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INTRODUCTION |
Our view of the lateral organization of plasma membrane
constituents has evolved in recent years from the conventional picture of membrane proteins diffusing freely in a sea of lipid (1). A large
body of evidence from studies using cell biological and biophysical
approaches suggests that there is selective confinement of lipids and
proteins in discrete regions of the membrane (2-4). These domains,
named lipid rafts, are rich in sphingolipids and cholesterol, and
appear to be a ubiquitous feature of mammalian cells. Lipid rafts are
likely to contribute to the structure and function of caveolae, plasma
membrane invaginations that are implicated in a variety of cellular
processes, including signal transduction, endocytosis,
transcytosis, and cholesterol trafficking. It has been proposed that
the spatial concentration of specific sets of proteins increases the
efficiency and specificity of signal transduction by facilitating
interactions between proteins and by preventing inappropriate
cross-talk between pathways.
Raft lipids have been proposed to exist in a separate phase from the
rest of the bilayer, in a state similar to the liquid-ordered (lo)1 phase
described in model membrane (5, 6). Acyl chains of lipids in the
lo phase are tightly packed and highly ordered and extended, similar to those in the gel phase. Thus, lipid structural features (such as saturated acyl chains) that enhance formation of the
gel phase can also enhance formation of the lo phase when these lipids are mixed with cholesterol. The presence of unusually long
saturated acyl chains on sphingolipids promotes phase separation and
formation of the lo phase in mixtures of phospholipids,
sphingolipids, and cholesterol at concentrations similar to those in
the plasma membrane at 37 °C (7), and is likely to do so in
biological membranes as well.
Cholesterol- and sphingolipid-rich detergent-resistant membrane (DRMs)
can be isolated from mammalian cells (8). Because there are good
correlations between onset of formation of an ordered phase and
acquisition of detergent insolubility in model membranes (7, 9), and
because DRMs isolated from cells are present in the lo
phase (10), DRMs are thought to be derived from rafts in living cells.
In contrast, cell membranes that are in the conventional disordered
phase are fully solubilized by non-ionic detergents.
Based on the structural model of rafts described above, it has been
postulated that proteins with a high affinity for an ordered lipid
environment are selectively recruited to rafts (6). These might be
expected to include proteins modified with saturated fatty acyl chains,
which could partition favorably into lo phase domains.
Indeed, the best characterized DRM targeting signals on proteins are
structures that include dual saturated acyl chains. These are
glycosylphosphatidylinositol membrane anchors (11, 12) (which contain
predominantly saturated fatty acids; Ref. 13), modification with tandem
amide-linked myristate and thioester-linked palmitate (14, 15), and
modification with tandem thioester-linked palmitate chains (16).
Palmitoylation is also required for DRM association of the integral
membrane proteins LAT (17) and influenza hemagglutinin (18). The
contribution of the lipid modification to raft recruitment is not
simply the addition of a hydrophobic moiety, as prenylated proteins are
not enriched in DRMs (18).
Sphingolipid- and cholesterol-rich liposomes (SCRL), constructed to
mimic the lipid composition of DRMs isolated from cells (8), contain
lo phase domains (7) and yield DRMs after detergent extraction (6). SCRL are useful for studying association of purified
proteins with DRMs in a defined model system. Purified glycosylphosphatidylinositol-anchored proteins incorporated into SCRL
are present in DRMs derived from the liposomes (6, 9), supporting the
model that acyl-chain interactions are important in targeting.
The heterotrimeric GTP-binding proteins (G proteins) are fatty-acylated
and prenylated, and thus are a useful model for examining the role of
lipid modifications in targeting proteins to rafts. All G protein 
subunits are fatty-acylated with amide-linked myristate,
thioester-linked palmitate, or both (19). G protein 
subunits are
modified by farnesyl or geranylgeranyl groups (19). In mammalian cells,
an unambiguous role for N-myristoylation and prenylation in
mediating membrane association of G protein subunits has been
established. Gi family members lacking
N-myristate or G subunits lacking prenylation are soluble
(19). Palmitoylation of G protein subunits does not appear to be a
major determinant of membrane avidity, at least in the presence of
G
subunits (20-22), but may play a role in targeting G
specifically to the plasma membrane (20, 21, 23, 24), and potentially
in targeting proteins to subdomains (25, 26). Palmitoylation-defective mutants of Gz, Go, and Gpa1p, a G protein
subunit in Saccharomyces cerevisiae, are mislocalized to
intracellular membranes (20, 22, 23). However, it is not possible to
discern whether mislocalization is attributable to the lack of
palmitate or mutation of the palmitoylated cysteine residue.
Biochemical and morphological evidence points to the organization of G
protein pathways in subdomains of the plasma membrane. All of the
components of the hormone-sensitive adenylyl cyclase system appear to
be enriched in preparations of low density plasma membrane fragments
(27). Gi and G subunits have a punctate appearance in
plasma membrane fragments when detected by immunofluorescence. This
punctate distribution of Gi is corroborated by its
clustered appearance when decorated with gold particles in electron
micrographs of plasma membrane (22). G subunits are enriched in DRM
isolated from cells, but there is disagreement as to the extent of
enrichment of G subunits in these preparations (18, 28, 29). Two nonexclusive mechanisms have been proposed for the targeting of G
subunits to lipid rafts; fatty acylation (25, 30), or interactions with
caveolin, a coat protein of caveolae that may function as a scaffolding
molecule (31, 32). Most Gi family members contain the
DRM-targeting signal, Met-Gly-Cys, that directs
N-myristoylation at Gly-2 and palmitoylation at Cys-3 (19).
As with similarly modified nonreceptor tyrosine kinases, mutation of
either site in Gi1 reduces association with DRM isolated
from transfected cells (25). It is assumed that the failure of the
mutant proteins to cofractionate with DRM is due to the loss of the
lipid modification. However, this has not been directly demonstrated. A
direct protein-protein interaction between Gi subunits
and caveolin has also been proposed as a mechanism for targeting
proteins to lipid rafts (33). Caveolin is reported to bind to an amino
acid motif in Gi2, fXfXXXf (where f represents aromatic amino acids Trp, Phe, or Tyr) (32), and also to
interact either directly or indirectly with the amino terminus of
Gi1 (30). The direct binding interaction between purified Gi and caveolin was not reproduced with
purified components (27), raising questions about the nature of the
interaction of caveolin with the fXfXXXf motif.
Co-immunoprecipitation of Gi2 with caveolin from
transfected cells requires only the NH2-terminal domain of
Gi2, is independent of the fXfXXXf
motif, and is dependent upon an intact palmitoylation site (30). The
studies published to date do not discriminate whether targeting of
proteins to rafts by the Met-Gly-Cys motif is mediated by interactions
of the protein's fatty acyl chains with membrane lipids, with other
proteins present in domains, or through a mechanism that is dependent
upon the primary amino acid sequence of the protein rather than the
lipid modification.
In this paper, we address the question of whether interactions between
the lipid modifications present on G proteins and raft lipids account
for their distribution in rafts in cells. The affinity of
differentially lipid-modified G proteins for rafts was examined by
reconstituting purified subunits into liposomes engineered to mimic
rafts. Membrane partitioning was evaluated using susceptibility of the
protein to Triton X-100 extraction. We present evidence that the
selectivity of protein targeting to rafts relies on the lipid structure
that modifies the protein.
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EXPERIMENTAL PROCEDURES |
Materials--
Phosphatidylcholine (liver),
phosphatidylethanolamine (liver), cerebrosides (brain), sphingomyelin
(brain), cholesterol (wool grease), and (16:0-18:0
(6-7Dibr)-phosphatidylcholine were obtained from Avanti Polar Lipids
Inc. [2-palmitoyl-9,10-3H]Phosphatidylcholine
and [3H]palmitate were purchased from NEN Life Science
Products. The myristoyl-CoA, palmitoyl-CoA, palmitoleoyl-CoA,
stearoyl-CoA, and linoleoyl-CoA lipid derivatives were purchased from
Sigma. The detergent Triton X-100 was purchased as an aqueous solution (10%) from Roche Molecular Biochemicals, CHAPS from Calbiochem, and
polyoxyethylene 10-lauryl ether from Sigma. The nucleotides guanosine
5'-diphosphate and guanosine 5'-triphosphate were obtained from Sigma.
GTPS was from Roche Molecular Biochemicals.
Gi Protein Expression and
Purification--
Recombinant nonlipidated Gi1
(Gi) was expressed and purified from Escherichia
coli (34). To obtain myristoylated Gi1 (MGi), the S. cerevisiae
N-myristoyltransferase was expressed together with
Gi in bacteria and MGi
purified as described (35). Activity of the different purified Gi preparations was evaluated by quantification of bound
GTPS (34). Recombinant G1 and hexahistidine-tagged
G2 subunits (G) were produced in Sf9 cells
and purified by sequential chromatography with nickel-nitrilotriacetic
acid (Qiagen) and Mono Q (Amersham Pharmacia Biotech) as described
(36). To evaluate the purity of the preparation, the proteins were
resolved by SDS-PAGE and stained with silver nitrate.
Fatty Acylation of MGi in
Vitro--
The procedure was adapted from the protocol of Duncan and
Gilman (37). Purified recombinant MGi (4 µM) was incubated in Buffer A (20 mM NaHepes
(pH 8.0), 2 mM MgCl2, 1 mM EDTA)
containing 7.5 mM CHAPS and 80 µM
myristoyl-CoA (C14:0), palmitoyl-CoA (C16:0), palmitoleoyl-CoA (C16:1),
stearoyl-CoA (C18:0) or linoleoyl-CoA (C18:2). For Gi
monomer, MGi was preincubated for 30 min at
30 °C in the presence of GDP (10 µM) and NaF (10 mM), or GTPS (10 µM). For experiments with trimeric G proteins, heterotrimer was formed by incubating
MGi (4 µM) with G (4 µM) in the presence of GDP (10 µM) for 30 min at 30 °C. The acylation reaction was started with the additions of a mix of the other reagents. The solution was then further incubated
for 90 min. All concentrations shown are the final concentrations in
the autoacylation reaction.
To monitor the efficiency of palmitoylation, GTPS-bound
MGi (1 µM) was incubated with
[3H]palmitoyl-CoA (20 µM, 3300 dpm/pmol) in
Buffer A containing 7.5 mM CHAPS.
[3H]Palmitoyl-CoA was prepared as detailed by Dunphy
et al. (24). Aliquots of the reaction (10 µl) were removed
at different time points and mixed with 30 µl of a solution
containing 1% SDS and 2 mg/ml aldolase. Samples were then precipitated
at room temperature by the addition of 500 µl of 15% trichloroacetic
acid, 2% SDS. Following a 45-min incubation, the samples were filtered
on BA85 nitrocellulose filters (Schleicher & Schuell) using a 10-place filter manifold (Hoefer). Each tube was rinsed twice with 4 ml of 6%
trichloroacetic acid, 2% SDS. The filters were washed twice with
2 × 2 ml of 6% trichloroacetic acid, 2% SDS, followed by 2 × 2 ml of 6% trichloroacetic acid. The filters were dried, placed in
4 ml of scintillation fluid, and counted by liquid scintillation spectrometry. To monitor the acylation of
MGi when using acyl-CoAs other than
palmitoyl-CoA, an indirect method of labeling was used.
MGi (1 µM) was acylated with
20 µM acyl-CoA (C14:0, C16:0, C16:1, C18:0, C18:2) as
described above. The solution was then spiked with
[3H]palmitoyl-CoA (20 µM, 6000 dpm/pmol)
and the incubation continued for 90 min. The reaction was stopped and
quantitated as above. Sequential acylation reactions allowed us to
estimate the level of acylation in the first reaction by measuring the
inhibition of incorporation of radioactive palmitate.
Liposome Preparation--
Lipids (6 mg) for making
phosphatidylcholine and cholesterol (PC:Chol, 7:1 mol:mol) liposomes or
phosphatidylcholine, phosphatidylethanolamine, sphingomyelin,
cerebrosides, and cholesterol (SCRL, 1:1:1:1:2) (6) were mixed in a
glass screw cap vial, dried under nitrogen gas, lyophilized for 1 h, and stored under argon gas at 
20 °C. For the sucrose gradient
sedimentation assay, the liposomes were brominated
(bromoPC:Chol, bromoSCRL) in order to create a
heavier liposome, by replacing half of the phosphatidylcholine with
brominated phosphatidylcholine (16:0-18:0(6-7Dibr)-PC). To make
[3H]phosphatidylcholine labeled PC:Chol or SCRL, 16 nCi/mg lipids of
[2-palmitoyl-9,10-3H]phosphatidylcholine was
incorporated in the lipid mixture prior to lyophilization.
To prepare liposomes, a vial of dried lipids was first brought to room
temperature and lyophilized for 30 min. The lipids were suspended in
buffer B (100 mM NaCl, 50 mM NaHepes (pH 8.0), 5 mM MgCl2, 2 mM EDTA) at 2 mg/ml,
transferred to a Corning glass screw cap tube, and the tube filled with
argon gas. All the buffers used for liposome experiments were bubbled
with nitrogen gas for a few minutes to reduce solubilized oxygen. The
lipid suspension was sonicated in a bath ultrasonicator (Laboratory
Supplies, Inc.) until the solution became partially clear (between 5 and 20 min). The suspension was then frozen and thawed three times by
alternative immersion of the tube in baths of methanol/dry ice and
water at 30 °C. The liposomes were recovered in the pellet by
ultracentrifugation for 30 min at 200,000 × g and
suspended in buffer B at 20 mg/ml. Liposomes were discarded after
36 h.
G Protein Reconstitution into Liposomes--
Freshly acylated G
proteins (40 pmol of M/PGi or
M/PGi), or mock-acylated
Gi, MGi, G, and
MGi were mixed with CHAPS (375 nmol) in a
total volume of 12 µl. The protein solution was then mixed with 500 µg of PC:Chol or SCRL, and incubated for 45 min at room temperature.
Under those conditions, the molar ratio of CHAPS:phospholipids is 0.72 and only partial solubilization of the liposomes occurs (38). The mixture was then diluted stepwise at room temperature with buffer B
containing either 10 µM GDP in the presence or the
absence of 10 mM NaF or 10 µM GTPS, until
the CHAPS concentration reached 2 mM. Equal volumes of
buffer were added to the partially solubilized liposomes every 2 min
and the solution gently vortexed. To recover the liposomes, the sample
was fractionated by centrifugation at 200,000 × g for
30 min at 4 °C and the pellet suspended in nucleotide-supplemented buffer B at 2.2 mg of lipids/ml. A sample of the dilution prior to the
fractionation, input (I), and the resulting supernatant (S) and
suspended pellet (P) were kept to evaluate the efficiency of G protein
incorporation into the liposomes. Quantification of G proteins in the
fractions was determined either by GTPS binding for
Gi-containing samples and/or by image analysis (NIH Image) of the gels resulting from protein resolution by SDS-PAGE and
stained with Coomassie Blue. Recoveries of the protein in the
supernatant (S) and the suspended pellet (P) following the fractionation averaged more than 90% of the starting material (I),
(S+P)/I. The reconstitution efficiencies of G protein was expressed as
the percentage of protein associated with the liposome pellet over the
total amount of protein recovered, P/(S+P).
Sedimentation Assay of Liposomes and Total Lipid
Analysis--
Sucrose gradients were prepared by freezing (80 °C)
a 15% sucrose solution in buffer B supplemented with 10 µM amounts of the appropriate nucleotide in
ultracentrifugation tubes (Beckman), and thereafter allowing the
solution to slowly thaw at room temperature. This method gives rise to
a linear gradient from ~5% to 20% sucrose, as determined by the
sucrose refractive index measured in the fractions recovered. A sample
of reconstituted protein in bromoPC:Chol and
bromoSCRL or protein alone in buffer B containing 7.5 mM CHAPS was applied to the top of the gradient and
centrifuged at 200,000 × g for 8 h at 4 °C.
Fractions were collected and analyzed for total lipid and protein
content. Lipids were isolated by chloroform extraction (1:1). Following
vigorous mixing, the solution was centrifuged in a tabletop centrifuge
and the organic phase was recovered and evaporated under nitrogen gas
to ~10 µl. The concentrated lipid extracts were spotted side by
side on a HPTLC plate (Whatman) and allowed to dry. Lipids were
revealed by charring (39). Proteins were detected by immunoblot and
visualized by chemiluminescence (Pierce).
Membrane Partitioning of G Protein--
Reconstituted G proteins
were chilled on ice for 10 min. Cold Triton X-100 was added to the
suspension at a final concentration of 1% and the preparation
incubated on ice for 25 min. The solution was then fractionated by
centrifugation at 200,000 × g for 30 min. An
equivalent sample from the resulting supernatant and the suspended
detergent-resistant fraction were resolved by SDS-PAGE, and proteins
were detected by Coomassie Blue staining of the gel. The gel was
scanned and the intensity of the bands evaluated by densitometry using
NIH Image. The amount of protein in the detergent-resistant fraction
was expressed as a percentage of the protein recovered following the
fractionation, P/(P+S).
Effect of Nucleotide on Partitioning of Gi
Subunits--
MGi was palmitoylated
in vitro in the presence of GDP and NaF and reconstituted in
SCRL as described above. To compare the effect of the activation state
of Gi on its membrane partitioning, NaF and
MgCl2 were removed from half of the preparation to return the protein to a GDP-bound inactive state. Accordingly, the solution of
reconstituted liposomes was divided, and fractionated by
ultracentrifugation. The resulting pellets were washed once in a large
volume of buffer B containing 5 µM GDP and 10 mM NaF or buffer B (lacking MgCl2) and 5 µM GDP. The washed SCRL pellets were then assayed for
membrane partitioning as described above. In parallel, the samples were subjected to a trypsin protection assay (36), to monitor the activation
state of Gi following the wash step. Gi
is substantially protected from tryptic proteolysis when activated with
either GTPS or GDP/NaF (40, 41). Briefly, aliquots (20 µl) of the reconstituted liposomes were mixed with 50 mM NaHepes (pH
8.0), 1 mM EDTA, 3 mM dithiothreitol, and
0.05% polyoxyethylene 10-lauryl ether, together with 150 ng of
tosylphenylalanyl chloromethyl ketone-treated trypsin, in a final
volume of 40 µl. The mixture was incubated at 30 °C and terminated
20 min later by the addition of 40 µg of soybean trypsin inhibitor
and then transferred to ice. The samples were subjected to SDS-PAGE on
14% acrylamide gel. The gel was stained with Coomassie Blue to
visualize the proteins.
| |
RESULTS |
Lipid Modifications Are Required for Reconstitution of G Proteins
in Liposomes--
An in vitro approach was chosen to
determine the minimal molecular requirement for targeting
membrane-anchored G proteins to lipid rafts. G proteins were
reconstituted into PC:Chol and SCRL. The former are present in a
disordered state and are fully solubilized by Triton X-100 (6). G
protein subunits were purified using recombinant expression systems to
permit manipulation of the lipid modifications. The experiments were
performed with Gi112 (42).
It has previously been reported that Go and
Gt subunits bind only weakly to liposomes unless
co-reconstituted with prenylated G subunits, suggesting that G
requires G as a membrane anchor (43, 44). However, the ability of
G modified with both myristate and palmitate to bind to liposomes
has not been tested. As predicted, nonacylated Gi did
not associate with either vesicle preparation (Fig.
1). The addition of myristate to
Gi increased its association with vesicles. However,
consistent with the previous reports, the reconstitution efficiency was
not as high as that of prenylated G subunits. Approximately 20%
of MGi was found in the pellet of PC:Chol or
SCRL, compared with more than 50% of prenylated G subunits. The
addition of the second lipid modification to
MGi increased the reconstitution efficiency
to levels comparable with G subunits. The efficiency of
reconstitution of the dually acylated proteins may be an underestimate,
as the stoichiometry of chemical acylation was approximately 75% (data
not shown). The reconstitution efficiency of the heterotrimer was
similar whether Gi was modified with one or two acyl
chains. To ensure that the protein remained in its native form during
the time-consuming palmitoylation and reconstitution processes, we
monitored the activity of Gi by GTPS binding at
different steps during the procedure. No significant binding activity
was lost (data not shown). These results show that functional G
proteins can be reconstituted efficiently into liposomes in a
lipid-dependent manner. Gi can associate
directly with liposomes if it is sufficiently acylated. The results
also argue that the liquid-ordered domains present in SCRL do not
interfere with the ability of the protein to become incorporated in the
membrane, as similar efficiency of protein reconstitution was obtained
with the single phase liquid-crystalline PC:Chol.
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Fig. 1.
Reconstitution of G proteins into PC:Chol and
SCRL. Monomeric Gi1 protein (unmodified
(Gi), myristoylated (MGi),
myristoylated and palmitoylated (M/PGi),
prenylated G (G)), or heterotrimeric G proteins
(MGi, M/PGi)
were reconstituted in PC:Chol (upper panel) or in
SCRL (lower panel) as described under
"Experimental Procedures." To evaluate the proportion of protein
reconstituted, a sample of the suspension, before (I, input)
and after isolation of the reconstituted vesicles by centrifugation
(S, supernatant; P, suspended pellet), was
subjected to SDS-PAGE and the proteins detected by Coomassie Blue
staining. A representative gel of each type of reconstitution is shown
on the left of the panels. For the trimeric G
protein reconstitutions, the upper band on the gels represents
Gi and the lower band, G. To quantitate the
reconstitution efficiency, the gels were scanned and the intensity of
the bands quantitated by densitometry. The amount of protein in the
pellet was expressed as a percentage of the total material recovered
from the starting material, P/(S+P) (right
panels; filled column,
Gi; hatched column, G). The
recovery of protein from the starting material, (S+P)/I, averaged
95.5% ±3.3 (PC:Chol) and 101.7% ±5.7 (SCRL). The results shown
represent the mean ± S.E. of 4-15 independent experiments.
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To confirm that the pelleted proteins were associated with the
liposomes, M/PGi in bromoPC:Chol
was subjected to sedimentation through sucrose gradients (Fig.
2). Most of the reconstituted
M/PGi (Fig. 2A, top
panel) sedimented in the same fractions as the lipids (Fig.
2A, bottom panel), in the middle of
the gradient. In contrast, when nonacylated Gi was
included in the reconstitution mixture (Fig. 2B), the
protein was found at the top of the gradient (Fig. 2B,
top panel), whereas the lipids were in fractions
4-6 (Fig. 2B, lower panel). As a
control, mock-reconstituted M/PGi was also
found in the fractions at the top of the gradient (Fig. 2C).
Similar results were obtained for proteins reconstituted into SCRL
(data not shown). The fact that Gi and the liposomes co-sedimented in the sucrose gradient only when Gi was
acylated strongly suggests a lipid-dependent anchoring of
the protein to liposomes.
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Fig. 2.
Lipid-dependent association of
Gi with PC:Chol.
Myristoylated and palmitoylated Gi
(M/PGi) or unmodified Gi
(Gi) were reconstituted in bromoPC:Chol
(A and B) as described under "Experimental
Procedures." An aliquot of liposomes with
M/PGi (A) or Gi
(B) and of mock-reconstituted
M/PGi (C) was applied to the top
of a 5-20% sucrose gradient and centrifuged at 200,000 × g for 8 h. Fractions of equal volumes were collected
and subjected to SDS-PAGE, followed by protein immunoblotting with
Gi antiserum (top panel,
A and B, and C). The lower
panels in A and B show the lipid
content of the fractions. An aliquot of each fraction was spotted on a
HPTLC plate, and lipids were detected by charring as described under
"Experimental Procedures." The results shown are representative of
two independent experiments.
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Only Fatty-acylated G Proteins Partition into Rafts--
We next
determined the Triton X-100 insolubility of
M/PGi, G,
MGi, and
M/PGi reconstituted in PC:Chol or SCRL
(Fig. 3) as a way of measuring the
membrane compartmentalization of these proteins. Only the amount of
insoluble Gi recovered from liposomes containing
M/PGi, MGi, and
M/PGi is shown in the figure, while the
amount of insoluble G in liposomes containing G alone is
shown. As predicted, all G proteins reconstituted in PC:Chol were
completely solubilized by the Triton X-100 treatment. No lipids (data
not shown) and only a marginal amount (less than 10%) of
M/PGi, G, MG, or
M/PGi could be detected in the Triton
X-100-insoluble pellet.
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Fig. 3.
Membrane partitioning of lipidated G proteins
in SCRL. G protein subunits (M/PGi,
G) or heterotrimeric G proteins
(MGi, M/PGi)
were reconstituted in PC:Chol (hollowed bars) or
in SCRL (solid bars). The reconstituted liposomes
were collected by centrifugation and treated with 1% ice-cold Triton
X-100 for 25 min. The mixture was fractionated by ultracentrifugation,
and the proteins in the detergent-resistant pellet and the supernatant
were subjected to SDS-PAGE and detected by Coomassie Blue staining. The
gels were scanned and the intensity of the bands analyzed by
densitometry. The percentage of Gi in the
detergent-insoluble fraction when liposomes containing
M/PGi, MGi, or
M/PGi were extracted is reported.
Quantitation of Gi in the detergent-insoluble fraction by
GTPS binding gave similar results to those by densitometry (data not
shown). The percentage of G in the detergent-insoluble fraction
when liposomes containing only G were extracted is reported. Less
than 10% of G was detergent-insoluble when liposomes containing
heterotrimers were extracted (data not shown). The results shown
represent the mean ± S.E. of three to eight independent
experiments.
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Different results were obtained when the G proteins were incorporated
into SCRL. When SCRL containing M/PGi alone
were extracted, more than 50% of the protein was found associated with
the Triton-insoluble pellet. In contrast, more than 90% of the
farnesylated G subunit was solubilized when it was incorporated
into SCRL in the absence of a Gi subunit. This striking
difference in the distribution between fatty-acylated Gi
and prenylated G points to selective lipid-lipid interactions as
a mechanism for targeting proteins to rafts.
Because M/PGi and G had different
solubility behavior when incorporated into SCRL independently, we then
looked at the compartmentalization of G protein heterotrimers (Fig. 3).
G was almost completely solubilized from SCRL (data not shown),
whereas approximately 25% of MGi or
M/PGi was found in the detergent-insoluble
pellet (Fig. 3), a substantial reduction from what was observed for
M/PGi reconstituted alone. These results
suggest a dominant effect of the G prenyl group over the fatty
acid(s) of Gi on partitioning of the heterotrimer.
Interestingly, both monoacylated and diacylated Gi had
the same partitioning. The presence of a fraction of Gi in the detergent-resistant fraction is a consequence of partial dissociation of the heterotrimer, as shown by the fact that the ratio
of Gi:G reconstituted in SCRL (1.0 for
MGi/G and 0.9 for
M/PGi/G) changed to 1.5 (MGi/G) and 1.4 (M/PGi/G) in the detergent-resistant
pellet. We were not able to find conditions where heterotrimer
dissociation was minimized and the liposomes were efficiently
solubilized. The fact that only ~25% "trimeric"
Gi compared with more than 50%
M/PGi alone remained with the
detergent-resistant pellet argues that G plays a significant role
in restraining fatty-acylated Gi from associating with
rafts. The results also suggest that MGi and
M/PGi, when released from G, can move
into rafts. However, this observation remains to be addressed clearly.
Detergent Treatment of SCRL Does Not Induce Diffusion of Lipids or
Proteins Containing Saturated Fatty Acids into DRM--
Triton
treatment of M/PGi reconstituted in SCRL
could have artifactually driven the protein into the
detergent-resistant fraction, even if it was not present in
lo phase domains before extraction. To address this
possibility, we determined whether either
M/PGi or a saturated chain phospholipid
could "hop" from Triton-soluble liposomes into DRMs derived from
separate liposomes, if the two liposomes were mixed during lysis. This
strategy was previously used to show that both sphingomyelin and a
glycosylphosphatidylinositol-anchored protein must be in membranes
containing lo-phase domains before detergent extraction to
be recovered in DRMs (9). Trace amounts of
[2-palmitoyl-9,10-3H]phosphatidylcholine were
incorporated into PC:Chol. These vesicles were treated with 1% Triton
X-100 in the presence of unlabeled PC:Chol or SCRL (Fig.
4, left panel). If
Triton X-100 induces an artifactual redistribution of lipids containing
saturated fatty acids into rafts, then the
[2-palmitoyl-9,10-3H]phosphatidylcholine
should be found in the detergent-resistant fraction of the PC:Chol/SCRL
solution. The results of such an experiment show that nearly 100% of
the [2-palmitoyl-9,10-3H]phosphatidylcholine
was solubilized by Triton upon mixing the labeled PC:Chol with PC:Chol
or SCRL, arguing that the saturated lipid did not diffuse in rafts upon
solubilization of the liposomes. However, when the
[2-palmitoyl-9,10-3H]phosphatidylcholine was
first incorporated into SCRL, and then mixed with unlabeled PC:Chol or
SCRL, most of the labeled phospholipid was resistant to solubilization,
suggesting that the labeled phosphatidylcholine was present in
lo-domains. Therefore, there appears to be little redistribution of lipids induced by detergent.
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Fig. 4.
Characterization of the Triton X-100
treatment of SCRL. Left panel, trace amounts
of [2-palmitoyl-9,10-3H]phosphatidylcholine
were incorporated into PC:Chol or SCRL (labeled) as described under
"Experimental Procedures." The radiolabeled liposomes were mixed
with equal amounts of the indicated unlabeled liposomes, and treated
with 1% ice-cold Triton X-100. The mixture was fractionated by
ultracentrifugation, and radioactivity present in the supernatant and
pellet was quantitated by liquid scintillation spectrometry. The
graph shows the percentage of radioactivity in the
detergent-resistant pellet. The figure shows the average ± S.D.
of two experiments. Right panel,
M/PGi was reconstituted in PC:Chol or SCRL
and the reconstituted liposomes isolated, as described under
"Experimental Procedures." The reconstituted liposomes or soluble
M/PGi in Triton X-100 (2%) were then mixed
with equal volume of PC:Chol or SCRL, as indicated, and treated with
ice-cold Triton X-100 for 25 min. The final Triton X-100 concentration
in all samples was 1%. The mixture was fractionated by
ultracentrifugation and the amount of M/PGi
quantitated in the supernatant and the detergent-resistant fraction by
GTPS binding. The graph shows the percentage of
M/PGi that is Triton-resistant. The results
shown represent the mean ± S.E. of 5-14 independent
experiments.
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Qualitatively similar results were obtained when using
M/PGi (Fig. 4, right
panel). In these experiments, 77% of
M/PGi was associated with DRM. In contrast,
the addition of a Triton solution containing
M/PGi to SCRL resulted in only 16%
incorporation of protein into the detergent-resistant pellet. This
fraction was increased to 36% when M/PGi
reconstituted in PC:Chol was treated with Triton in the presence of
empty SCRL. These results suggest that treatment with detergent induces
some transfer of M/PGi into DRM in the
presence of empty SCRL. However, the amounts of
M/PGi found in the the detergent-resistant
pellets under these conditions is significantly less than the amounts
associated with DRM when M/PGi is
reconstituted directly into SCRLs. The presence of
M/PGi in the detergent-insoluble pellet is
not likely to be due to nonspecific aggregation because the protein
retains GTPS binding activity. These results argue that proteins
exist in lo domains prior to treatment with Triton
X-100.
The Activation State of M/PGi Does Not
Influence Its Partitioning into Detergent-resistant Domains--
We
next tested the possibility that the activation state of
M/PGi might influence its distribution in
SCRL. In the previous experiments, M/PGi
reconstituted in SCRL was in the GTPS-bound form. The protein was
activated with GTPS to maintain its stability during the autoacylation and reconstitution protocols. To determine if GDP-bound M/PGi also partitioned into DRM, we took
advantage of the fact that Gi can be activated by NaF
and MgCl2 in a reversible manner, in the GDP-bound state
(45). GDP-bound MGi was palmitoylated and
reconstituted into SCRL in the presence of NaF and MgCl2.
Activation was reversed by washing the liposomes in buffer lacking NaF
and MgCl2. Recovery of Gi in the inactive GDP-bound state was confirmed by its susceptibility to proteolysis with
trypsin (data not shown). The activated form of Gi is
partially protected from tryptic digestion, whereas the GDP-bound form
is digested completely. The distribution of GDP-bound and NaF-activated M/PGi in detergent-resistant membranes
isolated from SCRL is shown in Fig. 5.
Approximately 50% of the protein in either state was resistant to
detergent extraction. Therefore, the activation state of the
M/PGi monomer does not influence its
association with rafts.
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Fig. 5.
Effect of
M/PGi activation state
on its membrane distribution in SCRL.
MGi was activated with NaF and
MgCl2 in the presence of GDP to stabilize the protein
during the autopalmitoylation reaction and reconstituted into SCRL. The
resulting liposome suspension was divided, and the liposomes were
washed twice in buffer containing GDP, NaF, and MgCl2 or in
buffer containing GDP but lacking MgCl2 to inactivate
Gi. The membrane distribution of
M/PGi under both conditions was evaluated by
treatment of the preparations with 1% ice-cold Triton X-100 for 25 min. The solution was fractionated by ultracentrifugation, and
M/PGi in the soluble (S), the
detergent-resistant pellet (P), and the total starting
material (T) was quantitated by GTPS binding. The results
shown represent the mean ± S.E. of four independent
experiments.
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Fatty Acid Saturation Influences Protein Targeting to
Detergent-resistant Membranes--
As shown in the previous
experiments, one of the determinants for association of lipidated
proteins with DRM is the structural compatibility of the lipid that
modifies the protein with membrane lipids. Only fatty-acylated proteins
were targeted to DRM in SCRL. To explore further the lipid-lipid
interactions between the modified protein and the rafts, membrane
partitioning of Gi modified with different isomers of
fatty acids was tested. We hypothesized that a protein modified with
unsaturated fatty acids would behave similarly to a prenylated protein
and be excluded from DRM. A cis-double bond results in an
increase in the cross-sectional area of the hydrocarbon chain (46),
making it more difficult to pack in the ordered lipid environment
present in rafts. MGi was acylated with
fatty acids of different chain length and degrees of
cis-unsaturation (C14:0, C16:0, C16:1, C18:0, C18:2) and
reconstituted in SCRL. The in vitro acylation method used allowed modification of MGi with an
efficiency similar to that of palmitate (C14:0 = 80.2% ±3.9,
C16:1 = 79.7% ±12.1, C18:0 = 74.8% ±8.2, C18:2 = 87.0% ±8.3). Unsaturation in the fatty acyl chain did not affect the
reconstitution of Gi in SCRL, as similar efficiencies of
reconstitution were obtained to that of
M/PGi (Table
I). DRM partitioning of the different
dually acylated Gi in SCRL are shown in Fig.
6. Similarly to C16:0-modified
MGi, 45-55% of the proteins acylated with
C14:0 and C18:0 were resistant to detergent extraction, suggesting that
acyl chain length does not significantly affect the distribution of
Gi. However, proteins modified with the unsaturated
lipids C16:1 and C18:2 were relatively less resistant to the Triton
extraction. Indeed, less than 30% of the protein was found in the
insoluble fraction. These results highlight the fact that structural
differences between lipids are at the origin of the selectivity for
raft association.
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Table I
Reconstitution efficiency of dually acylated Gi in SCRL
MGi was autoacylated with the acyl-CoAs shown below
and reconstituted in SCRL. In each experiments, the recovery of
material after the centrifugation was within 104.7 ± 2% of the
starting material. Results shown represent the mean ± S.E. of
four independent experiments.
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Fig. 6.
Effect of saturation of thioester-linked
fatty acids on membrane partitioning of
Gi.
MGi was fatty-acylated in vitro
(C14:0, C16:0, C16:1, C18:0, C18:2) and reconstituted in SCRL, as
described under "Experimental Procedures." The reconstituted
liposomes were isolated, treated with 1% ice-cold Triton X-100, and
processed for quantitation as described in the legend for Fig. 3.
The results shown represent the percentage of protein that remains
associated with the detergent-resistant fraction. The data shown are
the mean ± S.E. of three to four experiments.
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DISCUSSION |
In addition to their well characterized role as hydrophobic
anchors for otherwise soluble proteins, covalent lipid modifications appear to be important determinants of the lateral distribution of
proteins in subdomains of the plasma membrane (5, 47). In this study,
we have addressed the role of fatty acylation and prenylation of G
protein subunits in membrane binding and in membrane partitioning using
a reconstituted system with purified components. We confirmed previous
studies demonstrating that G and the G protein heterotrimer
associate with membranes, presumably through prenylation of G and
that myristoylated Gi binds relatively weakly to
liposomes in the absence of G (43, 44). Here we demonstrate that
Gi tandemly modified with myristate and palmitate binds
efficiently to the lipid bilayer in the absence of any other proteins.
These results are consistent with measurements of the affinities of
lipidated peptides for artificial membranes where the presence of
tandem acyl groups results in a very long-lived membrane association in
contrast to that mediated by a myristoyl group alone (48-50).
The effect of palmitoylation on membrane association of
Gi family members in cells has been studied using
palmitoylation-defective mutants. In stable cell lines expressing
modest amounts of palmitoylation-defective Gz (C3A), the
total membrane distribution of the mutant protein is not significantly
different from wild type, although a fraction of the mutant protein is
mislocalized to intracellular membranes (20, 21). G subunits
stabilize membrane association of the mutant protein in the inactive
state. Activation of the G protein results in subunit dissociation, but
this apparently does not result in release of the protein from
membranes. Native Gi in membranes that has been
depalmitoylated enzymatically remains associated with membranes (22).
This suggests that myristate is sufficient to hold Gi at
the membrane or that other protein interactions stabilize its membrane
association in the absence of G. Thus, in intact cells, the
contribution of palmitate to the membrane avidity of Gi
is probably secondary to other functions for this modification (21,
22). A role for palmitoylation in regulating G interactions with
G subunits and regulators of G protein signaling is indicated by
in vitro studies (51, 52). In addition, studies of
Gi (C3A) also point to a role for palmitate in targeting
proteins to rafts (25).
The reconstituted system was used to evaluate a model for how fatty
acylation facilitates protein association with sphingolipid cholesterol-rich domains. Lipids found in rafts are thought to exist in
a more ordered state than those in the surrounding fluid phase (5-7).
The physical parameters of raft lipids that govern the formation of
lo domains may also apply to lipidated proteins that are
present in rafts. Proteins modified with fatty acids could associate
with ordered domains by spontaneous insertion of the fatty acyl chains
into the lo phase (6). In this study, we present evidence
that directly supports this hypothesis. We report that
Gi modified with amide-linked myristate and
thioester-linked palmitate acquires detergent resistance when
reconstituted in liposomes that contain lipids in the lo
phase. These results establish that lipid-lipid interactions are
sufficient to mediate raft localization of proteins containing the DRM
targeting signal, Met-Gly-Cys. Thus, interactions with caveolin or
unidentified resident proteins of caveolae or rafts are not a
prerequisite for targeting dually acylated Gi to rafts,
although this does not exclude the possibility that these protein
interactions exist in rafts.
In the model membranes, fatty-acylated Gi exhibited
detergent resistance, whereas prenylated G was solubilized. This
result recapitulates the distribution of G protein subunits in DRM
isolated from Madin-Darby canine kidney cells (18). The branched and bulky prenyl group that modifies G is not predicted to fit easily in
the compact lipid organization of rafts (18), even though the
hydrophobicity of farnesyl and geranylgeranylated groups is similar to
that of myristate and palmitate (49). The finding that
Gi modified with unsaturated fatty acid was more easily solubilized than protein modified with its saturated counterpart supports the idea that the ability of the lipids to pack into an
ordered environment is a critical factor for sorting proteins into
liquid-ordered domains.
The solubility of G suggests that the protein resides outside
lo domains. However, we cannot rule out the possibility
that G binds to rafts with lower affinity, and this interaction
is susceptible to disruption by Triton X-100. Interestingly, the partitioning behavior of prenylated G was dominant when the heterotrimer was analyzed. The fraction of
M/PGi in DRM was significantly reduced from
that of M/PGi reconstituted alone. That
M/PGi remaining in DRM could be accounted
for by subunit dissociation, since little G was seen in DRM. A
possible explanation for the dominance of the prenyl group in membrane
partitioning is the preferential interaction of the fatty acyl chains
on M/PGi with the prenyl group on G. In
transducin, the T acyl chain binds cooperatively with the T
farnesyl chain in membrane lipids (44). This cooperativity could
represent lipid-lipid interactions between the farnesyl and myristoyl
moieties. The three-dimensional x-ray crystal structure of transducin
and of Gi112 has been solved using proteins that lack their respective lipid modifications (42, 53). The positions of the observed NH2 terminus of
Gi and the COOH terminus of G within the structures
are consistent with the hydrophobic modifications inserting
simultaneously at a single locus in the lipid bilayer. In SCRL, the
cooperativity between lipids may favor partitioning of the heterotrimer
into regions outside of lo domains. An alternative model
for the domain organization of G proteins that is also consistent with
our data is the disposition of the heterotrimer at the edge of rafts.
This would permit insertion of the lipids into their respective
domains. Whether the heterotrimer is located at the edge or outside of DRM, the different distribution of monomeric and trimeric
Gi in DRM raises the possibility that partitioning of
Gi into rafts is dynamic and regulated by its activation
state. This may represent another regulatory input into G protein signaling.
The behavior of Gi in this model system is likely to be
representative of a large number of proteins that undergo sequential modification by amide-linked myristate and thioester-linked palmitate and are enriched in DRM. These include other G protein subunits, many nonreceptor tyrosine kinases (NRTK), and endothelial nitric-oxide synthase (reviewed in (47)). Myristoylation is necessary, but not
sufficient for inclusion of these proteins in DRM. Abrogation of
palmitoylation by mutation decreases the enrichment of these proteins
in DRM. Furthermore, the introduction of a palmitoylation site (Cys-3)
into the nonpalmitoylated kinases, p60src or
p61hck, results in palmitate incorporation and
inclusion in DRM (15, 54). A function for Cys-3 independent of
palmitoylation could not be excluded as an alternative explanation
for the targeting of proteins to DRM, particularly for
p56lck, where there is disagreement as to
whether Cys-3 is the primary site of palmitoylation of the protein (12,
55). In the in vitro system, we have been able to manipulate
the status of fatty acylation without changing the primary amino acid
sequence of the protein. This allowed us to demonstrate directly that
fatty acylation at Cys-3 is the critical parameter for targeting
Gi to rafts. This finding is likely to apply to other
proteins that are similarly modified.
Evidence for the functional importance of compartmentalizing signaling
proteins into lipid rafts is accumulating. T cell activation leads to
the compartmentation of the T cell receptor with downstream NRTKs into
rafts (56). Disruption of raft structure by cholesterol-removing reagents or by internalization interferes with the earliest steps in T
cell activation (56). T cells treated with polyunsaturated fatty acids
also exhibit diminished calcium responses to activation of CD3 or CD59
(57). Loss of signaling is strictly correlated with the displacement of
p56lck and p59fyn from
detergent-resistant domains. Whether polyunsaturated fatty acid
disrupts raft structure through incorporation of unsaturated acyl
chains into NRTKs, raft lipids, or both is not known. Our finding that
introduction of an unsaturated acyl chain into Gi significantly reduces its partitioning into DRM demonstrates that changes in the acylation status of the protein are sufficient to shift
the distribution of the protein. The effect of acyl chain saturation on
partitioning of Gi into DRM may have other important biological correlates. Gi incorporates radiolabeled
arachidonic acid in platelets, suggesting that the protein is
posttranslationally modified with unsaturated as well as saturated
fatty acids (58). Our data suggest that the lateral distribution of
Gi in platelet membranes will be affected accordingly.
In this study, we have provided evidence using well defined, purified
components that selective interaction between saturated fatty acids on
lipid-modified proteins with lipids in DRM is a targeting mechanism for
localization in membrane subdomains. Our data are consistent with the
hypothesis that packing order conferred by the structure of the lipid
modification rather than hydrophobicity is the primary determinant of
whether a lipidated protein is targeted to DRM. The heterotrimeric G
protein is both prenylated and fatty-acylated, and thus harbors
conflicting signals. In the model system, this manifests as the
exclusion of heterotrimer from DRM and suggests that dynamic
association of fatty-acylated Gi with prenylated G
may regulate Gi targeting to rafts.
,
TOP
ABSTRACT
INTRODUCTION
