From the Department of Biochemistry and Cell Biology,
State University of New York at Stony Brook, Stony Brook, New York
11794-5215 and the ¶ Department of Biochemistry, University of
Texas Southwestern Medical Center, Dallas, Texas 75235-9038
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ABSTRACT |
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Sphingolipid and cholesterol-rich Triton
X-100-insoluble membrane fragments (detergent-resistant membranes,
DRMs) containing lipids in a state similar to the liquid-ordered phase
can be isolated from mammalian cells, and probably exist as discrete
domains or rafts in intact membranes. We postulated that proteins with
a high affinity for such an ordered lipid environment might be targeted to rafts. Saturated acyl chains should prefer an extended conformation that would fit well in rafts. In contrast, prenyl groups, which are as
hydrophobic as acyl chains but have a branched and bulky structure,
should be excluded from rafts. Here, we showed that at least half of
the proteins in Madin-Darby canine kidney cell DRMs (other than
cytoskeletal contaminants) could be labeled with [3H]palmitate. Association of influenza
hemagglutinin with DRMs required all three of its palmitoylated Cys
residues. Prenylated proteins, detected by [3H]mevalonate
labeling or by blotting for Rap1, Rab5, G Increasing evidence suggests that cholesterol and
sphingolipid-rich lipid microdomains or rafts exist in eukaryotic cell
membranes and have important functions there (1-3). These rafts are
likely to be important in the structure and function of caveolae,
plasma membrane invaginations that are implicated in signal
transduction (4, 5), endocytosis (6), transcytosis across endothelial cells (7, 8), and cholesterol trafficking (9-11). However, rafts are
not restricted to caveolae (2, 3, 12) and recent evidence suggests that
they act in signal transduction in cells that lack distinct caveolae,
such as T lymphocytes (13-16) and basophils (17-19). Rafts have also
been implicated in protein and lipid sorting in the secretory and
endocytic pathways (1, 20-22).
Cholesterol and sphingolipid-rich detergent-resistant membranes
(DRMs)1 can be isolated from
mammalian cells (23). DRM lipids are in a state similar to the
liquid-ordered (lo) phase (3, 24-26). The lo
phase, which requires cholesterol to form, is favored by lipids like
sphingolipids, whose long saturated acyl chains give them a high degree
of order and a high acyl-chain melting temperature (3). Acyl chain
order explains the detergent-insolubility of DRMs (3). We hypothesize
that DRMs are an in vitro correlate of rafts in intact
membranes. It is important to note that detergent insolubility can
underestimate the association of proteins and lipids with the
lo phase; some proteins and lipids that are in rafts can be
solubilized (25). Nevertheless, DRM association provides a powerful
tool for identifying molecules that are likely to have a high affinity
for rafts.
DRMs isolated from cells contain a number of proteins (27-29) which
are undoubtedly crucial for the function of the domains in
vivo. For this reason, it is important to determine how proteins associate with DRMs. Three DRM targeting signals have been defined. First, glycosylphosphatidylinositol (GPI)-anchored proteins are targeted to DRMs through acyl chain interactions (23-25, 30). An
N-terminal Met-Gly-Cys motif that is present in some Src family kinases
and heterotrimeric G protein The finding that DRM lipids are in an lo-like phase
suggests a unifying mechanism for targeting of proteins to DRMs.
Proteins with a high affinity for the ordered lipid environment of the lo phase might spontaneously partition into the domains. In
agreement with this model, all three of the DRM targeting signals
listed above contain two closely spaced acyl chains. Myristate and
palmitate, as well as most of the acyl chains on GPI-anchored proteins
(34) are saturated, and thus should fit well into ordered lipid
domains. This suggests that acylation, especially multiple acylation,
may be a general DRM targeting signal.
Alternatively, however, it might be imagined that lipid modifications
could target proteins to DRMs simply through hydrophobic interactions.
Both models predict that many DRM proteins would be linked to lipids.
The behavior of prenylated proteins should distinguish between the
models, because prenyl groups are as hydrophobic as acyl chains, but
have a bulky branched structure that should not fit well into the
lo phase. Because of the possibility that multiple
hydrophobic modifications are required for DRM targeting, the behavior
of dually or multiply lipid-modified proteins that are prenylated is
especially informative in this regard. To test these models, we
examined the lipid modifications of DRM proteins and the DRM
association of prenylated proteins.
Materials--
Mouse monoclonal anti-Rab5 antibodies were the
gift of A. Wandinger-Ness or were from Transduction Laboratories
(Lexington, KY). Mouse monoclonal anti-Rap1, anti-Ras, and
anti-caveolin antibodies (Ig fraction) were from Transduction
Laboratories. Rabbit polyclonal anti-influenza hemagglutinin (HA)
antibodies (35) were used. Rabbit anti-G Cells and Plasmids--
MDCK strain II (40), COS-1 (33), and CV1
cells (35) were maintained as described previously. MDCK cells stably
expressing PLAP (detected without butyrate induction) have been
described (41). Met-18b-2 cells (Ref. 42; the gift of J. Faust) were grown in Ham's F-12 medium containing 5% iron-supplemented calf serum. Mutant HA cDNAs have been described (35). Briefly,
oligonucleotide-directed mutagenesis was used to mutate Cys residues at
positions 536, 543, and/or 546 near the C terminus of HA. Mutants were
named by three letters referring to the amino acids at these positions, respectively; e.g. SCC contains Ser at 536 and Cys at 543 and 546. All mutants incorporate less [3H]palmitate than
wild type, and SSS incorporates none (35). Wild-type and mutant HA
proteins were expressed in CV1 cells as described (35). A cDNA
encoding H-Ras in the pEXV-3 expression vector (43) was the gift of K. Cadwallader (Addenbrooke's Hospital, Cambridge, United Kinddom). H-Ras
was expressed transiently in COS-1 cells as described (44).
Protein Radiolabeling--
Metabolic labeling of MDCK cell
proteins with [35S]methionine (50 µCi/ml) to
steady-state was as described (29). CV-1 cells expressing HA mutants
were starved in media lacking Met and Cys for 30 min, pulse labeled for
20 min in media containing 0.6 mCi/ml Tran35S-label, and
chased for 40 min in complete medium. Lysis in 1% Triton X-100,
immunoprecipitation, analysis, and quantitation were as described (45).
For labeling with [3H]palmitic acid, MDCK cells were
incubated for 2 h in media supplemented with 5% calf serum, 5 mM pyruvic acid, and 1 mCi/ml [3H]palmitic
acid. Short labeling periods were used to minimize conversion of the
label to other products. Prior to [3H]mevalonate
labeling, cells were incubated for 30 min at 37 °C with 20 µM de-lactonized compactin, an inhibitor of mevalonate synthesis (42). Cells were then incubated with 78.5 µCi/ml (MDCK) or
31.3 µCi/ml (met-18b-2) [3H]mevalonolactone for
3.5 h at 37 °C in the presence of 20 µM compactin.
Preparation of Total Cell Membranes and DRMs--
For total cell
membranes, cells were scraped from dishes, washed in phosphate-buffered
saline (150 mM NaCl, 20 mM sodium phosphate, pH
7.4) and then in hypotonic buffer (10 mM Hepes, pH 7.4, 0.5 mM EDTA), resuspended in 1 ml of hypotonic buffer, and
broken by passage through a 25-gauge needle 40 times. Nuclei and debris were removed by centrifugation at 300 × g for 10 min
at 4 °C, and light membranes were collected from the supernatant by
centrifugation at 120,000 × g for 1 h. Membranes
were solubilized directly in gel loading buffer for analysis by
SDS-PAGE. For DRMs, cells in 1 confluent 10-cm dish were lysed in 1 ml
of TNE buffer (25 mM Tris-Cl, pH 7.5, 150 mM
NaCl, 5 mM EDTA) containing 1% Triton X-100 (TNE/Triton
X-100), and DRMs were isolated by flotation on sucrose gradients as
described (40). Unless otherwise indicated, the lysis buffer and all
sucrose gradient solutions were adjusted to pH 11 with 0.1 M sodium carbonate. In most cases, the floating membrane
band was harvested and diluted to about 12 ml with TNE. Membranes were
harvested by centrifugation for 1 h at 120,000 × g. Alternatively, where indicated 1-ml fractions were
collected from the bottom of the sucrose gradient with an ISCO
(Lincoln, NB) Model 185 density gradient fractionator. All buffers were ice-cold and contained the following protease inhibitors: 0.5 µg/ml
leupeptin, 0.7 µg/ml pepstatin, and 0.2 mM
phenylmethylsulfonyl fluoride.
Analysis of DRM Proteins, SDS-PAGE, and Blotting--
For
identification of GPI-anchored proteins, DRMs recovered from sucrose
gradients were solubilized in TNE containing 1% Triton X-114.
GPI-anchored proteins were detected as described (46). Briefly, phase
separation was induced by warming lysates to 37 °C (47). The aqueous
phase was removed, and the detergent phase containing hydrophobic
proteins was mixed with fresh TNE and incubated with 10 units/ml
phosphatidylinositol-specific phospholipase for 1 h at 37 °C.
The phase separation was repeated, and proteins in the aqueous phase
were incubated overnight with phenyl-Sepharose and then precipitated
with 15% trichloroacetic acid. DRMs harvested from sucrose gradients
and collected by centrifugation as described above were incubated with
1 ml of TNE/Triton X-100 at 37 °C for 5 min. Lysates were subjected
to centrifugation at top speed in a microcentrifuge for 5 min. Proteins
in the supernatant were precipitated with 15% trichloroacetic acid.
Precipitates were washed with acetone and diethyl ether to remove all
lipids, solubilized in gel loading buffer, and analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Ref. 48)
on 11% acrylamide gels. Where indicated, replicate lanes from the same
gel were incubated with either buffer containing 1 M
hydroxylamine and 1 M Tris-Cl (pH 7.5), or 1 M
Tris-Cl (pH 7.5) alone for 4 h with one change of reagent before
processing for fluorography. When gradients were fractionated, 75 µl
of each 1-ml fraction was mixed with 25 µl of 4 × gel loading
buffer and subjected to SDS-PAGE and Western blotting as described
(25). Quantitation of signals in the linear range of the film was
performed using a Bio-Rad GS-670 imaging densitometer as follows: % of
protein in DRMs = cpm in DRMs (gradient fractions 9-12))/cpm in
lysate (fractions 1-5) + cpm in DRMs. Although only the region of the
blot showing the protein of interest is shown, no other bands were
present. Two-dimensional gel electrophoresis, using non-equilibrium pH gradient electrophoresis in the first dimension and SDS-PAGE in the
second dimension, was performed according to Jones (49, 50) after
precipitation of proteins with acetone. In some cases, radiolabeled
proteins on two-dimensional gels were transferred to nitrocellulose.
After detection of Yes or caveolin by Western blotting, the ECL signal
was quenched with 0.05% sodium azide. Blots were then air dried,
sprayed with Amplify, and exposed to film to detect the
[35S]methionine signal.
Acylated Proteins in DRMs--
To determine how many DRM proteins
from MDCK cells were palmitoylated, cells were incubated with
[3H]palmitic acid before preparation of DRMs. For
comparison, DRMs were also prepared from cells incubated with
[35S]methionine to metabolically label all proteins. DRM
proteins were separated by SDS-PAGE and visualized by fluorography
(Fig. 1). As previously reported (29),
about 20-25 major proteins were observed (lane 1). Many of
the proteins were labeled with palmitate (lane 2). The same
pattern of [3H]palmitate-labeled proteins was also
observed in DRMs prepared without sodium carbonate at pH 7.5 (data not
shown).
In initial experiments, we also labeled cells with
[3H]myristate using a similar protocol, in an attempt
to visualize N-myristoylated proteins in DRMs. Many of the
proteins that were labeled with [3H]palmitate also
incorporated [3H]myristate (not shown). However,
S-acylated proteins can be labeled artifactually with
[3H]myristate (51), and we were unable to show that
[3H]myristate labeling of DRM proteins was specific. For
this reason, these studies were not pursued further.
It was important to verify that the [3H]palmitate label
was present as thioester-linked fatty acid. Thus, proteins labeled with
either [3H]palmitate or [35S]methionine
were separated on the same gel and treated with or without
hydroxylamine, which cleaves thioester bonds (52), as shown in Fig.
2. As expected, the
[35S]methionine label was resistant to hydroxylamine
(compare lanes 1 and 2) while the
[3H]palmitate label was removed (compare lanes
3 and 4).
Not All Acylated Proteins Are in DRMs--
We next determined how
many of the palmitoylated proteins in the cell were present in DRMs.
Cells were incubated with [3H]palmitate and lysed. DRMs
were then prepared after saving a small fraction of the whole cell
lysate. Both fractions were analyzed by SDS-PAGE and fluorography (Fig.
3). Very different patterns of labeled
proteins were seen in whole cell lysate and DRMs. Thus, only a subset
of palmitoylated proteins associated efficiently with DRMs.
Two-dimensional Gel Analysis of DRM Proteins--
We extended our
analysis of DRM proteins using two-dimensional gels, separating
[35S]methionine-labeled proteins in the first dimension
by non-equilibrium pH gradient electrophoresis, and in the second by
size by conventional SDS-PAGE. We first compared total cell proteins
(Fig. 4A) with DRM proteins
prepared from cells with or without carbonate during detergent
extraction (Fig. 4, B and C). For orientation,
MDCK cells stably expressing the GPI-anchored protein PLAP were used in
this experiment. PLAP was prominent among the DRM proteins (Fig. 4,
B and C), and could be detected in the whole cell
lysate after long exposure (not shown).
Ten proteins that were especially abundant in the whole cell lysate
(Fig. 4A, arrows) were detected in DRMs prepared without carbonate (Fig. 4B, asterisks). These are likely to be
cytoskeletal or other structural proteins. Consistent with this
possibility, fibrous material could sometimes be observed in DRM
preparations by electron microscopy (not shown). Most of these proteins
were present in lower abundance in DRMs prepared with carbonate (Fig. 4C, asterisks). Actin, the most abundant cellular protein,
was among these proteins. Actin could also be detected in DRMs on one-dimensional gels by Western blotting (not shown). Although these
abundant proteins may associate specifically with DRMs, it is likely
instead that they adhere nonspecifically during DRM preparation.
Finding these 10 abundant proteins in DRMs raised the possibility that
most of the spots on our two-dimensional gels were contaminants.
Proteins that associated specifically with DRMs, and were enriched
there, might be present at such low levels that they could not be seen.
To test this idea, as described next, we identified several proteins
that are known to associate specifically with DRMs. Our ability to
detect these proteins suggested that many of the other spots also
corresponded to bona fide DRM proteins.
We first examined [35S]methionine-labeled GPI-anchored
proteins released from DRMs by phosphatidylinositol-specific
phospholipase treatment (Fig.
5B). (No proteins were
observed when phosphatidylinositol-specific phospholipase was omitted
from the reaction (not shown).) These were analyzed in parallel with
total [35S]methionine-labeled DRM proteins (Fig.
5A). (For orientation, major cell proteins identified in
Fig. 4 are labeled with asterisks.) As found by others (53),
GPI-anchored proteins of 50 and 80 kDa could be detected in the whole
DRM pattern by alignment of the films. These are labeled GPI in Fig. 5,
A and C.
We next identified two known DRM proteins on two-dimensional gels by
Western blotting. DRM proteins from
[35S]methionine-labeled MDCK cells were separated on
two-dimensional gels and transferred to nitrocellulose. The positions
of the Src family non-receptor tyrosine kinase Yes and of caveolin, a
marker for caveolae (4, 54), were determined by Western blotting (not
shown). Blots were then exposed to film for detection of all
[35S]methionine-labeled DRM proteins. Alignment of the
films allowed identification of Yes and caveolin in the
[35S]methionine-labeled pattern. Spots corresponding to
Yes and caveolin (labeled Cav) are labeled in Fig. 5, A and
C. (The caveolin spot is faint because much of the caveolin
was in a high molecular weight oligomeric form (55). Oligomeric
caveolin was detected by Western blotting, but could not be aligned
unambiguously with an [35S]methionine-labeled spot.)
Thus, although these proteins are not the most abundant DRM proteins,
they are easily detectable, increasing our confidence that many of the
unidentified proteins are also specific.
We next separated DRM proteins from MDCK cells labeled with
[3H]palmitate (Fig. 5D, contrast enhanced by
image processing) or [35S]methionine (Fig. 5C)
on parallel two-dimensional gels. The
[3H]palmitate-labeled proteins were numbered, both on the
[3H]palmitate-labeled gel (Fig. 5D) and the
corresponding [35S]methionine-labeled gel (Fig.
5C). The positions of GPI-anchored proteins, Yes, and
caveolin were indicated (Fig. 5C). Finally, unidentified
[35S]methionine-labeled proteins not labeled with
[3H]palmitate were marked with letters (Fig.
5C).
As expected, except for faint labeling of a protein of about 30 kDa
(Fig. 5C, marked 10,*), the major cell proteins
in DRMs were not labeled with [3H]palmitate. Neither
could we detect [3H]palmitate labeling of the two
GPI-anchored proteins. Although palmitate is present in GPI anchors,
the anchors are preassembled and added to proteins en bloc
(56). Thus, a 2-h labeling period was probably not sufficient to allow
detectable labeling of these proteins. However, in agreement with data
from the one-dimensional gels, a large fraction of the other proteins
were labeled with [3H]palmitate. Twenty-three
palmitoylated proteins (including caveolin) and only 12 non-palmitoylated proteins (other than cytoskeletal or GPI-anchored
proteins) are labeled in Fig. 5C, the former with numbers
corresponding to [3H]palmitate-labeled proteins in Fig.
5D, and the latter with capital letters. The fact that we
did not detect [3H]palmitate labeling of caveolin,
although it is triply palmitoylated (57), suggests that other
less-abundant DRM proteins might also be palmitoylated, but not
detectable in Fig. 5D.
Finding that so many DRM proteins were palmitoylated was consistent
with the idea that palmitoylation is a DRM targeting signal. Thus,
we next examined several known palmitoylated proteins for DRM
association, in order to test the role of palmitoylation in DRM
targeting directly. Vesicular stomatitis virus glycoprotein does not
associate with DRMs (23, 33). Similarly, we found that endogenous MDCK
cell transferrin receptor (another palmitoylated transmembrane protein
(58)) was not in DRMs (data not shown). In contrast, influenza HA,
which is triply palmitoylated (35), associates with DRMs in several
cell types (45, 59, 60). In agreement with this result, we found that
50% of HA expressed in MDCK cells associated with DRMs (not shown).
DRM Association of Influenza Hemagglutinin Requires Palmitoylated
Cys Residues--
To test the role of the palmitoylated Cys residues
in DRM targeting, HA proteins mutated in one, two, or all three Cys
residues were expressed in CV1 cells. After detergent extraction and
separation of soluble and insoluble fractions, HA was recovered from
both fractions by immunoprecipitation as described (60), and the percent insoluble was determined. Using this procedure, 29% of wild-type HA was insoluble. (The difference between this and the 50%
of HA found in DRMs in transiently transfected MDCK cells may reflect
cell type or other procedural differences. It is also possible that the
exogenously expressed HA was incompletely palmitoylated.) Mutation of
any Cys, or any combination of Cys, essentially abolished detergent
insolubility (Fig. 6). Thus, all three
palmitoylated Cys residues are essential for targeting HA to DRMs.
Prenylated Proteins in DRMs--
We examined DRM proteins for
possible prenylation by incubating MDCK cells with
[3H]mevalonate, a precursor of prenyl groups. Cells were
then lysed as usual, except that sodium carbonate was omitted and lysis
was performed at pH 7.5.
To detect all cellular prenylated proteins, proteins in 10% of the
lysate were precipitated with trichloroacetic acid and analyzed by
SDS-PAGE (Fig. 7A, WCL). DRMs
were isolated from the remaining 90% of the lysate (Fig. 7A,
DRM). Although [3H]mevalonate-labeled proteins were
easily seen in whole cell lysates, they were virtually undetectable in
DRMs.
Most mammalian cells take up mevalonate poorly, making it difficult to
detect prenylated proteins by [3H]mevalonate labeling.
For this reason, we repeated the experiment shown in Fig. 7A
using met-18b-2 cells (42), which express a mutant mevalonate
transporter that allows faster uptake of mevalonate (61). Results are
shown in Fig. 7B. As expected, [3H]mevalonate
labeling was more efficient than in MDCK cells. As was seen for MDCK
cell DRMs, very few met-18b-2 cell DRM proteins were labeled.
In contrast to our findings, three prenylated proteins, Rab5 (62), Rap1
(27, 62), and the
The prenylated G
Most members of the Ras family are both farnesylated and palmitoylated
(43). Thus, if all lipid modifications enhance DRM targeting, then Ras
should be highly enriched there. In contrast, if packing into
lo phase rafts is important, then the prenyl group might
inhibit association of Ras with DRMs. In agreement with the latter
model, Ras is excluded from mouse lung DRMs (62). We repeated the
gradient fractionation procedure used for G
Although H-ras, N-ras, and K-rasA are both palmitoylated and
prenylated, K-RasB is not palmitoylated, but is targeted to membranes by prenylation in combination with a polybasic domain (43). Because we
do not know how much MDCK cell Ras is of this type, we confirmed that
H-Ras is excluded from DRMs by expressing the protein exogenously in
COS-1 cells. Western blotting of 1% of a whole cell lysate or DRMs
prepared from the remaining 99% of the lysate showed that less than
1% of the total was in DRMs (Fig. 9B).
Several findings suggest that cholesterol and sphingolipid-rich
lo phase microdomains or rafts can exist in cell membranes and can be isolated as DRMs (1, 3). First, GPI-anchored proteins and
gangliosides, which are enriched in DRMs, can exhibit a clustered
distribution in the plasma membrane (64-69). Furthermore, the physical
properties of DRMs isolated from cells are very similar to those of the
lo phase (24). In a complementary approach, we demonstrated
the plausibility of phase separation in biological membranes by showing
that lo phase microdomains form spontaneously at 37 °C
in liposomes containing physiologically reasonable levels of
sphingolipids and cholesterol (26).
Emerging functions for rafts in the structure and function of caveolae,
in signal transduction, and in sorting in the secretory and endocytic
pathways (1, 2, 5) highlight the importance of determining how lipids
and proteins associate with them. If rafts are lo phase
microdomains, as we propose, then acyl chain order should be a key
determinant of their formation. As expected, lipids with saturated acyl
chains, whose extended structure fits well into an ordered environment,
are enriched in DRMs isolated from cells (23) and model membranes (24,
25). Proteins, too, might be targeted to DRMs by modification with
saturated acyl chains. In agreement with his idea, the best defined
DRM-targeting signals (GPI anchorage (30, 39), tandem myristoylation
and palmitoylation (31, 32), and dual palmitoylation (15, 33) all
consist of saturated acyl chains. The role of palmitoylation in
targeting proteins to DRMs was tested further in this paper. An
important finding of this work is that a high fraction of the proteins
in DRMs is acylated. This suggests that acylation is a commonly used
signal for DRM targeting of proteins. Other proteins might be targeted
to DRMs indirectly, by binding to more tightly associated proteins or lipids.
It is important to note, however, that not all palmitoylated proteins
are targeted to DRMs. This suggests that although palmitoylation can
increase the affinity of proteins for DRMs, this effect is not always
strong enough to mediate stable association. Other structural features
(for instance, prenylation or membrane spans) that prefer a disordered
environment will also contribute to the overall affinity of the protein
for DRMs. The degree of partitioning of a protein into DRMs will
reflect all these interactions. Thus, modification with multiple acyl
chains should enhance DRM association. By contrast, a membrane-spanning
peptide might not pack easily into such an environment. In agreement
with this idea, to our knowledge all proteins examined to date that
lack membrane-spanning domains but are modified with dual saturated
acyl chains are targeted to DRMs. (This includes the myristoylated and
palmitoylated protein endothelial cell nitric oxide synthase, although
it associates with DRMs less efficiently than other such proteins
(70).)
The "rules" for how palmitoylation can target transmembrane
proteins to DRMs are less straightforward. We showed here that dual
palmitoylation is not sufficient for targeting of vesicular stomatitis
virus glycoprotein G or the transferrin receptor to DRMs, and that
three palmitate groups are required to target HA to DRMs. As HA is
trimeric, each molecule is modified with a total of nine palmitate
chains. This high concentration of saturated acyl chains may be
required to overcome packing difficulties and allow efficient targeting
to DRMs. In addition, transmembrane domain sequences undoubtedly play
an important role in determining the affinity for DRMs, as has been
shown for HA (60) and the hyaluronan receptor CD44 (71, 72). (CD44 is
palmitoylated (73), and the role of this modification in its DRM
association has not been explored.) However, Zhang et al.
(74) have shown that dual acylation is sufficient for targeting of LAT,
an membrane-spanning adaptor protein that plays an important role in T
cell signaling, to DRMs (15).
An alternative model is that lipid modifications target proteins to
DRMs through hydrophobic interactions that do not depend on the
structure of the lipid. Prenyl groups are hydrophobic, but would not be
expected to fit in an lo domain. Thus, the finding that
prenylated proteins are excluded from DRMs provides important support
for our model. Nevertheless, it might be imagined that prenyl groups
could aid in targeting proteins to DRMs via hydrophobic interactions,
but that DRM targeting requires two lipid modifications. This model
would predict that most Rab proteins would be enriched in DRMs, as most
of them (including Rab5) are dually geranylgeranylated (75). However,
we found that Rab5 was excluded from DRMs (Fig. 8), and we found no
enrichment there of [3H]mevalonate-labeled proteins in
the 20-25 kDa range that is characteristic of Rabs (Fig. 7). Thus,
dual geranylgeranylation does not target Rab proteins efficiently to DRMs.
Such a model would also predict an enrichment of Ras in DRMs, as most
forms of Ras are both prenylated and palmitoylated. Our finding (in
agreement with others) that Ras is excluded from DRMs supports the
model that lipid modifications target proteins to DRMs via packing
order, not hydrophobicity. Exclusion of prenylated proteins from rafts
may have important functional consequences.
The high frequency of palmitoylation among DRM proteins suggests that
acylation is a common means of targeting proteins to rafts. However,
there must be additional targeting signals, as at least one protein,
caveolin, associates with DRMs even after removal of palmitoylation
sites by mutagenesis (57). Caveolin, which binds cholesterol tightly
(76) and can induce the formation of caveolae when expressed in
caveolae-negative cells (77), appears to be an unusual protein that may
interact with rafts in a unique manner.
In summary, GPI-anchored proteins, Src family kinases, GAP-43, HA, and
LAT are now known to be targeted to DRMs via saturated acyl chains, and
the list continues to grow. The affinity of these lipid groups for an
ordered environment, and not simply their hydrophobicity, is required
for targeting. A substantial fraction of DRM proteins is acylated.
Thus, acylation appears to be a common mechanism of increasing the
affinity of proteins for DRMs, and may be the primary targeting
mechanism for proteins without membrane spans. As the role of rafts in
cellular function is becoming increasingly clear (1, 2, 15, 16), it is
becoming increasingly important to understand in molecular detail how
proteins and lipids are organized into these domains.
, or Ras, were
excluded from DRMs. Rab5 and H-Ras each contain more than one lipid
group, showing that hydrophobicity alone does not target multiply
lipid-modified proteins to DRMs. Partitioning of covalently linked
saturated acyl chains into liquid-ordered phase domains is likely to be
an important mechanism for targeting proteins to DRMs.
INTRODUCTION
Top
Abstract
Introduction
References
subunits, in which Gly is
myristoylated and Cys is palmitoylated, can also serve as a DRM
targeting signal (31, 32). Third, dual palmitoylated Cys residues are
required for raft association of the T cell adaptor protein LAT (15)
and the neuronal protein GAP-43 (33).
EXPERIMENTAL PROCEDURES
antibody B600,
against a C-terminal G
peptide, (36) was the gift of S. Mumby. Mouse monoclonal anti-transferrin receptor antibodies were a
gift of I. Trowbridge. Rabbit polyclonal antibodies to
p62yes (Yes) were generated by immunization of rabbits with
a TrpE-Yes fusion protein as described (37). A purified Ig fraction was obtained using an immobilized Protein A column according to
instructions from the supplier (Pierce, Rockville, IL). Rabbit
anti-placental alkaline phosphatase (PLAP; Ig fraction) was from Dako
(Carpinteria, CA). Horseradish peroxidase-conjugated goat anti-mouse
Ig(G + M) was from Jackson Labs (West Grove, PA), and horseradish
peroxidase goat anti-rabbit IgG was from Sigma. The enhanced
chemiluminescence (ECL) reagent and Amplify (fluorography enhancement
reagent) were from Amersham. Ampholytes were from Bio-Rad and
prestained protein molecular weight standards were from Bio-Rad or Life
Technologies (Gaithersberg, MD). EXPRE35S35S
protein labeling mixture (>1000 Ci/mmol; referred to as
"[35S]methionine") was from NEN Life Science Products
(Boston, MA). (Labeling media for MDCK cells lacked Met but not Cys,
and [35S]cysteine in the labeling mixture probably
accounted for only a small fraction of total protein labeling.)
Tran35S-label was from ICN (Conta Mesa, CA).
[3H]Palmitic acid (50 Ci/mmol) and
[3H]mevalonolactone (60 Ci/mmol) were from American
Radiolabeled Chemicals (St. Louis, MO). Compactin was the gift of R. Simoni (Stanford, CA). Recombinant Bacillus cereus
phosphatidylinositol-specific phospholipase C was purified from
overexpressing E. coli (the gift of J. Volwerk) according to
Koke et al. (38) with modifications as described (39). All
other materials were purchased from Sigma or Fisher Scientific
(Pittsburgh, PA).
RESULTS
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Fig. 1.
Many DRM proteins are acylated. MDCK
cells in two 10-cm dishes were incubated with either
[35S]methionine (lane 1) or
[3H]palmitate (lane 2). DRMs were prepared,
and proteins were separated by SDS-PAGE, and visualized by
fluorography. Both lanes are from the same gel. Molecular weight
markers are indicated. Exposure times; lane 1, 1 day;
lane 2, 28 days.
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Fig. 2.
Sensitivity of [3H]palmitate
label to hydroxylamine. MDCK cells were incubated with
[35S]methionine (lanes 1 and 2, one
10 cm dish/lane) or [3H]palmitate (lanes 3 and
4, two 10-cm dishes/lane). DRMs were prepared and proteins
were resolved by SDS-PAGE. Gels were incubated with (+) or without ( )
hydroxylamine and processed for fluorography. Exposure times:
lanes 1 and 2, 3 days; lanes 3 and
4, 28 days.
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Fig. 3.
Not all [3H]palmitate-labeled
proteins associate with DRMs. [3H]Palmitate-labeled
MDCK cells were either lysed directly in gel loading buffer
(WCL, whole cell lysate) or used to prepare DRMs. Proteins
were separated by SDS-PAGE and visualized by autoradiography. Sample
volumes (containing about 17 µg of WCL and 10 µg of DRM protein)
were adjusted to load approximately equal numbers of counts in each
lane.
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Fig. 4.
Comparison of whole cell proteins and DRM
proteins on two-dimensional gels.
[35S]Methionine-labeled MDCK cells expressing PLAP were
lysed with 1 ml of TNE + 2% Triton X-100 with or without 0.1 M sodium carbonate (one 10-cm dish each). 1% of the
no-carbonate lysate was reserved, and DRMs were prepared from the
remainder of both lysates for analysis on two-dimensional gels with the
acidic end on the left. A, whole cell lysate; B,
DRMs without carbonate; C, DRMs with carbonate. Proteins
that were very prominent in the whole cell lysate pattern and were
detected in DRMs are labeled (arrows in A; * in
B and C). Positions of PLAP, actin, and molecular
weight markers are indicated. Exposure times: A, 4 h;
B and C, 16 h.
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Fig. 5.
Detection of Yes, caveolin, GPI-anchored, and
palmitoylated DRM proteins on two-dimensional gels. DRMs were
prepared from MDCK cells in two 10-cm dishes labeled with
[35S]methionine (A-C) or
[3H]palmitate (D). Total DRM proteins
(A, C, and D) or GPI-anchored proteins
isolated from DRMs (B) were analyzed on two-dimensional gels
(acidic end on left). *, major cell proteins identified in
Fig. 4. Proteins labeled with [3H]palmitate
(D) and the corresponding
[35S]methionine-labeled spots (C) are
numbered. Proteins labeled by [35S]methionine but not by
[3H]palmitate are identified by capital
letters in C. Yes, actin, GPI-anchored proteins, and
caveolin (Cav) are indicated. Exposure times: A, 7 days;
B, 14 days; C, 4 days; D, 6 months.
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Fig. 6.
HA requires three palmitoylated Cys for
targeting to DRMs. Wild type (CCC) or mutant HA
proteins expressed in CV1 cells were labeled with
[35S]methionine and subjected to extraction with Triton
X-100. Detergent-soluble and insoluble fractions were separated and
wild-type or mutant HA was recovered from each by immunoprecipitation.
The insoluble fraction of each is shown.
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Fig. 7.
Prenylated proteins are largely excluded from
DRMs from MDCK cells and met-18b-2 cells. MDCK cells
(A) or met-18b-2 cells (B) in one 10-cm dish were
incubated with [3H]mevalonate. Cells were lysed in
TNE/Triton X-100 on ice. Proteins in 10% of the whole cell lysate
(WCL) and in DRMs isolated from the remaining 90% of the
lysate were separated by SDS-PAGE and visualized by fluorography.
Exposure times: A, 6 months; B, 3 months.
component of heterotrimeric G proteins (63),
have been detected by others in DRMs. For this reason, we next examined
MDCK cell DRMs for the presence of these proteins. Proteins in total
cell membranes or in DRMs prepared from at least 10 times as many cells
were separated by SDS-PAGE and transferred to nitrocellulose. Although
both Rab5 and Rap1 were easily detected in total cell membranes (Fig.
8, WM), they were barely
detected in DRMs (Fig. 8, DRM). Approximately 1% of the
total cellular Rap1 was present in DRMs.
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Fig. 8.
Rab5 and Rap1 are excluded from DRMs.
DRMs (from ten 10-cm dishes in the Rap1 experiment; 14 dishes in the
Rab5 experiment) or total cell membranes (WM: from one 10-cm
dish in each case) were prepared from MDCK cells without carbonate.
Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and
detected by Western blotting and ECL.
subunit is responsible for membrane
targeting of the G
complex. 30% of this complex was
found in DRMs isolated from a neuroblastoma cell line grown with serum (63). In contrast, very little G
was found in chicken gizzard DRMs (27). We subjected lysates of MDCK cells stably expressing
PLAP to sucrose gradient ultracentrifugation, fractionated the
gradients, and examined the distribution of G
and PLAP between the Triton-soluble lysate fractions and floating DRMs
(Fig. 9A, panels 1 and
2). Although 85% of PLAP was recovered in the DRM
fractions, G
was barely detectable. Similar results
were obtained whether or not the Triton X-100 lysis buffer contained
sodium carbonate.
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Fig. 9.
G and
Ras are excluded from DRMs. A, DRMs were prepared from
MDCK cells stably expressing PLAP in one 10-cm dish without carbonate
by fractionation of sucrose gradients. Lysate-containing fractions at
the bottom of the gradient and DRM-containing fractions near
the top are indicated. PLAP and either G
or
Ras (separate experiments) in each fraction were detected by Western
blotting and ECL. B, COS1 cells transiently expressing H-Ras
(one 10-cm dish) were lysed in 1 ml of TNE/1% Triton X-100. Proteins
in 10 µl of the lysate (WCL) and in DRMs prepared from the
remainder were separated by SDS-PAGE. H-Ras was detected by Western
blotting and ECL. Endogenous COS-1 cell Ras was not detectable under
these conditions (not shown). The doublet in the WCL may result from
incomplete lipid modification.
to show
that Ras is also excluded from MDCK cell DRMs (Fig. 9A, panels
3 and 4).
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank S. Mumby, I. Trowbridge, and A. Wandinger-Ness for antibodies, K. Cadwallader for pEXV-3-H-Ras, J. Faust for met-18b-2 cells, R. Simoni for compactin, and B. Haltiwanger for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM47897 (to D. A. B.) and GM37547 (to M. G. R.).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.
§ Present address: Dept. of Biology, Johns Hopkins University, Baltimore, MD 21218.
To whom correspondence should be addressed. Tel.:
516-632-8563; Fax: 516-632-8575; E-mail:
dbrown{at}mcbsgi.bio.sunysb.edu.
The abbreviations used are: DRMs, detergent-resistant membranes; MDCK, Madin-Darby canine kidney; GPI, glycosylphosphatidylinositol; PLAP, placental alkaline phosphatase; ECL, enhanced chemiluminescence; PAGE, polyacrylamide gel electrophoresis; HA, influenza hemagglutinin.
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REFERENCES |
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