We recently reported that the nonionic detergents Brij-96 and Triton X-100 isolated different lipid raft microdomains from rat basophilic leukemia RBL-2H3 cells [37].. In addition, we fo
Trang 1membrane microdomains distinct from classical lipid rafts and caveolar domains
Galina Radeva, Jocelyne Perabo and Frances J Sharom
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
In recent years, intense interest has been focussed on
the properties and biological functions of specialized
membrane domains known as lipid rafts [1,2] Rafts
consist of cholesterol–sphingolipid-rich regions within
the plasma membrane, stabilized by interactions
between cholesterol and the long saturated acyl chains
of sphingolipids They are thought to exist in the liquid-ordered phase, which has properties intermedi-ate between those of the liquid-crystalline and gel phases [3,4] Acylated and lipid-modified proteins are
Key words
ABC transporter; caveolin-1;
detergent-resistant membranes; lipid rafts;
P-glycoprotein
Correspondence
F J Sharom, Department of Molecular and
Cellular Biology, University of Guelph,
Guelph, Ontario, Canada, N1G 2W1
Fax: +519 837 1802
Tel: +519 824 4120; ext 52247
E-mail: fsharom@uoguelph.ca
(Received 11 May 2005, revised 27 July
2005, accepted 4 August 2005)
doi:10.1111/j.1742-4658.2005.04905.x
P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) super-family responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid–cholesterol microdomains in cell membranes
We have used nonionic detergent extraction and density gradient centrifu-gation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CHRB30, to address this question Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-Brij-96-insoluble membrane microdomains We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM1 ganglioside- and caveolin-1-enriched caveolar domains We found that the buoyant density
of the Brij-96-based Pgp-containing microdomains was sensitive to choles-terol removal by methyl-b-cyclodextrin The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1⁄ GM1 microdomains did not Using confocal fluor-escence microscopy, we determined that caveolin-1 and GM1 colocalized, while Pgp and caveolin-1, or Pgp and GM1, did not Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in inter-mediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96
Abbreviations
ABC, ATP-binding cassette; BSS, buffered saline solution; CTB, cholera toxin B subunit; CTB–HRP, cholera toxin B–horseradish peroxidase conjugate; DRM, detergent-resistant membranes; ECL, enhanced chemiluminescence; MbCD, methyl-b-cyclodextrin; MDR, multidrug resistance ⁄ resistant; MEM, minimal essential medium; MRP, multidrug-resistance-associated protein; NaCl ⁄ P i , phosphate-buffered saline; Pgp, P-glycoprotein.
Trang 2often sequestered into lipid rafts, probably as a result
of their acyl chain properties; GPI-anchored proteins
are found in the outer leaflet, and Src-family tyrosine
kinases are found in the inner leaflet Substantial
evi-dence supports the existence of lipid raft microdomains
in model membrane systems in vitro, and in intact cells
in vivo[5–7], although there is still controversy
regard-ing their size and dynamic properties [8]
Pgp (P-glycoprotein, MDR1, ABCB1) is an
energy-dependent drug efflux pump that is a member of the
ATP-binding cassette (ABC) family of proteins [9]
Pgp decreases the intracellular concentration of a wide
variety of drugs and hydrophobic molecules by actively
transporting them across the plasma membrane,
pow-ered by ATP hydrolysis at two cytoplasmic
nucleotide-binding domains Pgp has been proposed to act as a
drug flippase or a hydrophobic ‘vacuum cleaner’ [10]
Under normal physiological conditions Pgp is involved
in cellular detoxification leading to cell survival;
however, in cancer cells its overexpression confers a
multidrug-resistant (MDR) phenotype thus causing
chemotherapy failure [11] An accompanying change in
many cells expressing MDR transporters, including
Pgp, is elevated levels of certain sphingolipids [12–14]
The ATPase activity of Pgp is modulated by lipids
[15–17], and its interaction with drug substrates
depends on the lipid surroundings [18] Pgp has also
been associated with active cholesterol redistribution
across the plasma membrane [19], and cholesterol
affected its drug binding [18,20], transport and ATPase
activity [21–24] Pgp is functional when reconstituted
into a sphingomyelin-cholesterol mixture that mimics
lipid rafts [25]; however, it can carry out both ATP
hydrolysis and drug transport in bilayers of only
phos-phatidylcholine [17,26], so cholesterol is not required
for its function As sphingolipids and cholesterol are
both components of lipid rafts, the fine interplay
between lipid environment and Pgp function may be
linked to the membrane microdomain organization of
the protein
Raft domains have been isolated from intact cells
based on their insolubility in cold nonionic detergents,
especially Triton X-100, and their low buoyant density
in sucrose gradients The resulting detergent-resistant
membranes (DRM) are believed to arise from the
coalescence of smaller raft structures on the cell
surface Caveolin-1, a 21 kDa transmembrane
choles-terol-binding protein, is the primary constituent of
invaginated plasma membrane structures called
caveo-lae Caveolar and noncaveolar DRM microdomains
represent distinct plasma membrane regions [27,28]
Caveolin-1, GM1 ganglioside and cholesterol are
believed to be hallmarks of caveolae which are distinct
from the classical lipid rafts that are enriched in GPI-anchored proteins, cholesterol and GM1, but do not contain caveolins [29] Up-regulation of caveolin-1 and caveolae has been observed in MDR cells expressing Pgp, suggesting a functional link between them [30,31] Interestingly, Pgp was reported to appear in the low density membrane fractions in Triton X-100 extracts [32], as well as in detergent-free cell extracts [21] Demeule and coworkers found that Pgp was contained
in the caveolae in MDR cells and blood–brain barrier endothelial cells [33,34] In contrast, Hinrichs et al determined that Pgp was localized in the noncaveolar rafts [35], while flow cytometry and confocal
microsco-py showed that a substantial fraction of Pgp was asso-ciated with lipid rafts and the cytoskeleton in human colon carcinoma cells [36]
We recently reported that the nonionic detergents Brij-96 and Triton X-100 isolated different lipid raft microdomains from rat basophilic leukemia (RBL-2H3) cells [37] We therefore employed these detergents
to investigate the microdomain localization of Pgp in the MDR cell line CHRB30 In the present work, we showed that this ABC transporter is localized in inter-mediate-density membrane microdomains that are dis-tinct from caveolar domains and Src kinase-containing classical lipid rafts We also showed that these domains are differentially extracted by Brij-96, but not
by Triton X-100 In addition, we found that Brij-96 segregates caveolar domains from Src kinase-based classical lipid rafts, leading to distinct sets of fractions containing each class of raft Triton X-100 extraction apparently leads to the copartitioning of different types
of membrane microdomains ino a common pool Tri-ton X-100 rafts are disrupted by cholesterol removal, whereas the Brij-96 rafts change their buoyant density, but maintain their structural integrity
Results
Pgp is localized in intermediate-density membrane microdomains
DRM are commonly isolated by cold nonionic deter-gent extraction followed by sucrose density centrifuga-tion We previously showed that Brij-96 and Triton X-100 isolate lipid rafts with different physical and biochemical properties from RBL-2H3 cells [37] In this work, we used a similar approach to investigate the membrane domain localization of Pgp Brij-96 or Triton X-100 extracts of CHRB30 cells expressing Pgp were subjected to sucrose density gradient flotation, and the distribution of Pgp among the fractions was determined by western blotting
Trang 3Triton X-100 extraction of CHRB30 cells yielded a
bimodal distribution for Pgp (Fig 1A, right panel, top
row) This type of distribution is typical for the protein
constituents of classical lipid rafts, such as
GPI-anchored proteins and the Src tyrosine kinases A
significant amount of Pgp was observed in the
low-density lipid raft fractions 4–6, while the majority of
the transporter remained in the high-density fractions
9–11 However, in the density gradient fractions from
Brij-96 extracts, Pgp displayed a continuous
distribu-tion in fracdistribu-tions 2–10, peaking in fracdistribu-tions 5 and 6
(Fig 1A, left panel, top row) This pattern of Pgp
par-titioning along the sucrose density gradient is quite
unlike the picture observed for the known constituents
of lipid rafts, such as GPI-anchored proteins or Src
kinases In our previous work, we showed that under
the same conditions, the lipid raft protein Thy-1 of
RBL-2H3 cells is concentrated entirely in the lowest
density lipid raft fractions 2–4 [37] We have therefore
termed the fractions in which Pgp is incorporated, as
intermediate-density fractions The data indicated that
all of the Pgp in CHRB30 cells is localized in Brij-96-based domains that are completely resistant to solubili-zation with this detergent Furthermore, Pgp is only partially located in the Triton X-100-resistant DRM, and about half of it can be solubilized by extraction with this detergent
Pgp is an N-glycosylated protein [38], and because glycosylation may affect the membrane domain local-ization of proteins, we examined whether the glycosy-lation status of Pgp had any bearing on its distribution
in the density gradient after extraction using Brij-96
To address this, we used the CHRPHAR cell line (a lectin-resistant variant of the parental line used to derive CHRB30), which is deficient in N-linked glyco-sylation The results presented in Fig 1A show that the profile for Pgp localization in the sucrose density gradient is very similar when either cell line is used with each of the detergents (compare first and second rows) We conclude that glycosylation does not play
a role in the partitioning of Pgp into intermediate-density microdomains
A
B
Fig 1 Sucrose density gradient partitioning
of P-glycoprotein (Pgp) and markers of clas-sical lipid rafts (A) CHRB30 cells or
CH R PHA R cells (second row only) were lysed in either 0.5% (w ⁄ v) Brij-96 or 1% (w ⁄ v) Triton X-100 at 4 C, and postnuclear lysates were fractionated by ultracentrifuga-tion on a discontinuous sucrose gradient A total of 13 fractions was collected from the top of the gradient tube and an aliquot from each fraction was run on SDS ⁄ PAGE Separ-ated proteins were transferred to a nitrocel-lulose membrane and the presence of Pgp, Yes, caveolin-1 (Cav-1), and CD71 was observed by western immunoblot analysis and enhanced chemiluminescence (ECL) detection, as described in the Experimental procedures (B) CH R B30, RBL-2H3 and Jur-kat cells were lysed in Triton X-100 at 4 C Lysates were precleared by centrifugation at
10 000 g for 5 min An aliquot from each
extract was run on SDS ⁄ PAGE, and the separated proteins were transferred to a nitrocellulose membrane and analyzed for Src-family kinases (Lck, Lyn, and Yes) by western immunoblot analysis and ECL.
Trang 4Comparison of the density gradient partitioning
of Pgp and markers of classical lipid rafts
CHRB30 cells have little or no expression of the most
common GPI-anchored proteins, such as Thy-1,
alka-line phosphatase, decay accelerating factor, etc We
therefore employed Src family tyrosine kinases for the
identification of classical lipid rafts and comparison
with the intermediate-density Pgp membrane domains
First, we determined which members of the Src
tyro-sine kinase family are expressed in CHRB30 cells, using
extracts from RBL-2H3 and Jurkat cells as positive
controls (Fig 1B) Of the three proteins we tested for
(Lck, Lyn and Yes), Lyn was expressed only in
RBL-2H3 cells, and Lck only in Jurkat cells, whereas Yes
was seen in both of these cell lines In CHRB30 cells
we found that only Yes kinase was detectable
Next, we investigated how the distribution of Yes
kin-ase along the density gradient compared with that of
Pgp When Triton X-100 was used, Yes kinase resided
in almost the same fractions as Pgp A portion of Yes
kinase partitioned into the low-density sucrose fractions
3–6, while a significant amount (about half) remained
in the high-density fractions 10–13 (Fig 1A, third row,
right panel) When Brij-96 was used, Pgp and Yes kinase
did not copartition, as determined by density
centrifuga-tion (Fig 1A, third row, left panel) Yes kinase was
localized exclusively in the lowest-density fractions 1–4
This localization is similar to that obtained for another
Src-family tyrosine kinase, Lyn, whose sucrose density
gradient partitioning was examined in RBL-2H3 cells
following extraction with Brij-96 [37] The data
presen-ted in Fig 1A indicate that Pgp is localized in
mem-brane microdomains that are distinct from classical lipid
rafts containing Src-family tyrosine kinases The
mem-brane microdomains containing Pgp displayed an
inter-mediate density in the sucrose gradient when Brij-96 was
used They can be separated from classical lipid rafts if
extracted with Brij-96, but not with Triton X-100
The total protein content of each fraction was
meas-ured by the bicinchoninic acid assay, as shown in
Fig 2 (lower panel) Both detergents solubilized the
majority of cellular proteins, leaving them in the
high-density soluble fractions 11–13 Brij-96 extraction
resulted in small, but detectable, amounts of protein in
the low density fractions, whereas Triton X-100
extrac-tion resulted in virtually no protein in these fracextrac-tions
Relationship of the intermediate-density
Pgp-containing microdomains to caveolae
It is well-documented that at least two types of
deter-gent-insoluble membrane microdomains exist The
first class encompasses the classical lipid rafts (or noncaveolar lipid rafts), which contain GPI-anchored proteins and Src-family kinases, while the other class represents the caveolar raft microdomains, with cave-olin as a hallmark protein We examined the possible relationship between the intermediate-density mem-brane structures in which Pgp is found, and caveolae structures, by assessing copartitioning of Pgp and caveolin-1 in the sucrose density gradient fractions Caveolin-1 was concentrated in fractions 4–8 in both
of the detergent extracts, although in the case of Tri-ton X-100 there was tailing out to fractions 11–12 (Fig 1A, fourth row) Importantly, the localization of caveolin-1 displayed a significant overlap with that of Pgp in both the low-density fractions from Triton X-100 extracts and in the intermediate-density frac-tions from Brij-96 extracts (Fig 1A, compare the first row with the third row)
These results suggest two interesting possibilities First, Brij-96 appears to differentially isolate the caveolar (fractions 4–8, caveolin-1 marker) from
Fig 2 Protein and GM 1 profile of Triton X-100 and Brij-96 sucrose density gradients Post-nuclear lysates of detergent extracts of
CH R B30 cells were run on sucrose gradients, and the gradient frac-tions were assayed for the distribution of total protein and GM 1
ganglioside, as described in the Experimental procedures The pro-tein content is shown for a 20 lL aliquot of each gradient fraction from 5–10 · 10 8
cells lysed in 1 mL of buffer, and the activity of cholera toxin B–horseradish peroxidase conjugate (CTB–HRP) in a
50 lL aliquot of each gradient fraction from 2–3 · 10 8 cells is indi-cated Data are displayed as the mean ± range; where error bars are not visible, they are contained within the symbols.
Trang 5noncaveolar (fractions 1–4, Yes marker) lipid rafts.
Such a distribution was not observed in the sucrose
density fractions from the Triton X-100 extracts The
observed differences were not an artefact of the
deter-gent used because the integral membrane protein,
CD71 (transferrin receptor), was fully solubilized by
both Brij-96 and Triton X-100 (Fig 1A, bottom row)
Second, the Pgp distribution profile partially
over-lapped with that of caveolin-1, but not with that of
Yes kinase, when Brij-96 was used This observation
suggests that the intermediate-density fractions
con-taining Pgp might represent caveolar membrane
struc-tures Such an idea is in agreement with previous
reports suggesting a close interaction between Pgp and
caveolin-1 [33,34] However, this observation does not
necessarily signify molecular colocalization of Pgp and
caveolin-1 For example, all three proteins – Pgp, Yes,
and caveolin-1 – segregate into membrane domains of
similar density when Triton X-100 is used to prepare
the cell extracts, but exhibit different distribution
patterns in the case of Brij-96 detergent extraction
(Fig 1A) The potential colocalization of Pgp and
caveolin-1 was therefore further examined directly by
confocal fluorescence microscopy and
immunoprecipi-tation experiments, as described below
Identification of lipid raft microdomains by
detection of lipid raft-associated GM1ganglioside
GM1 ganglioside is a known marker of both classical
lipid rafts and caveolae This glycosphingolipid has
been shown to cofractionate not only with markers of
various detergent-insoluble microdomains (such as
caveolae, GPI-anchored protein-enriched rafts, and
glycosphingolipid-enriched domains), but also to
colo-calize with caveolin-1 [39] We employed a cholera
toxin B–horseradish peroxidase conjugate (CTB–HRP)
enzyme assay to identify the fractions into which lipid
raft-associated GM1 partitions (Fig 2, upper panel)
For Triton X-100, these were fractions 5, 6, and 7 In
the density gradient of Brij-96 extracts, GM1 was
detected in fractions 2–5 This pattern is very similar
to that observed for GM1 in RBL-2H3 cells [37];
how-ever, the peak seen for GM1 localization in the
CHRB30 gradient fractions is somewhat broader The
gradient partitioning of GM1(Fig 2, upper panel)
par-tially overlaps with the Yes kinase classical lipid rafts
fractions on the one hand, and with the
caveolin-1-enriched raft fractions on the other (Fig 1A) This
broader profile can be explained by the fact that GM1
is a constituent of both classical lipid rafts and
caveo-lae The high level of GM1 apparently present in the
high density fractions of the gradient in Fig 2 (upper
panels), which is not observed in the dot-blots (Fig 6, panel B) is probably spurious This was also reported
by Blank et al [40], and could arise from the presence
of soluble HRP-like activity in the cells
Examination of Pgp and caveolin-1 localization
by confocal fluorescence microscopy and immunoprecipitation
We wanted to determine whether the intermediate-density fractions containing Pgp in the Brij-96 extract represent caveolar membrane microdomains Demeule
et al reported that Pgp and caveolin-1 coimmunopre-cipitated in extracts from Pgp-expressing CHRC5 cells and brain capillary membranes [33] We tested the coimmunoprecipitation of Pgp and caveolin-1 using the pooled lipid raft fractions from the Brij-96 and Triton-100 extracts However, we were unable to detect coimmunoprecipitation between the two pro-teins under these conditions We decided therefore to examine their potential association using total cell extracts because these would contain the entire pool
of Pgp and caveolin-1 Cell extracts were prepared using various lysis buffer conditions Combinations of different detergents were used to establish whether the choice of detergent plays a role in the observation of coimmunoprecipitation of the two proteins Hinrichs and coworkers had already reported a weak associ-ation of multidrug resistance-associated protein 1 (MRP1) with caveolin-1 when Lubrol was used, but saw no such association in the presence of Triton
X-100 [35] In our experiments, all buffers contained sufficient detergent to disrupt the vesicles previously observed to exist in the DRM fractions [37] Other-wise, a false impression of coimmunoprecipitation would be obtained if the two proteins were simply located in the same vesicular structure Under these conditions, only a very faint band of Pgp was seen in the caveolin-1 immunoprecipitates (Fig 3A, top), but
no signal for caveolin-1 was detected in the Pgp immune complexes (Fig 3A, bottom), suggesting that there is no significant coimmunoprecipitation between the two proteins A signal for caveolin-1 in Pgp immu-noprecipitates was seen only after prolonged overnight exposure (Fig 3B, bottom), while an enhanced Pgp band was seen in the caveolin-1 immunoprecipitates when the film was overexposed (Fig 3B, top) We sug-gest that either a very small fraction of the two pro-teins is associated with each other, or that they are located close together in the membrane, but not directly interacting with one another This result agrees with the results of confocal immunofluorescence analysis (see below) and is in accordance with the
Trang 6work of Hinrichs et al [35], who also reported no
coimmunoprecipitation between the two proteins in
2780AD human ovarian carcinoma cells
We further examined the possible cellular
colocaliza-tion of the two proteins by confocal fluorescence
microscopy We first examined the colocalization of
GM1 and caveolin-1 in CHRB30 cells The individual
staining patterns for GM1 and caveolin-1 were very
similar; bright punctuate spots were observed, mainly
on the plasma membrane (Fig 4A,B) When the
sig-nals from the two dyes, Alexa488 and Alexa594, were superimposed, there were several areas of clear lap, as indicated by the yellow colour (Fig 4C, over-lay) This observation indicates a close colocalization
of GM1 and caveolin-1 in some regions of the cell, probably in the caveolar raft domains Next we investi-gated the localization of Pgp and GM1 (Fig 4D,E)
We found that the cellular localization patterns of Pgp and GM1 were distinct and did not overlap, indicating that the two molecules do not directly interact (Fig 4F) When the localization of caveolin-1 and Pgp was compared, the signals for these proteins were, once again, very distinct (Fig 4G,H) Both caveolin-1 and Pgp maintained the pattern described above Pgp displayed staining at the plasma membrane but also
A
B
Fig 3 Immunoprecipitation of P-glycoprotein (Pgp) and caveolin-1.
(A) Lanes 1 and 6, Brij-96 extracts; lanes 2 and 7, Triton X-100
extracts; lanes 3 and 8, Nonidet P-40 ⁄ Triton X-100 ⁄ octylglucoside
extracts; lanes 4 and 9, Brij-96 ⁄ radioimmunoprecipitation assay
(RIPA) extracts; lanes 5 and 10, Triton X-100 ⁄ RIPA extracts One
microgram of each anti-Pgp or anti-(caveolin-1) immunoglobulin was
added to 500 lL of cell extracts Immune complexes were
collec-ted on Protein-A–Sepharose beads and washed twice in the
appro-priate buffer The immunoprecipitated proteins were extracted in
Laemmli’s sample buffer One half of each immunoprecipitation
sample was run on 7.5% nonreducing SDS ⁄ PAGE for Pgp analysis
(A and B top) The other half of each sample was run on 12%
non-reducing SDS ⁄ PAGE for caveolin-1 analysis (A and B, bottom)
Sep-arated proteins were transferred to a nitrocellulose membrane and
analysed by western immunoblot (IB) analysis and enhanced
chemi-luminescence The film exposure time in (A) was 5 min; (B) is an
overnight exposure of (A).
Cav-1
overlay
GM 1
Fig 4 Confocal fluorescence microscopy analysis of P-glycoprotein (Pgp) and caveolin-1 localization CH R B30 cells grown in monolayer culture were first labelled with cholera toxin B–horseradish peroxi-dase conjugate (CTB–HRP), as described in the Experimental procedures Cells were then fixed in 4% paraformaldehyde in phos-phate-buffered saline (NaCl ⁄ P i ), pH 7.4, permeabilized in 0.1% (v ⁄ v) Triton X-100 and blocked in 5% (w ⁄ v) skim milk Pgp and caveolin-1 (Cav-1) proteins were detected with mouse and rabbit immunoglob-ulin, respectively, and localization was revealed with anti-species immunoglobulin conjugated to either Alexa 488 (green) or Alexa 594
(red) fluorophores CTB–HRP was conjugated to the Alexa 488 fluoro-phore The overlay image was produced by superimposing the image from the green and red channels, using LCS Lite software.
Trang 7showed intracellular and perinuclear staining When an
overlay image from the two proteins was produced, no
association between Pgp and caveolin-1 was observed,
as indicated by the absence of any yellow colour
(Fig 4I)
Cholesterol distribution in sucrose density
fractions following treatment of CHRB30 cells
with methyl-b-cyclodextrin
Cholesterol is a component of classical lipid rafts that
is proposed to be required for their structural integrity
The removal of cholesterol by various agents often
leads to the disruption of microdomain structures,
which is manifested by detergent extraction of
mole-cules residing there or by altered activity of signalling
components We have demonstrated previously that
Pgp is localized in intermediate-density membrane
microdomains, which are distinct from classical lipid
rafts and caveolar rafts Our next step was to examine
whether cholesterol plays a role in the formation and
stabilization of these domains One commonly used
agent for the depletion of cholesterol is
methyl-b-cyclo-dextrin (MbCD) We tested various concentrations of
MbCD (5–50 mm) with CHRB30 cells, and found that
treatment with 20 mm MbCD for up to 1 h produced
the maximal cholesterol depletion while still preserving
cell viability (G Radeva & S J Sharom, unpublished
data)
We measured the cholesterol content of the density
gradient fractions of extracts from untreated CHRB30
cells and from cells treated with MbCD, and found
that cholesterol was effectively removed from the lipid
raft fractions obtained using both detergents (Fig 5)
In untreated cells, cholesterol displayed a bimodal
dis-tribution profile when the extracts were prepared with
Triton X-100 One peak of cholesterol was seen
around fractions 4–6 and another in fractions 9–13
(Fig 5, bottom) This pattern corresponds to the lipid
raft marker protein distribution for this detergent
(Fig 1A) This cholesterol distribution pattern is also
similar to that reported in our previous study in the
RBL-2H3 cell line [37] When CHRB30 cells were
trea-ted with MbCD and then extractrea-ted with Triton X-100,
the cholesterol content was dramatically reduced in
lipid raft fractions 4–6, and to a lesser extent in
fractions 9–13 When lipid rafts were isolated using
Brij-96, cholesterol partitioned into a single peak
exclusively in fractions 1–5, which falls into the region
where protein markers of classical lipid rafts segregate
(Fig 1A) Upon treatment with MbCD, cholesterol
was significantly depleted from these fractions (Fig 5,
top)
Effect of cholesterol depletion on Pgp, caveolin-1 and GM1distribution in the sucrose density gradient
After we determined that cholesterol was effectively depleted from lipid raft fractions upon treatment with MbCD, we examined whether cholesterol removal had
an effect on the distribution of Pgp, caveolin-1 and
GM1 in the sucrose density gradient We found that Pgp located in the low-density raft fractions 4–6 in untreated cells was shifted slightly towards the high-density fractions when cells were extracted with Triton X-100 (Fig 6A, right panel) In addition, more Pgp appeared in the high-density soluble fractions relative
to those of low density upon cholesterol depletion
Fig 5 Cholesterol distribution in sucrose density fractions follow-ing treatment of CH R B30 cells with methyl-b-cyclodextrin (MbCD).
CH R B30 cells treated with 20 mM MbCD (grey bars) or untreated control cells (black bars) were lysed in either 0.5% (w ⁄ v) Brij-96 or 1% (w ⁄ v) Triton X-100 Post-nuclear cell extracts were then run on sucrose gradients, and the separated gradient fractions were assayed for the distribution of cholesterol as described in the Experimental procedures The cholesterol content is shown for the entire gradient fraction from 1–2 · 10 8 cells lysed in 300 lL of buf-fer, as the mean ± range The results shown in Figs 5 and 6 were obtained using the same set of gradient fractions.
Trang 8Interestingly, in Brij-96 extracts, there was a significant
shift in the Pgp distribution after cholesterol depletion
Pgp was located primarily in fractions 6–11 following
cholesterol depletion, as compared to fractions 3–10
for the untreated cells (Fig 6A, left panel) This
indi-cates a substantial change in the buoyant density of
the intermediate-density fractions upon cholesterol
depletion GM1 distribution also shifted towards the
higher-density fractions of the gradient after
choles-terol was removed (Fig 6B), for rafts isolated
using Brij-96 However, no Pgp was found in the
high-density fractions, suggesting that these microdomains
retain their structural integrity on cholesterol
deple-tion However, when rafts were extracted with Triton
X-100 following cholesterol removal, GM1 was not
only shifted to slightly higher density in the raft
frac-tions but could also be seen in the high-density soluble
fractions 10–11 (Fig 6B) Caveolin-1 also showed
dif-ferent behaviour in the two detergent extracts The
protein shifted towards the higher-density fractions
upon treatment with MbCD in Brij-96 cell extracts
(Fig 6C, left panel) However, if cholesterol-depleted
cells were extracted with Triton X-100, a large fraction
of the caveolin-1 partitioned into the high-density sol-uble fractions 10–12 (Fig 6C, right panel), while the remaining protein remained localized in fractions 5–7 This behaviour is similar to that seen for GM1 under the same conditions These results suggest that the domains in which GM1 and caveolin-1 are located prior to cholesterol depletion, corresponding to the low density fractions, require cholesterol for their sta-bilization and are disrupted when it is removed
Discussion
The lipid raft hypothesis proposes the existence of discrete microdomains in cellular plasma membranes, which arise from the specific interactions of sphingo-lipids, glycosphingolipids and cholesterol Pgp has recently been proposed to mediate active cholesterol redistribution in the plasma membrane [19] It has also been reported that MDR cells display differential expression and accumulation of glycosphingolipids [12–14] These observations were suggestive of a
speci-fic membrane domain organization for Pgp, prompting
us to examine this issue using techniques commonly
A
B
C
Fig 6 Effect of cholesterol removal on the
distribution of P-glycoprotein (Pgp), GM1
and caveolin-1 in the sucrose density
gradi-ent CHRB30 cells treated with 20 mM
methyl-b-cyclodextrin (MbCD) or untreated
control cells were lysed in either 0.5%
(w ⁄ v) Brij-96 or 1% (w ⁄ v) Triton X-100.
Post-nuclear lysates were fractionated on a
5–30% discontinuous sucrose gradient, and
13 fractions were collected An aliquot from
each fraction was run on SDS ⁄ PAGE, and
the separated proteins were transferred to a
nitrocellulose membrane and analysed for
Pgp (A) and caveolin-1 (C) by western
immunoblot (IB) analysis and enhanced
chemiluminescence (ECL) detection GM1
(B) detection was carried out by dot-blot
analysis The results in Figs 5 and 6 were
obtained using the same set of gradient
fractions Note that these experiments were
carried out under somewhat different
condi-tions from Fig 1; as a result, the distribution
of caveolin-1 in the sucrose gradient is
slightly narrower.
Trang 9employed to study lipid rafts, namely cold nonionic
detergent extraction and sucrose density centrifugation
We have recently demonstrated that in RBL-2H3 cells,
Brij-96 and Triton X-100 isolate DRM with different
physical and biochemical properties [37] Here we
present evidence that Pgp is localized in
intermediate-density membrane microdomains that are completely
insoluble in Brij-96, but partially soluble in Triton
X-100 Other ABC transporter proteins appear to
reside in Lubrol WX-resistant domains, but not in
Tri-ton X-100-resistant domains [35,41,42] Two yeast
ABC transporters have been reported to be involved in
trafficking cholesterol specifically from lipid raft
micro-domains in the plasma membrane to the endoplasmic
reticulum, thus facilitating exogenous sterol uptake
into the cell [43]
We used Yes kinase as a marker for classical lipid
rafts and found that Pgp does not segregate with this
protein upon extraction with Brij-96 Thus, the
inter-mediate-density domains containing Pgp generated
using Brij-96 are distinct from classical lipid rafts
There have been other reports of the existence of
non-classical rafts For example, hepatitis C core protein
was associated with DRM that did not colocalize with
GM1 or caveolin-1, and Drobnik et al found that the
GPI-anchored molecules CD14 and CD55 did not
colocalize with ABCA1 after isolation of Lubrol rafts
[41] In polarized HepG2 cells, Lubrol WX-insoluble
and Triton X-100-insoluble domains with differing
properties were functionally linked to distinct
traffick-ing pathways in the apical targettraffick-ing of proteins [42]
Lubrol WX-based rafts were also described where
var-ious ABC proteins were entirely recovered in the
Lubrol-insoluble fractions and only partially (or not at
all) in the Triton X-100-insoluble fractions [35,41,42]
These observations are consistent with our findings
that Pgp extracted from CHRB30 cells is partially
solu-bilized by Triton X-100 but is completely resistant to
Brij-96 solubilization We previously showed that the
degree of enrichment of microdomain constituents in
various regions of the density gradient depends on the
ratio of cell number to detergent [37] The observed
differences in microdomain localization of ABC
pro-teins might therefore reflect variations in the amount
of cellular starting material relative to detergent
Indeed, we found it necessary to double the
cell : detergent ratio when using CHRB30 cells,
com-pared to RBL-2H3 cells, in order to detect the protein
constituents of lipid rafts in the sucrose gradient
frac-tions
Proteins that partition into lipid rafts are generally
those with lipid modifications, such as GPI-anchored
proteins, or acylated proteins that are members of the
Src tyrosine kinase family, while many integral mem-brane proteins appear to be excluded Recent data, including the present work, points out that multispan-ning proteins of the ABC transporter superfamily may display lipid raft domain localization [35,41,42] Cyc-lic-nucleotide-gated channels also appear to be targeted
to lipid rafts [44] It is conceivable that some proteins with transport functions may be organized into mem-brane microdomains, probably together with regula-tory molecules, thus providing an additional level of control over the entry and exit of their substrates One of the apparent differences between the Brij-96 and Triton X-100-insoluble microdomains in CHRB30 cells is their buoyant density, which is determined by lipid composition and protein content Cholesterol
is often required for maintaining lipid rafts but may also modulate Pgp catalytic and transport activity [19,21,36] We found that the Brij-96-insoluble mem-branes contain most of the cellular cholesterol, while the Triton X-100-insoluble domains comprise only a fraction of total cholesterol, the remainder of which is located in the high-density soluble fractions However,
in RBL-2H3 cells, most of the cholesterol in Triton X-100 extracts was detected in the low-density frac-tions [37], indicating that cell-specific differences exist
in raft microdomain detergent solubility Drobnik
et al also detected a lower percentage of cellular cho-lesterol in the low-density fractions of Triton X-100 lysates, as compared to high-density fractions, in human skin fibroblasts but not in monocytes [41] Their data corroborate our current findings and sug-gest that the ratio of cholesterol in the low-density vs high-density fractions in Triton X-100 extracts is a cell type-specific phenomenon
Upon depletion of cellular cholesterol by MbCD treatment, the Pgp-containing intermediate-density domains isolated using Brij-96 showed a shift to higher buoyant densities However, the domains retained their structural integrity as no Pgp was solubilized into the high-density fractions Cholesterol may not be neces-sary for the maintenance of some types of membrane microdomains, for example those containing K-ras [45] and galectin-4 [46] In contrast, the Pgp-containing Tri-ton X-100 microdomains remaining after cholesterol depletion showed only a small shift in buoyant density However, a significant fraction of these domains appeared to have been disrupted, so that more Pgp appeared in the soluble high-density fractions, indica-ting a strong cholesterol requirement for maintenance
of their integrity This finding also suggests that the reason only a fraction of the cellular Pgp is observed
in the Triton X-100-insoluble fractions may be that cholesterol is removed from these domains upon
Trang 10detergent treatment Indeed, a larger proportion of
cel-lular cholesterol is seen in the high-density fractions of
Triton X-100 extracts, in contrast to Brij-96 extracts
Interestingly, the effect of cholesterol removal on GM1
and caveolin-1 distribution was far more profound in
the Triton X-100 rafts than in the Brij-96 rafts The
GM1- and caveolin-1-containing domains not only
showed a shift to higher density, but significant
amounts of GM1 and caveolin-1 were also detected in
fractions 10–12, indicating that cholesterol depletion
leads to their solubilization GM1 and caveolin-1 do
not behave identically to Pgp, probably because the
critical level of cholesterol required to maintain their
raft association is different, so their sensitivity to
cho-lesterol depletion varies
Possible associations between Pgp, GM1 and
caveo-lin-1 were investigated by confocal fluorescence
micros-copy Clear colocalization was seen between caveolin-1
and GM1, consistent with fact that caveolar fractions
are enriched in GM1[39] However, we did not observe
colocalization between Pgp and caveolin-1, or Pgp and
GM1 Our findings agree with those of Hinrichs et al
who reported that the ABC transporter MRP1 does
not colocalize with caveolin-1 and is enriched in
non-caveolar detergent-insoluble domains [35] However,
Demeule et al reported coimmunoprecipitation of Pgp
and caveolin-1 in CHRC5 cells and brain endothelial
cells [33,34] We were unable to see any interaction
between Pgp and caveolin-1 by coimmunoprecipitation
under conditions where the raft vesicles are solubilized
by detergent It is therefore possible that Pgp and
cave-olin-1 are localized in neighbouring raft domains at the
plasma membrane and copartition into the same DRM
after detergent extraction, but there is no direct, strong
association between them Alternatively, preservation
of their interaction is highly dependent on the ratio of
cell lipid⁄ protein : detergent employed during
extrac-tion The association between Pgp and caveolin-1 may
be cell type-specific, but the CHRB30 cell line used in
this study was derived from CHRC5, so this seems
unlikely
Lipid raft microdomains are proposed to exist in the
more highly ordered lo phase, compared to the bulk
membrane lipids, which are in the liquid-disordered ld
phase Work with the fluorescent probe, merocyanine
540, showed that increasing Pgp expression in MDR
cells correlated with an increase in the packing density
of the plasma membrane outer leaflet, relative to that of
the drug-sensitive parent [47], perhaps reflecting larger
numbers of raft microdomains containing Pgp Unlike
many membrane transporters, which often cease to
function in rigid gel phase bilayers, the rate of
Pgp-mediated drug transport remained high in the gel phase
[26], suggesting that ordered microdomains may be help-ful to the function of the protein Pgp-mediated ATP hydrolysis was also efficient in the gel phase, with a lower activation energy, Eact, than in the liquid-crystal-line phase [17] The intermediate density microdomains
in which Pgp is located may therefore provide a suitable environment for the protein to function optimally
Experimental procedures
Materials The anti-Pgp monoclonal antibody, C219, was supplied by
ID Laboratories (London, ON, Canada) Anti-Lyn, anti-Yes, anti-Lck, anti-caveolin-1 and anti-CD71 (transferrin receptor) mouse monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) CTB–HRP, MbCD, Protein A–agarose, Protein G–agarose, phenylmethanesulfonyl fluoride, Nonidet P-40, DNase, pepstatin A, n-octylglucoside and leupeptin were purchased
HRP-labelled goat anti-rabbit and goat anti-mouse immu-noglobulin were purchased from Jackson Immunoresearch Laboratories (Mississauga, ON, Canada) Triton X-100 was supplied by Roche Diagnostics (Laval, QC, Canada),
Brij-96 was obtained from Fluka (Oakville, ON, Canada), and SDS was purchased from Fisher Scientific (Whitby, ON, Canada)
Cells The highly MDR Chinese hamster ovary cell line, CHRB30, and a glycosylation deficient lectin-resistant variant,
CHRPHAR, were as described previously [48] Cells were grown as monolayers in a-minimal essential medium (a-MEM) containing 10% (v⁄ v) fetal bovine serum (Hy-clone, Logan, UT, USA) supplemented with 2 mm glutamine and 2 mm penicillin⁄ streptomycin, at 37 C in a humidified atmosphere of 5% (v⁄ v) CO2in the presence of 30 lgÆmL)1 colchicine Typically, cells were harvested using 0.25% (w⁄ v) trypsin or 5 mm EDTA in phosphate-buffered saline (NaCl⁄ Pi, pH 7.4) The RBL-2H3 cell line was cultured as described previously [37] Jurkat cells were grown using the same culture medium and conditions as CHRB30 cells
Isolation of lipid raft microdomains using sucrose gradient centrifugation
Lipid rafts were isolated from either freshly harvested or frozen cells, using Triton X-100 or Brij-96, as described pre-viously for RBL-2H3 cells [37] About 5–10· 108 cells (200–250 lL cell pellet) were washed twice in NaCl⁄ Pi,
pH 7.4 or Tris-buffered saline (TBS; 25 mm Tris⁄ HCl,
140 mm NaCl, pH 7.5) and then treated on ice with 1 mL