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INTRODUCTION |
Recent evidence suggests that lipids in mammalian membranes are
not uniformly miscible, but that lateral separation of specific lipid
species leads to the formation of specialized phase domains called
rafts. In mammals, the association of membrane proteins with raft lipid
microdomains has emerged as an important regulator of polarized
intracellular sorting and signal transduction (1, 2).
Raft formation is based on the tendency of cholesterol to
organize the bilayer into cholesterol-rich liquid ordered and
cholesterol-poor liquid disordered domains (3), a process that is
enhanced by the preferential interaction of cholesterol with
sphingolipids and the fact that sphingolipids have higher melting
temperatures than phospholipids (Ref. 4; reviewed in Ref. 5). Rafts
form when the sphingolipid/cholesterol-rich phase separates from the phospholipid-rich phase that constitutes the rest of the membrane. In
model membranes, the formation of the liquid ordered phase correlates
with the acquisition of insolubility in the nonionic detergent Triton
X-100 (6). Insolubility in Triton X-100 or in Triton X-114, a related
detergent, has been used as a criterion for isolation of rafts from
cellular membranes (7, 8).
We decided to establish a system to study rafts in a genetic model
organism with well characterized development: Drosophila melanogaster. Genetics would provide a powerful tool with which to
identify molecules involved in raft formation, trafficking, and
function. Furthermore, since raft formation is thought to play
important roles in cell polarization and signal transduction, examining
their functions in Drosophila may provide insights into the
control of important developmental processes.
We began by asking whether Drosophila membranes contained
membrane domains similar to mammalian rafts. Drosophila
cannot synthesize sterols and require a dietary source. Furthermore,
their membranes must remain fluid at lower temperatures than
those of mammals; thus, the biophysical properties of their lipids
might be expected to differ. We wondered whether raft formation would
occur under these conditions and, if so, to what extent
Drosophila rafts would resemble their mammalian
counterparts. Our data show that, despite differences in the chemical
structure of their lipids, Drosophila membranes
contain rafts with a similar protein and lipid composition.
We then examined membrane-associated proteins of well characterized
developmental pathways to establish which of them might constitute a
suitable system in which to study raft function. In particular, we
wished to examine whether the N-terminal fragment of the Hedgehog
protein might be raft-associated. Hedgehog protein undergoes
autocatalytic cleavage that results in covalent linkage of its
N-terminal fragment to cholesterol (9). Since cholesterol is enriched
in raft membranes, it seemed possible that cholesterol linkage might be
a raft-targeting signal.
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EXPERIMENTAL PROCEDURES |
Preparation of Drosophila Embryonic
Membranes--
Drosophila were reared on yeast-based
medium, and adults in population cages (10) were fed fresh yeast on
apple juice/molasses agar plates twice/day. Flies were allowed to lay
eggs for 14 h overnight, and embryos from eight population cages
were collected onto stacked coarse, medium, and fine wire mesh screens
with a paint brush. The embryos were washed in water and with 0.9%
NaCl + 0.1% Triton X-100 (embryo wash) to remove debris and yeast and then dechorionated in 250 ml of 20% bleach and 80% embryo wash for 3 min. After decanting onto the fine wire mesh, the embryos were sprayed
vigorously with water to remove residual chorion and collected into
50-ml Falcon tubes (~25 ml of packed embryos/tube). The embryos were
then washed twice in embryo wash, three times in 0.9% NaCl, twice in
TNE buffer (100 mM Tris (pH 7.5), 150 mM NaCl,
and 0.2 mM EGTA) + 0.3 M sucrose, and
resuspended in 40 ml/tube TNE buffer + 0.3 M sucrose + 0.001 volume of CLAP (10 mg/ml each chymostatin, leupeptin, antipain,
and pepstatin in Me2SO). Embryos were broken in a
Potter-Elvehjam and then in a Dounce homogenizer with a loose pestle
followed by a tight pestle until the pestle moved smoothly
(approximately five times). The embryo homogenate was spun at 5000 rpm
for 10 min at 4 °C to pellet nuclei, and the post-nuclear
supernatant was removed, adjusted to 1.4 M sucrose, and
distributed into SW 27 centrifuge tubes. Post-nuclear supernatants were
overlaid with 10 ml of 1.22 M sucrose in TNE buffer and 10 ml of 0.1 M sucrose in TNE buffer and then spun for 18 h at 25,000 rpm at 4 °C. Membranes with a white, flocculent appearance were observed floating above the 1.22/0.1 M
interface, and membranes with a more yellowish homogeneous appearance
were observed at the 1.4/1.22 M interface and occasionally
at the 1.22/0.1 M interface. The heavier membranes at the
1.4/1.22 M interface were enriched in endoplasmic reticular
and mitochondrial NADH-cytochrome c reductase, whereas the
lighter membranes above the 1.22/0.1 M interface were
enriched in plasma membrane proteins. The Golgi enzymes
galactosyltransferase and N-acetylglucosaminyltransferase were found in both fractions (data not shown). Membranes floating above
the 1.22/0.1 M interface were collected, diluted three
times in TNE buffer, and spun onto a 62% sucrose cushion in an SW 27 rotor for 20 min at 25,000 rpm. The membranes were then resuspended in
TNE buffer, aliquoted, and stored at
80 °C. These membranes contained ~33% of the total membrane protein.
Purification of Detergent-insoluble Embryonic
Membranes--
Membranes were thawed and washed in 3 volumes of TNE
buffer to remove residual sucrose. They were then resuspended in 350 µl of ice-cold TNE buffer to a phospholipid concentration of 880 nmol/ml. An equal volume of prechilled 2% Triton X-114 or Triton X-100
was added, and the membranes were solubilized at 0 °C for 30 min in
SW 60 tubes. After solubilization, the membranes were brought to a
final concentration of 24% OptiprepTM (Nycomed Pharma AS);
overlaid with 1 ml of 21, 15, and 6% Optiprep in TNE buffer; and then
spun for 5 h at 36,000 rpm at 2 °C. Insoluble membranes could
be observed between the 6% and the 15% layers.
Western Blotting--
After polyacrylamide gel electrophoresis,
proteins were transferred to nitrocellulose at 4 °C in 40 mM Tris/glycine (pH 8.8) + 20% methanol for 2 h at
0.7 mA. Nitrocellulose blots were blocked with 5% powdered milk in
Tris-buffered saline (10 mM Tris-HCl (pH 7.2), 150 mM NaCl, and 0.1% Tween 20) for 20 min and then incubated
with primary antibody overnight at 4 °C. The dilutions of antibodies
used were as follows: anti-Hedgehog, 1:2000; anti-Notch, 1:1000;
anti-fasciclin-1, 1:2000; and anti-Drac1, 1:2000. Blots were washed
three times in Tris-buffered saline for 15 min each and then bound to
horseradish peroxidase-conjugated secondary antibodies (goat
anti-rabbit, goat anti-rat, and goat anti-mouse; Bio-Rad) at a 1:1000
dilution for 2 h at room temperature. After washing three times in
Tris-buffered saline for 15 min each, the signal was detected with an
ECLTM kit (Amersham Pharmacia Biotech).
Analysis of Lipids by TLC--
Phospholipids and sterols were
prepared from membranes by Bligh and Dyer extraction (11).
Phospholipids were quantified according to Rouser et al.
(12). Sphingolipids were prepared by a Folch total lipid extraction
(13), followed by mild alkaline hydrolysis of the glycerophospholipids
for 2 h at 30 °C in methanolic 0.1 M NaOH. The
base-resistant sphingolipids were desalted on an RP18 reversed-phase
column. Lipid samples in chloroform (phospholipids) or
chloroform/methanol (1:2; glycolipids) were applied to activated high
performance TLC plates (Merck) and developed in
chloroform/methanol/water (60:35:8; phospholipids) or
chloroform/methanol/water (60:40:10; glycolipids) in a Whatman lined
tank. The plates were dried at room temperature and stained with iodine
vapor for detection of phospholipids and sterol or with ninhydrin spray
reagent (Sigma) to detect amino phospholipids. Glycolipids were stained
with orcinol/sulfuric acid reagent (14).
Lipid Analysis with Electrospray Ionization Tandem Mass
Spectrometry--
Sphingolipids were extracted and purified as
described above and stored as a dried lipid film. Just prior to
analysis, the lipids were dissolved in methanol containing 5 mM ammonium acetate and centrifuged for 15 min in a
tabletop centrifuge at full speed. Sterols were identified and
quantified after sulfation in the presence of
[3,4-13C2]cholesterol as an internal
standard. After addition of standard to membranes and subsequent
extraction in 1,4-dioxane, sulfation was performed by adding sulfur
trioxide-pyridine complex in absolute pyridine to the dried lipid
samples. The reaction was quenched with barium acetate, and the lipids
were diluted with methanol prior to
measurement.1 Mass
spectrometric analysis was performed with a triple quadrupole instrument equipped with a nanoelectrospray ionization interface and a
Dynolyte TM detector system (Micromass Quattro II). The first and third
quadrupoles are used as independent analyzers, whereas the second
serves as a hexapole collision cell. Ceramides and glycosphingolipids
were detected in the positive ion mode, and phospholipid-derived fatty
acids as well as the sulfated sterols were detected in the negative ion
mode. The spray was induced with a capillary voltage of ±0.8-1.2 kV.
The tandem mass spectrometric precursor scans were performed with argon
as a collision gas at a nominal pressure of 2 millitorrs. Spectra were
obtained by averaging 30-100 repetitive scans of 4 s.
Assaying Membrane Proteins for
GPI2 Linkage--
Floating,
detergent-insoluble membranes were washed once in PIPES (pH 6.8), 0.5 mM CaCl2, and 150 mM NaCl and
resuspended in 300 µl of 25 mM Tris-HCl (pH 7.5) + 0.5 mM CaCl2 + 0.001 volume of CLAP. Half of each
sample was treated with 0.75 units of phosphatidylinositol-specific phospholipase C (Sigma) for 3 h at 30 °C. The other half was
mock-treated for the same length of time under the same conditions.
Samples were then subjected to Triton X-114 phase separation (16), and proteins in the pellet, detergent, and aqueous fractions were precipitated by adding 4 volumes of methanol + 1 volume of chloroform, mixing, adding 3 volumes of H2O, spinning for 2 min at
14,000 rpm, removing the upper phase, adding 3 of volumes methanol,
spinning as before, and removing the supernatant from the protein
pellet. Proteins were then analyzed by two-dimensional gel
electrophoresis (8), and gels were stained with a
SilverXpressTM kit (Novex).
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RESULTS |
Drosophila Lipids Are Shorter than Mammalian Lipids, but Retain the
Structural Properties Required for Raft Formation--
To identify the
lipids present in Drosophila embryonic membranes, we used
TLC analysis with standards (data not shown) and mass spectrometry
(Fig. 1A). These data are
summarized in Fig. 1 (B-D). We found that both the
sphingolipids and phospholipids were shorter than those found in
mammals. The sphingolipids were based on a tetradeca-4-sphingenine
backbone (C14) rather than the C18 backbone
found in mammals. The amide-linked fatty acids present in the
sphingolipids were also shorter; the most abundant fatty acid was
arachidic acid (C20:0) (Fig. 1, A and
B), shorter than the lignoceric acid (C24:0)
often found in mammalian sphingolipids. Consistent with this, the free
ceramide precursors of the sphingolipids contained the same fatty acids
(Fig. 1, A, B, and D). The fatty acids
in Drosophila sphingolipids were completely saturated (Fig. 1, B and D). The most abundant glycosphingolipids
were glucosylceramide and mannosylglucosylceramide. The only
phosphosphingolipid in mammalian membranes, sphingomyelin, was not
present in Drosophila; instead, like other insects (17),
Drosophila membranes contained phosphoethanolamine ceramide
(PECer).

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Fig. 1.
Structure of Drosophila
sphingolipids and phospholipids. A, sphingolipid
classes in a total sphingolipid extract from Drosophila
embryos were detected by positive mode electrospray ionization tandem
mass spectrometry. [M + H]+ ions were detected by
precursor scanning for m/z 208 with a collision
offset of 40 eV. Fragment 208 is a typical derivative of the
hexadeca-4-sphingenine backbone. The peaks at 779 and 808 m/z are unidentified non-ceramide-related
compounds, based on daughter ion analysis (data not shown).
B, shown is the assignment of peaks shown in A.
C, fatty acid length and degree of saturation of the major
phospholipids were analyzed qualitatively by identification of intact
molecular ions and collision-induced daughter ions (data not shown).
D, quantitative analysis of fatty acid composition of the
free ceramides was based on relative peak intensities in the mass
spectra of intact ceramide molecular ions.
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Drosophila membranes contained phosphoglycerolipids with the
same head groups as those of mammalian cells (phosphocholine, phosphoethanolamine, phosphoserine, and phosphoinositol); however, they
differed in having shorter fatty acyl chains. Whereas the longest fatty
acid found in Drosophila phosphoglycerolipids was C18 (Fig. 1C), mammalian phosphoglycerolipids
contain up to C24 (18, 19). Approximately 60% of the fatty
acids were unsaturated (Fig. 1C), similar to ratios found in
several mammalian cells (19).
In summary, both the sphingolipids and phosphoglycerolipids are shorter
than those found in mammals and would therefore be predicted to have
lower melting temperatures, consistent with the requirement that
Drosophila membranes remain fluid at lower temperatures.
Despite these differences, sphingolipids are still longer and more
saturated than phosphoglycerolipids, as they are in mammals, and would
therefore be predicted to have higher melting temperatures than
phosphoglycerolipids. Thus, the structural properties of
Drosophila sphingolipids and phosphoglycerolipids are
consistent with the ability to separate into liquid ordered and
disordered phases.
Ergosterol Is the Predominant Membrane Sterol in Drosophila Fed
on Yeast--
In model membranes, cholesterol must be present at
between 7 and 30 mol % to induce the formation of the liquid ordered
phase (3, 4). Consistent with this, cholesterol is essential for raft
formation in vertebrate cells (20, 21). To determine the levels and
types of sterols in Drosophila membranes, we performed mass
spectrometry on lipid extracts and quantified sterols after sulfation
in the presence of an internal 13C-labeled
standard.1 The relative percentages of the different types
of sterols observed are shown in Table I.
Ergosterol is the most abundant sterol (69% of the total). Ergosterol,
along with campesterol and sitosterol, could be absorbed directly from
the diet in population cages (wet yeast on apple juice/beet syrup/agar
plates). 24-Methylenecholesterol and fucosterol are intermediates in
the conversion of campesterol and sitosterol, respectively, to
cholesterol. Although the position of the double bond in
dehydrocholesterol cannot be determined from this analysis, this sterol
may be
7-dehydrocholesterol, an intermediate in the conversion of
ergosterol to cholesterol (22). Cholesterol itself represents only 3%
of the total membrane sterol. Taken together, sterols represent 18 mol
% relative to phospholipids, similar to levels of cholesterol found in
vertebrates. Because ergosterol, campesterol, and sitosterol are even
more efficient than cholesterol at ordering acyl chains (23, 24), these
levels should be sufficient to induce formation of the liquid ordered
phase.
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Table I
Sterol composition of Drosophila embryonic membranes
The sterols in Drosophila embryonic membranes were
identified and quantified after sulfation in the presence of 200 pmol
of [13C2]cholesterol. Sulfated sterols were
detected by negative mode electrospray ionization tandem mass
spectrometry precursor scanning for m/z 97 with a
collision offset of +60 eV. The values were corrected for differences
in sulfation and ionization efficiencies with respect to the standard
by measuring known amounts of pure sterol in the presence of
[13C2]cholesterol standard (data not shown).
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The Lipid Composition of Drosophila Detergent-insoluble Membranes
Resembles That of Mammalian Rafts--
Mammalian raft membranes, rich
in cholesterol and sphingolipids, can be isolated on the basis of their
insolubility in the detergents Triton X-100 and Triton X-114 at low
temperature (7, 8). To determine whether similar membrane domains form
in Drosophila, we isolated detergent-insoluble membranes and
analyzed their lipid composition. Drosophila embryonic
membranes were treated with 1% Triton X-114 or Triton X-100 for 30 min
at 0 °C, and insoluble membranes were purified from solubilized
material by their ability to float through a density gradient. We
separated lipids derived from the insoluble fraction or from membranes
that had not been solubilized by TLC and stained them with iodine vapor
(Fig. 2A). This showed that
sterols were clearly enriched relative to the phospholipids (PI, PC,
and PE) in detergent-insoluble membranes. The mole percent sterol
relative to phospholipids in the Triton X-114-insoluble membranes
(30.5%) is similar to what has been reported for cholesterol in
mammalian rafts (31%) (7). The relative enrichment over the amount in
unsolubilized membranes was less than that observed when solubilization
is performed on whole Madin-Darby canine kidney cells, however. We
began with a membrane fraction that was already enriched in plasma
membrane with respect to the endoplasmic reticulum and mitochondria
(see "Experimental Procedures"). TLC analysis showed that this
fraction already contains proportionally more sterol than the total
membrane fraction (data not shown) and would be expected to contain
more sphingolipid as well (25, 26). Therefore, the final levels of
enrichment of both sterol and sphingolipid should be lower than if
rafts were isolated from whole cells.

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Fig. 2.
Sterol, phosphoethanolamine ceramide, and
glycolipids are enriched in insoluble membranes. A,
membranes were solubilized with the detergents indicated at either 0 or
29 °C, and insoluble membranes were collected by flotation through a
density gradient. Lipid extracts from insoluble membranes or from half
of the corresponding amount of starting material were separated on TLC
plates and stained with iodine vapor. The positions of PE, PECer
(PEC), PC, PI, and sterols (S) are indicated. The
identities of the lipids were determined by running cholesterol,
sitosterol, campesterol, ergosterol, PE, PI, and PC standards. All
sterols migrated indistinguishably from each other. PECer was
identified by its reactivity with ninhydrin and resistance to mild base
cleavage (data not shown). B, the mole percent of PECer was
calculated by determining the moles of phosphate in the PECer, PC, and
PE spots in A and expressing PECer as a percent of the
total. The mole percent sterol relative to phospholipid in
unsolubilized membranes was determined by quantifying the amount of
each sterol by mass spectrometry, as shown in Fig. 2, and comparing the
sum to the total moles of phospholipid. The mole percent sterol in
Triton X (TX)-114-insoluble membranes was determined
enzymatically using lipids from unsolubilized membranes as a standard.
C, lipids from Triton X-114-insoluble membranes or
unsolubilized membranes were analyzed by TLC and stained with orcinol.
Triton X-114-insoluble membranes had 69 and 66% of the
glucosylceramide (G) and mannosylglucosylceramide
(MG), respectively (determined by scanning band
intensities), but 24% of the phospholipid (determined by quantifying
moles of phosphate) present in unsolubilized membranes. Embr.
membr., embryonic membranes; enrichm.,
enrichment.
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PECer, like its mammalian counterpart, sphingomyelin, also appeared to
be enriched in insoluble membranes (Fig. 2A). To determine the mole percent of PECer relative to other phospholipids, we determined the number of moles of PECer, PE, and PC present in the TLC
spots shown in Fig. 2A. PECer was present at 14.7 mol % in
insoluble membranes, comparable to the level of sphingomyelin found in
mammalian rafts (14.2%) (7). PECer was 2.8-fold more abundant in the
Triton X-114-insoluble membranes than in unsolubilized material (Fig.
2B).
Next, we investigated whether glycosphingolipids were enriched in the
insoluble fraction. We performed TLC on mild base-resistant lipids
prepared from either total membranes or Triton X-114-insoluble membranes and stained them with orcinol (Fig. 2C). The
intensity of the glucosylceramide and mannosylglucosylceramide bands
before and after solubilization was quantified, and the percent that remained insoluble was compared with the percent of phospholipid that
remained insoluble. By this estimate, these glycosphingolipids are
enriched by ~3-fold relative to phospholipids in detergent-insoluble membranes. The orcinol-stained bands migrating closer to the origin represent more complex glycolipids (27), which have not been further
identified by mass spectrometry. These glycolipids also appear to be
enriched in the detergent-insoluble fraction. These experiments show
that detergent solubilization can be used to isolate a
Drosophila membrane fraction that is rich in sterol and
sphingolipids, like mammalian rafts.
Drosophila Detergent-insoluble Membranes Are Rich in GPI-linked
Proteins--
In mammals, linkage to GPI targets proteins to raft
membranes, and GPI-linked proteins are an abundant component of
mammalian rafts (7, 28). To determine whether the Drosophila
detergent-insoluble membrane fraction was similar to mammalian raft
membranes in this regard, we analyzed its content of GPI-linked
proteins. The Triton X-100 detergent-insoluble membrane fraction was
isolated by solubilization in 1% Triton X-100 at 0 °C and flotation
through a density gradient. These membranes were treated with
PI-specific phospholipase C to release the lipid moiety from GPI-linked
proteins. As a control, Triton X-100-insoluble membranes were
mock-treated under the same conditions in the absence of PI-specific
phospholipase C. Membranes were then dissolved in 1% Triton X-114
first at 4 °C and then at 29 °C. At 29 °C, solutions of Triton
X-114 separate into detergent and aqueous phases; lipid-linked proteins
and transmembrane proteins partition into the detergent phase, and
other proteins partition into the aqueous phase (16). We analyzed the
proteins present in the detergent and aqueous phases, as well as those
that remained insoluble in Triton X-114 at 29 °C, by two-dimensional
gel electrophoresis. Treatment of Triton X-100-insoluble membranes with
PI-specific phospholipase C caused most major protein constituents to
shift from the detergent to the aqueous phase of Triton X-114,
indicating that a lipid anchor had been removed (Fig.
3, compare A and B with C and D). Because GPI-linked proteins are so
abundant in Drosophila rafts, we conclude that, as in
mammals, GPI linkage constitutes a specific targeting signal for these
membrane domains in Drosophila.

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Fig. 3.
GPI-linked proteins are abundant in
detergent-insoluble membranes. Triton X-100-insoluble membranes
were either treated with PI-specific phospholipase C (PIPLC;
A, C, and E) or mock-treated
(B, D, and F) and then subjected to
phase separation in Triton X-114 and two-dimensional gel analysis.
A and B, proteins in the aqueous phase;
C and D, proteins in the detergent phase;
E and F, the insoluble pellet. Arrows
indicate proteins that shift from the detergent to aqueous phase after
PI-specific phospholipase C treatment.
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We were surprised at how few raft proteins were left in the Triton
X-114 detergent phase (and by implication, associated with raft
membranes) after PI-specific phospholipase C treatment. Taken at face
value, this would indicate that almost all of the non-peripherally associated membrane proteins in Drosophila rafts were either
GPI-linked or linked to insoluble cortex. It has been reported that a
fraction of raft membrane lipid remains insoluble in Triton X-100 at
temperatures between 13 and 37 °C (28). This led us to wonder
whether some raft membranes might have remained insoluble in Triton
X-114 even at the higher temperature required for phase separation of
the detergent and thus constitute part of the "pellet" fractions
shown in Fig. 3 (E and F). To address this
possibility, we subjected membranes solubilized in Triton X-114 at the
higher temperature to flotation through a density gradient. Floating
insoluble membranes were observed just as when solubilization was
performed at 0 °C. When we examined their lipid composition by TLC,
we found that it was similar to that of raft membranes isolated at
0 °C (Fig. 2). This shows that a subfraction of raft membranes are
resistant to Triton X-114 solubilization even at 29 °C. The protein
and lipid composition of Triton X-100- and Triton X-114-insoluble membranes indicates that these detergents can be used to isolate Drosophila membranes that resemble mammalian rafts.
The Hedgehog N-terminal Fragment Associates Specifically with
Detergent-insoluble Membranes--
To determine whether the Hedgehog
N-terminal fragment associated with Drosophila rafts, we
solubilized embryonic membranes in 1% Triton X-114 at 0 °C for 30 min, floated the material though a density gradient, and investigated
whether Hedgehog was found in the floating insoluble membranes. Western
blotting of gradient fractions showed that a significant amount of the
N-terminal fragment of Hedgehog remained associated with insoluble
membranes (Fig. 4A). Although
no lipid modification has yet been found on the Hedgehog C-terminal
fragment, it copurifies with embryonic membranes, and it partitions
almost exclusively into the detergent phase of Triton X-114 when the
temperature is raised to 29 °C, suggesting that it is significantly
hydrophobic (data not shown). Nevertheless, unlike the N-terminal
fragment, it is completely solubilized by Triton X-114 at 0 °C (Fig.
4A).

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Fig. 4.
Hedgehog and fasciclin-1 associate
specifically with detergent-insoluble membranes.
Drosophila embryonic membranes were solubilized with 1%
Triton X-114 and then floated through a density gradient. Western blots
of identical gradient fractions are shown probed with antibodies to
detect Hedgehog N- and C-terminal fragments (A);
fasciclin-1, a GPI-linked protein (B); Drac1, an
isoprenylated protein (C); or Notch, a transmembrane protein
(D). We detected very low levels of Notch in insoluble
membranes after longer exposures, but not comparable to the level of
Hedgehog N-terminal fragment.
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To show that insoluble Hedgehog was found in the same fractions as
raft-associated GPI-linked proteins, we probed the gradient fractions
with an antibody to fasciclin-1 (41). We found this GPI-linked protein
in the same low density gradient fractions as Hedgehog (Fig.
4B). This indicates that Hedgehog associates with raft membranes.
To demonstrate that the conditions we used were sufficiently stringent
to solubilize other membrane proteins, the same gradient fractions were
probed with antibodies to Drac1 (42) and Notch (43). Drac1 attaches to
membranes via an isoprenyl group. Drac1 did not float out of the loaded
volume of the gradient, indicating that it is completely solubilized by
this treatment (Fig. 4C). Furthermore, the transmembrane
protein Notch also remained predominantly in the fractions containing
solubilized material (Fig. 4D). These results show that the
association of Hedgehog with insoluble membranes is specific and takes
place under conditions that solubilize other membrane proteins.
The proportion of Hedgehog present in detergent-insoluble membranes,
although significant compared with that of Drac1 and Notch, does not
appear to be as great as that of fasciclin-1. To determine whether this
might be due to the existence of a non-sterol-linked population of
Hedgehog molecules, we examined the sterol linkage of Hedgehog in
embryonic membranes. Removing covalently linked cholesterol by base
treatment causes Hedgehog to migrate more slowly on polyacrylamide gels
(29). Base treatment of embryonic membranes caused all of the Hedgehog
N-terminal fragment to shift to a lower mobility, indicating that all
the Hedgehog in embryonic membranes is linked to sterol (data not
shown). Failure of a fraction of Hedgehog molecules to associate with
rafts cannot therefore be due to lack of sterol linkage (see
"Discussion").
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DISCUSSION |
Our data show that Drosophila membranes contain raft
lipid microdomains that resemble their mammalian counterparts in both lipid and protein composition. They further demonstrate that the cholesterol-linked Hedgehog N-terminal fragment and GPI-linked fasciclin-1 associate specifically with Drosophila rafts.
Analysis of the lipids present in Drosophila membranes shows
that, despite differences in chemical structure between mammalian and
Drosophila lipids, the properties of sterols, sphingolipids, and phosphoglycerolipids that allow formation of the liquid ordered (raft) phase have been preserved. One factor that allows the formation of laterally separated liquid ordered and disordered phases is the
different melting temperatures of sphingolipids versus
phosphoglycerolipids. Mammalian sphingolipids have a higher melting
temperature than phosphoglycerolipids in part because their fatty acyl
chains are longer and more saturated. We find that the fatty
acids present in Drosophila sphingolipids are longer and
more saturated than those in their phosphoglycerolipids, even though
both sphingolipids and phosphoglycerolipids are shorter in
Drosophila than in mammals (consistent with
Drosophila membranes remaining fluid at lower temperatures).
Thus, as in mammals, these lipids would be predicted to have
different melting temperatures.
Despite being unable to synthesize sterols, Drosophila
accumulate 18 mol % sterol in their membranes, relative to
phospholipids, a level comparable to that of cholesterol in mammalian
membranes. Dietary sterol appears to be incorporated largely without
modification (ergosterol, campesterol, and sitosterol together account
for 79% of the total membrane sterol). Nevertheless, small amounts of
cholesterol (along with intermediates in the conversion of the dietary
sterols to cholesterol) are observed. It will be interesting to
determine whether these small amounts of cholesterol are necessary for
modification of Hedgehog, or whether Hedgehog is modified by other
sterols in vivo, as can apparently occur in vitro
(30).
The concept of raft association as an important mediator of protein
localization has been developed based on experiments in mammalian
tissue culture cells. We have shown that similar membrane domains rich
in sterol and sphingolipid exist in D. melanogaster, an
organism at some evolutionary distance from mammals, with different lipid classes and species. Detergent-insoluble membranes enriched in
proteins similar to those found in mammalian rafts have also been
recovered from Dictyostelium (31) and Saccharomyces
cerevisiae (32). The preservation of the biophysical lipid
properties allowing raft formation in such widely separate phyla
suggests that these phase domains perform important cellular functions.
The functional importance of sterol in fly membranes has been
questioned because fly tissue culture cells can be maintained in media
depleted of sterols (33) and because adults survive when fed a
sterol-free diet (although the efficiency of sterol depletion in adults
is unknown) (34). Nevertheless, when Drosophila feed on an
ergosterol-deficient strain of yeast, they produce embryos that fail to
develop (34). This indicates that sterols are essential for the more
complex cellular functions involved in embryonic development.
Our data show that the sterol-linked N-terminal fragment of the
Hedgehog protein associates with raft membranes, suggesting that sterol
linkage may be a raft-targeting signal. Cholesterol prefers to
associate with sphingolipids rather than phospholipids due in part to
the higher degree of saturation of the fatty acids in sphingolipids,
which allows for a more favorable packing of the planar rings of
cholesterol (35, 36). Cholesterol that is covalently linked to Hedgehog
should retain its ability to associate with sphingolipids in this
manner; this property would be predicted to confer affinity for the
sphingolipid-rich raft membranes.
Although a significant amount of Hedgehog is present in
detergent-insoluble membranes, it does not resist solubilization as efficiently as GPI-linked fasciclin-1 (Fig. 4). This indicates that a
subpopulation of Hedgehog may not associate with rafts, although all
Hedgehog in our membrane preparation is sterol-linked. Recent evidence
indicated that a variable proportion of the sonic Hedgehog N-terminal
fragment is also covalently linked to palmitic acid (37). Double
acylation is known to be a raft-targeting signal for Src-related
kinases, whereas modification by a single palmitoyl group is
insufficient (38). If Drosophila Hedgehog is also
palmitoylated, it will be interesting to determine whether sterol or
palmitate or both are required to direct Hedgehog to raft membranes.
A question of converging interest for cell and developmental biologists
is how proteins that pattern developing tissues move within and between
cells, and how the regulation of this traffic contributes to the
activity and distribution of morphogens. The limited spread of Hedgehog
protein from a spatially restricted subset of producing cells is
critical for patterning a variety of tissues. Covalent modification of
Hedgehog by cholesterol has been postulated to influence the activity
of the protein by conferring a general membrane affinity and thereby
limiting its diffusion (29). Our data raise the possibility that sterol
modification may play a more specific role by targeting Hedgehog to
raft membranes. Rafts have been shown to play important roles in axonal
trafficking of proteins in neurons and apical trafficking in epithelia
(1). The Hedgehog protein moves axonally in photoreceptor neurons (39), and recent evidence suggests that epithelial cells actively transport Hedgehog protein through the plane of the epithelium (40); it will be
interesting to determine whether proper cellular trafficking of
Hedgehog depends on its association with raft membranes.
Rafts are also thought to play roles in signal transduction. Many G
protein-coupled receptors, upon binding to their ligands, acquire
affinity for raft membranes where mediators of signal transduction such
as G proteins and adenylate cyclase are concentrated (15). Hedgehog
signals through Smoothened, a putative G protein-coupled receptor.
Although Hedgehog that is not modified by cholesterol is capable of
signaling (29), modification of Hedgehog by cholesterol increases its
potency in signal transduction by 30-fold (37). This raises the
possibility that the efficiency of Hedgehog signaling might depend on
its localization to raft membranes. The identification of rafts in a
genetic model organism and the finding that Hedgehog associates with
them will foster new approaches to understanding raft membrane
microdomains and the role of polarized protein trafficking in development.