the sum of the rates of flip and flop across the plasma
membrane (38, 39). By labeling yeast at low temperature, we
demonstrated (Figs. 1 and 2) that NBD-phospholipid flip can be
monitored independently of flop. This approach has allowed us to
characterize more fully flip and thereby determine that it is an
energy-requiring process that depends on the proton-electrochemical gradient across the plasma membrane. We have shown that
NBD-phospholipid net accumulation is dramatically reduced by culture
density-dependent nutrient starvation (Fig. 4A)
resulting from the down-regulation of flip (Fig. 4B) without
affecting the rate of flop (Fig. 4C). We also uncovered a
new role for PDR1 and PDR3, transcription factors
previously identified based on their ability to confer drug resistance
by up-regulating drug efflux (39). In addition to confirming their role
in up-regulating NBD-phospholipid flop, we have shown that
PDR1 and PDR3 also down-regulate NBD-phospholipid flip.
Previous work demonstrated that the net accumulation of NBD-PC and
NBD-PE at 30 °C (38, 39) as well as their low temperature flip
(38-40) are inhibited by ATP-depletion. In contradiction to these
results, Marx et al. (29) found that the low temperature flip of NBD-PC and NBD-PtdSer was unaffected by ATP depletion. The
reason for this discrepancy is not clear. Here we demonstrated that the
low temperature flip of both NBD-phospholipids was also inhibited more
than 94% by collapse of the proton electrochemical potential across
the plasma membrane with the protonophore, CCCP (Fig. 2). These results
suggest that inhibition of flip by ATP depletion reflects a requirement
for ATP to maintain the proton electrochemical potential rather than a
direct coupling of ATP hydrolysis to NBD-phospholipid flip. Based on
the observations that NBD-PE and NBD-PC net accumulation at 30 °C is
also blocked by CCCP2 and that NBD-PE internalization is
reduced to the same extent at 2 °C as at 30 °C in response to
nutrient starvation (Fig. 4A), we inferred that the same
mechanism of transport is responsible for the majority of NBD-PE and
NBD-PC flip at both temperatures.
These results suggest that the transporter responsible for
NBD-phospholipid flip in yeast differs significantly from that responsible for their flip in mammalian cells in two important respects. The most obvious is the difference in energy coupling. In
mammalian cells, NBD-labeled and spin-labeled phospholipids are
internalized by a transporter directly coupled to ATP-hydrolysis (19,
20, 53) as opposed to secondary active transport dependent on the
proton electrochemical gradient observed here for yeast. Second, the
yeast transporter internalizes NBD-PC slightly faster that NBD-PE,
whereas in most mammalian cells studied, the aminophospholipid analogues, NBD-PE and NBD-PS, are transported much faster than NBD-PC
(20, 37, 54). However, these results are not completely unprecedented
in mammalian cells. The WI-38 cell line transformed with the SV40 virus
differs from its parent in that it rapidly transports NBD-PC across its
plasma membrane (55). Furthermore, spin-labeled phosphatidylcholine was
found to be rapidly internalized in an ATP-dependent,
N-ethylmaleimide-sensitive manner in Madin-Darby canine
kidney II cells (16). However, based on the differences in energy
coupling, the yeast transporter responsible for NBD-PC and NBD-PE
transport is unlikely to be a structural orthologue of the mammalian
aminophospholipid translocase thought to be responsible for
establishing phospholipid asymmetry across the plasma membrane.
Previously, we determined that the loss of NBD-PE internalization
observed in gain-of-function mutants, PDR1-11 and
pdr3-11, correlated with an increase in the amount of
endogenous PtdEtn exposed to the outer leaflet of the yeast plasma
membrane detected by covalent labeling with the impermeant reagent,
TNBS (39). We concluded that the decreased net inward flux of NBD-PE
reflected the behavior of endogenous PtdEtn, which resulted in an
increase in PtdEtn exposed to the outer surface. Given that NBD-PC
(Fig. 6) and NBD-PS accumulation (data not shown), which are also
internalized predominantly by flip, are also almost completely
inhibited in PDR1-11 and pdr3-11 strains, it is
unlikely that the loss of accumulation reflects an increased net
outward flux of all three of these phospholipids, which make up the
majority of the plasma membrane phospholipids. More likely, the short
chain, NBD-labeled phospholipids have different affinities for their
transporters than their endogenous counterparts, and therefore the
extent of their accumulation does not quantitatively reflect the
endogenous phospholipid distribution. Although we cannot rule out the
possibility that the NBD-phospholipids are transported either inward or
outward by mechanisms that are different than those of endogenous
phospholipids, some degree of specificity for the phospholipid
structure is observed for the flip pathway, since NBD-PE flip is
dramatically inhibited during nutrient starvation, whereas that of
NBD-ceramide and rhodamine 123 is not.2 Therefore, the
NBD-phospholipids may provide information regarding the mechanisms of
the transbilayer transport of endogenous phospholipids while not being
strictly representative of their quantitative distribution.
On the other hand, the NBD-phospholipids may be more representative of
the behavior of short chain diacyl or long chain lysophospholipids. Phospholipase B molecules secreted into the periplasm turnover roughly
6% of the total PtdCho per doubling period based on the amount of
glycerophosphocholine released into the medium (56). Given this rapid
turnover of PtdCho presumably from the outer leaflet of the plasma
membrane, it would be metabolically advantageous for some fraction of
the lysophosphatidylcholine produced in the first step of this two-step
reaction to be transported inward across the plasma membrane for
reutilization. Transport of the short-chain NBD-phospholipids may
reflect such a pathway.
The observed down-regulation of NBD-phospholipid flip as cells progress
through the diauxic shift and enter stationary phase is consistent with
the nutrient retrieval hypothesis, since yeasts are known to remove the
class of constitutive permeases from their cell surface upon nutrient
starvation when metabolic demands are reduced (57). However, we cannot
rule out the possibility that the decreased inward movement of the
NBD-phospholipids in response to nutrient starvation reflects
alterations in the distribution of endogenous phospholipids between the
leaflets of the plasma membrane. If such were the case, regulation of
phospholipid asymmetry would provide a means to regulate numerous
cellular functions (e.g. enzyme, transporter, and channel
activity as well as membrane fusion and fission events required for
vesicular trafficking) in unicellular organisms and single cells in
multicellular organisms in addition to its well defined role in
intercellular signaling (20, 58). Regardless of the interpretation, the
significance of the regulation of NBD-phospholipid flip by nutrient
starvation clearly requires further investigation.
In addition to its regulation with growth phase, we have shown here
that flip is regulated by PDR1 and PDR3, thus
identifying a novel role for these transcription factors. It is well
established that gain-of-function mutants in PDR1 and
PDR3 produce drug resistance by up-regulating the expression
of a variety of ABC transporters as well as members of the major
facilitator superfamily, many of which have been convincingly shown to
efflux drugs (41-44). The role of ABC transporters to efflux/flop
NBD-phospholipids is confirmed by the fact that deletion of the ABC
transporters PDR5 and YOR1, individually or in
combination, partially rescues the NBD-PE accumulation defect of a
PDR1 gain-of-function mutant (45). Furthermore, transfection
of epithelial cells with human MDR3 specifically increases
the flop rate of endogenously synthesized NBD-PC (59), and
overexpression of human MDR1 and mouse
mdr1a (mdr3) P-glycoprotein results
in increased flop of NBD-PC and other NBD lipid analogues (59). Thus,
several, but not all, ABC transporters have the ability to flop
NBD-phospholipids across the plasma membrane, and it was previously
assumed that the NBD-lipid accumulation defect seen in some
PDR1 and PDR3 gain of function mutants was due
solely to up-regulated NBD-phospholipid flop. Our data indicate that
Pdr1p and Pdr3p regulate the net accumulation of NBD-phospholipids by
down-regulating flip in addition to up-regulating flop. The
similarities between the regulation of NBD-phospholipids and drug
accumulation by these transcription factors suggest that down-regulation of unidirectional amphipathic drug uptake may be a
major mechanism for reducing net intracellular drug accumulation. If
so, this strategy for evading cytotoxic compounds could prove to be an
even more difficult problem to circumvent than up-regulated efflux when
addressing pleiotropic drug resistance.
Given the dual nature of the NBD-phospholipids (structural phospholipid
analogues with drug-like behavior), unraveling the relationship between
phospholipid flip and flop and drug influx and efflux remains a
challenge (19). These studies provide the basis for identification of
the transporter responsible for NBD-phospholipid flip that will be
necessary to further characterize its physiological function and
regulation. There appear to be at least two regulatory pathways: one
that responds to nutrient starvation and another mediated by
PDR1 and PDR3. Interestingly, strains carrying
disruptions of either PDR1, PDR3, or both still
respond to the diauxic shift by down-regulating flip (data not shown).
This observation argues that these two regulatory pathways are
independent but converge on the same downstream targets, presumably
NBD-phospholipid transporters at the plasma membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Yeast strains used in this study
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Fig. 1.
Inhibition of NBD-PE flop by low temperature
and azide treatment. Cells (CRY2) were labeled on ice with
Me2SO-solubilized NBD-PE, washed with either cold SDC or
cold-SC-azide, and then either warmed to 30 °C or kept at 2 °C.
Aliquots of cells were removed at the given time points, washed with
cold SC-azide, and analyzed by flow cytometry. , SDC, 30 °C;
,
SDC, 2 °C;
, SC-azide, 30 °C;
, SC-azide, 2 °C. Data
reported are the mean and S.D. of at least three independent
experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
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Fig. 2.
Inhibition of low temperature
NBD-phospholipid flip by CCCP. Early log phase cells (CRY2) were
transferred to either fresh SDC or SDC plus 50 µM CCCP.
After a 10-min incubation under these conditions, cells were chilled in
an ice bath and subsequently labeled with 5 µM
Me2SO-solubilized NBD-PE or NBD-PC. At the indicated times,
an aliquot of cells was removed and washed with cold SC-azide prior to
flow cytometric analysis. , NBD-PC, control;
, NBD-PE, control;
, NBD-PC plus CCCP;
, NBD-PE plus CCCP. Data reported are the
mean and S.D. of at least three independent experiments.
strains did
not grow on YP-glycerol, indicating that the petite phenotype resulted
from a respiratory defect. An almost complete reduction in the
accumulation of the mitochondrial potential-sensitive dye,
3,3'-dihexyloxacarbocyanine iodide (DiOC6)
(51), confirmed the disruption of mitochondrial potential in these
strains (Fig. 3A). Despite
these mitochondrial defects, NBD-PC and NBD-PE uptake at low
temperature (Fig. 3A) remained essentially unaffected, indicating that fluorescent phospholipid flip is not dependent on the
mitochondrial membrane potential. Thus, the inhibition of
NBD-phospholipid flip by CCCP results from collapse of the electrochemical potential across the plasma membrane rather than the
mitochondria.
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Fig. 3.
Mitochondria membrane potential is not
required for the localization or retention of NBD-phospholipids in the
mitochondria. A, CRY2 cells and
rho petite cells derived from the CRY2 strain
(LKY165) were labeled with 0.1 µM
3,3'-dihexyloxacarbocyanine iodide (DiOC6) at room
temperature for 15 min and harvested by centrifugation prior to
analysis by flow cytometry. These strains were also labeled with 5 µM Me2SO-solubilized NBD-PC or NBD-PE at
2 °C as described above. Mean fluorescence of the CRY2 strain
(black bars) was normalized to 1. Relative
fluorescence of the petite strains (gray bars)
was plotted as the mean and S.D. of three experiments. B,
rho
petite cells (LKY165) were labeled with
DAPI for 15 min. at 30 °C prior to chilling to 2 °C and labeling
with 5 µM Me2SO-solubilized NBD-PC or NBD-PE
as described above. Cells were washed thoroughly with SC-azide and
imaged by fluorescence microscopy as described under "Experimental
Procedures." m, mitochondria. C, DAPI-labeled
diploid cells (CRY3) were labeled for 2 h on ice with 10 µM Me2SO-solubilized NBD-PE, washed
thoroughly with ice-cold SDC, and resuspended in either SDC or SDC plus
50 µM CCCP at room temperature. After a 15-min incubation
under these conditions, cells were washed once with ice-cold SC-azide
and observed by fluorescence microscopy. m, mitochondria.
Calibration bar = 10 µm.
View larger version (19K):
[in a new window]
Fig. 4.
NBD-PE net uptake, flip, and flop as a
function of growth phase. A, yeast (CRY2) were grown to
different culture densities, as indicated by optical density at 600 nm.
Cultures at each density were harvested by centrifugation and diluted
to an A600 = 0.1 in their respective
nutrient-depleted media. Cells were then labeled for 30 min with 40 mol
% of NBD-PE-containing vesicles at 30 °C ( ) or labeled at low
temperature for 60 min. as described for B (
) prior to
washing and flow cytometric analysis. Data presented are representative
of the results of at least three experiments. B, time
courses of NBD-PE uptake at 2 °C into yeast grown to different
culture densities. Cells (CRY2) grown and resuspended as described
above were incubated in an ice bath with 5 µM
Me2SO-solubilized NBD-PE. At each time point, an aliquot of
cells was removed and washed with ice-cold SC-azide before analysis by
flow cytometry.
, A600 = 0.1;
,
A600 = 0.5;
, A600 = 1.0;
, A600 = 2.0. C, cells (CRY2)
in early log and late diauxic phase were labeled with NBD-PE at low
temperature as described for B, and flop of the fluorescent
lipid at 30 °C was monitored as described in the legend to Fig. 1.
, A600 = 0.1;
,
A600 = 2.0. Data are the means and S.D. of at
least three independent experiments.
View larger version (25K):
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Fig. 5.
NBD-PE flop is up-regulated in the drug
resistant mutant, pdr3-11. The
pdr3-11 strain (LKY156) and its PDR3 parent
(CRY2) were labeled on ice with Me2SO-solubilized NBD-PE,
washed with either ice-cold SDC or ice-cold SC-azide, and then either
warmed to 30 °C or kept at 2 °C. Aliquots of cells were removed
at the given time points, washed with ice-cold SC-azide, and analyzed
by flow cytometry. , pdr3-11, 30 °C;
,
pdr3-11, 30 °C with azide;
, pdr3-11,
2 °C;
, pdr3-11, 2 °C with azide;
,
PDR3 control, 30 °C. Data reported are the mean and S.D.
of at least three independent experiments.
View larger version (23K):
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Fig. 6.
Down-regulation of NBD-phospholipid flip by
PDR1 and PDR3. Low temperature NBD-PE internalization of
PDR1 and PDR3 gain-of-function and null strains
was characterized as described in the legend to Fig. 2.
Closed symbols (A and
C), NBD-PC; open symbols
(B and D), NBD-PE. and
, PDR1
PDR3 (LKY118 or AGY72);
and
, PDR1-11 PDR3
(LKY154);
and
, PDR1 pdr3-11 (LKY156);
and
,
pdr1
pdr3
(AGY75). Data reported are the
mean and S.D. of at least three independent experiments.
View larger version (22K):
[in a new window]
Fig. 7.
Passive uptake of NBD-PE. Early log
phase cells grown in SDC were switched to either medium
containing 50 µM CCCP or to medium lacking glucose but
containing azide and fluoride. After a 10-min incubation under these
conditions, cells were chilled on ice and then labeled with 5 µM Me2SO-solubilized NBD-PE for 30 min. After
thorough washing with cold SC-azide, cells were analyzed by flow
cytometry. Strains were the same as in Fig. 6.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Robert Fuller (University of Michigan) and John McCusker (Duke University) for the generous gift of mutant yeast strains.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant GM52410 and a grant from the University Research Committee of Emory University (to J. W. N.).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.
Supported by a National Institutes of Health training grant GM08367.
§ To whom correspondence should be addressed: 1648 Pierce Dr., Dept. of Physiology, Emory University School of Medicine, Atlanta, GA 30322. Tel.: 404-727-7422; Fax: 404-727-2648; E-mail: wnichols@ physio.emory.edu.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M009065200
2 P. K. Hanson and J. W. Nichols, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: PtdSer, phosphatidylserine; PtdEtn, phosphatidylethanolamine; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DAPI, 4',6-diamidino-2-phenylindole; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; NBD-PC, 1-myristoyl-2-[6-(NBD)-aminocaproyl]-phosphatidylcholine; NBD-PE, 1-myristoyl-2-[6-(NBD)-aminocaproyl]-phosphatidylethanolamine; PtdCho, phosphatidylcholine; PDR, pleiotropic drug resistance; ABC, ATP binding cassette.
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REFERENCES |
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