From the
Department of Medical Biochemistry and
the Wallenberg Laboratory for Cardiovascular Research, the
Swegene Proteomic Center, and the
¶Department of Anatomy and Cell Biology,
Göteborg University, SE-413 45 Göteborg, Sweden
Received for publication, February 10, 2003 , and in revised form, April 29, 2003.
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ABSTRACT |
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INTRODUCTION |
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ADRP and perilipins are abundant on lipid droplets. ADRP is found mostly on smaller droplets; when overexpressed, it stimulates lipid droplet formation (7), suggesting a role in the assembly process. Perilipins are present on large lipid droplets (7) and have a central role in the turnover of TG within these structures (1, 8, 10), perhaps affording protection against hormone-sensitive lipase (11). Perilipin-null mice have smaller lipid droplets and a higher rate of basal lipolysis than wild-type mice and are resistant to diet-induced obesity (12).
Caveolin is a 21-kDa membrane protein with a hairpin structure whose N and C termini face the cytosol (13, 14). In mammals, there are three caveolin genes. Caveolin 1 and 2 are expressed in adipocytes, whereas caveolin 3 is present in muscle. Caveolin plays a key role in intracellular lipid transport (15, 16), binding fatty acid (17), and transporting cholesterol (16) in a manner reminiscent of the way plasma apolipoproteins transport lipids in the blood (16, 18).
Phospholipase D (PLD) catalyzes the conversion of phosphatidylcholine to phosphatidic acid (PA) and appears to be important in intracellular transport and sorting processes (1922), either as an intracellular messenger or as a cone-shaped lipid that alters the curvature of the membrane (2325). The formation of PA is important for the fission of transport vesicles (26, 27). Two mammalian isoforms of phosphatidylcholine-specific PLD (PLD1 and PLD2) have been identified and extensively investigated (2830). Although they exhibit about 50% identity, the two enzymes have different biochemical properties and depend on different cofactors (2831). Our studies (32) suggest that PLD is important for the second step in the assembly of very low density lipoproteins (reviewed in Refs. 33 and 34), which appears to involve the formation of a lipid droplet in the microsomal lumen (35).
Cell-free systems are extremely useful for studies of sorting processes, such as the budding of transport vesicles (reviewed in Refs. 36 and 37) and are the only method for determining the mechanism by which such complex structures are formed. We developed a microsome-based, cell-free system to study the transport of TG from microsomes to the cytosol. This system assembles TG-containing lipid droplets similar to the small lipid droplets isolated from 3T3-L1 cells. Here we report that caveolin, ADRP, vimentin, and the 78-kDa glucose-regulated protein (GRP-78) are present on these lipid droplets and that the release of the droplets from microsomes is dependent on PLD and the formation of PA.
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EXPERIMENTAL PROCEDURES |
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AntibodiesRabbit immunoglobulin was purchased from Dako (Glostrup, Denmark). Caveolin antibodies were from Transduction Laboratories (Lexington, KY) and Affinity Bioreagents (Golden, CO). Adipophilin (ADRP) antibodies and antibodies to perilipins were obtained from Research Diagnostics (Flanders, NJ). Antibodies to Na/K-ATPase and vimentin were from Abcam (Cambridge, UK). Antibodies to GRP-78 were from Affinity Bioreagents (Golden, CO). Golgin antibodies were from Molecular Probes (Leiden, Netherlands). Gold-conjugated anti-rabbit antibodies were from British Biocell International (Cardiff, UK).
Isolation of Microsomes from 3T3 Cells3T3-L1 cells were cultured and differentiated for 2 days (unless stated otherwise) as described (38). The cells (2 x 106 cells/incubation) were homogenized (30 strokes in a Dounce glass homogenizer in 10 mM Tris-HCl, pH 7.5, containing 250 mM sucrose, 1 mM EDTA, 100 KIU/ml Trasylol, 0.1 mM leupeptin, 0.1 µM pepstatin, and 1 mM phenylmethylsulfonyl fluoride), and the microsomes were isolated essentially as described (39). To remove loosely bound (nonintegral) proteins from the microsomes, the pellet was resuspended in 500 mM Tris-HCl, pH 7.5, containing 1.2 M potassium chloride (high salt wash; modified from Ref. 40) and incubated for 30 min at 4 °C with head-over-tail mixing. The microsomes were then recovered by ultracentrifugation at 160,000 x g for 70 min at 12 °C. The washed microsomes were resuspended in 10 mM Tris-HCl, pH 7.5, containing 250 mM sucrose.
The Cell-free SystemThe in vitro system used to investigate the formation of lipid droplets was based on microsomes that had been subjected to a high salt wash, an activator from the 160,000 x g supernatant from homogenized rat adipocytes, and a substrate for DGAT.
To prepare the DGAT substrate, 0.25 mg of diacylglycerol was dissolved in 40 µl of 10 mM Tris-HCl, pH 7.5, with 1.0 mM palmitoyl-CoA by gentle vortexing; 1.25 µl of [14C]palmitoyl-CoA (55 mCi/mmol; total 0.06 µCi) and 360 µl of 10 mM Tris-HCl, pH 7.5, were added, and the mixture was vortexed again. In some experiments the [14C]palmitoyl-CoA was replaced with diacylglycerol (1-stearoyl, 2-[1-14C]arachidonyl-sn-glycerol).
To start the reaction, 600 µl of the partially purified cytosolic activator in 10 mM Tris-HCl, pH 7.5, with 250 mM sucrose, 150 µl of 1.4 M MgCl2 (in the same buffer), and 50 µl of the microsome solution (0.51 mg of microsomes) was added to the DGAT substrate. Incubation was carried out at 37 °C for 60 min (the production of lipid droplets plateaued between 30 and 60 min).
After the incubation, the total production of radioactive triglycerides was measured, and the incubation mixture was subjected to gradient ultracentrifugation as described below. Before centrifugation, the mixture was supplemented with (final concentrations) Trasylol (100 KIU/ml), leupeptin (0.1 mM), phenylmethylsulfonyl fluoride (1 mM), pepstatin A (1 µM), N-acetyl-Leu-Leu-norleucinal (5 µM), and EDTA (0.5 mM).
Partial Purification of the Cytosolic ActivatorTo form lipid droplets, the cell-free system needed an activator present in the cytosol of rat adipocytes. Adipocytes were prepared essentially as described (41), suspended in 10 mM Tris-HCl, pH 7.4, with 250 mM sucrose, homogenized by 15 strokes in a Dounce homogenizer, and centrifuged at 160,000 x g for 2 h at 4 °C. The centrifuge tube was punctured 1 cm from the bottom, and the supernatant was aspirated, carefully avoiding contamination from the fat cake and the pellet. The supernatant was concentrated (final volume, 0.5 ml) and subjected to gel chromatography on a Superdex 200 column (HR 10/30; Amersham Biosciences) equilibrated with 10 mM Tris-HCl, pH 7.4, containing 250 mM sucrose, using an Äkta prime chromatography system (Amersham Biosciences). The flow rate was 0.3 ml/min, and the fraction size was 0.5 ml. The optical density (280 nm) was recorded, and the eluted peaks were assayed for their ability to induce lipid droplet formation. This activity eluted from the column with an apparent molecular mass of 180 kDa.
This 180-kDa fraction was concentrated (final volume, 2 ml) and subjected to anion-exchange chromatography with a Q-resource column (and the Äkta prime system) equilibrated with 10 mM Tris-HCl, pH 8.0. The column was eluted first with 18 ml of the same buffer and then with a 00.5 M NaCl gradient (in 10 mM Tris-HCl, pH 8.0; total volume 37 ml). Optical density (280 nm) and conductivity were recorded, and all fractions were analyzed for their ability to induce lipid droplet formation. Fractions containing this activity (eluted between 0.17 and 0.23 M NaCl) were combined, desalted, and concentrated. They are referred to as the Q-fraction.
The Q-fraction was adjusted to pH 7.0 and chromatographed on an S-resource column using the Äkta prime system. The column was equilibrated with 10 mM Tris-HCl, pH 7.0. The elution was started with 30 ml of the same buffer, followed by a 00.5 M NaCl gradient (in 10 mM Tris-HCl, pH 7.0; total volume 20 ml). Optical density and conductivity were recorded, and each fraction (2 ml) was analyzed for its ability to induce lipid droplet formation. The activity was eluted between 0.1 and 0.5 M NaCl. This fraction is referred to as the partially purified cytosolic activator or S-fraction.
Gradient UltracentrifugationFor gradient I the samples were
adjusted to 25% sucrose, and 1 ml was layered under 2 ml of 50 mM
Tris-HCl, pH 7.5, with 10% sucrose and 10 mM EDTA, which in turn
was overlaid with 2 ml of 50 mM Tris-HCl, pH 7.5, and 10
mM EDTA. Centrifugation was carried out at 160,000 x
g for 17 h at 4 °C in a Beckman SW55 Ti rotor. Homogenates of
3T3-L1 cells (induced for 10 days) were used to establish the gradient. The
gradient was divided into four fractions with mean densities of 1.018 (2
ml), 1.034 (1 ml), 1.055 (1 ml), and 1.098 (1 ml) g/ml, respectively. The
major amount of TG (62.6 ± 9.7%) was recovered in the d
1.018 g/ml fraction; 17.7 ± 3.0% was recovered in the 1.034 g/ml
fraction; 8.3 ± 2.0% was recovered in the 1.055 g/ml fraction, and 9.7
± 9.2% was recovered in the 1.098 g/ml fraction. The pellet (microsomes
and cell debris) contained only 2.7 ± 0.5% of the total amount of TG
recovered from the gradient (all data are mean ± S.D.; n = 3).
Caveolin was present in all fractions and in the pellet.
For gradient II the samples were adjusted to 40% sucrose, and 1.2 ml was
layered under 3 ml of 50 mM Tris-HCl, pH 7.5, with 25% sucrose and
10 mM EDTA, which was in turn overlaid with 1 ml of 50
mM Tris-HCl, pH 7.5, and 10 mM EDTA. Centrifugation was
carried out at 160,000 x g for 17 h at 4 °C in a Beckman
SW55 Ti rotor. The gradient was arbitrarily divided into five fractions with
mean densities of 1.055 (1.2 ml), 1.099 (1 ml), 1.112 (1 ml), 1.141 (1 ml),
and 1.161 (1 ml) g/ml, respectively. Most (75%) of the TG was present in the
top fraction (d 1.055 g/ml). Caveolin was present in the top
fraction and in the two bottom fractions (mean densities, 1.141 and 1.161
g/ml, respectively).
Lipid AnalysisLipids were extracted as described
(42), with modifications
(43). The lipid extract was
separated by TLC using a two-solvent system:
chloroform/methanol/H2O (60:30:5 by volume) followed by petroleum
ether/diethyl ether/acetic acid (80:20:1.5 by volume). Lipids were visualized
by iodine vapor and identified by standards run in parallel. Radioactive TG
was separated, counted as described
(43), and expressed as a
percentage of the total amount of radioactive TG recovered in the d
1.055 g/ml fraction.
The PLD assay was performed as described (44), except that labeled 1-butanol was used. The labeled phosphatidylbutanol formed was isolated by TLC with ethyl acetate/trimethyl pentane/acetic acid (8:5:2 by volume) as the solvent. Using the partially purified cytosolic activator, we observed a substrate-dependent reaction velocity with a Vmax at 420 mM 1-butanol (not shown). We therefore carried out the assay at that concentration.
Other MethodsSamples for electron microscopy were pelleted and fixed in 2.5% glutaraldehyde in 0.05 M sodium cacodylate, pH 7.2. The fixed sample was postfixed in 1% osmium tetroxide in 0.1 M sodium cacodylate and stained with 0.5 M uranyl acetate. Immunoelectron microscopy was performed with a Zeiss transmission electron microscope (model 902A) as described (45).
Treatment with 1% Triton X-100 was carried out essentially as described
(46). The Triton-treated
d 1.055 g/ml fraction from the cell-free system was centrifuged
(46) in a Beckman TLS-55 rotor
at 55,000 rpm (258,000 x g) at 4 °C for 13 h. Seven
fractions (300 µl each) were recovered from the top and analyzed for
caveolin. The Triton-treated d
1.018 fraction from the
homogenized 3T3-L1 cells was centrifuged
(46) at 35,000 rpm in a
Beckman SW40 rotor at 4 °C for 12 h. Fractions of 2.4 ml were collected
and analyzed for caveolin.
Immunoprecipitation was performed with the µMacs system essentially as recommended by the manufacturer, except that the beads were recovered by centrifugation for 15 min at 16,000 x g after the incubation with the antibody-reacted sample. The bound material was eluted with hot sample buffer. To analyze the phospholipids present on the immunoaffinity-purified lipid droplets, the beads were removed from the column and extracted with chloroform.
Protein was quantified with the BCA kit (Pierce). SDS-PAGE and immunoblotting were carried out as described (43, 47). For analysis by mass spectrometry, the gels were silver-stained as described (48). Protein bands were cut out, treated with trypsin, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) as described (47), except that Mascot software (available at www.matrixscience.com) was used for protein assignment. If the spectra were not good enough, the samples were cleaned up and concentrated with a ZipTip containing C18 material (Millipore) before the MS run. When the MALDI-TOF analysis was inconclusive, tandem MS/MS was performed with the quadrupole time-of-flight technique as described (49), and proteins were identified with Mascot software.
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RESULTS |
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Upon incubation of the complete cell-free system (high salt-washed
microsomes, DGAT substrate, and partially purified activator), newly formed
(labeled) TG and caveolin were released from the microsomes and appeared in
the less dense fractions obtained by gradient ultracentrifugation
(Fig. 3A). Most of the
TG and the caveolin were in the three bottom fractions of the gradient;
relatively small amounts were present in the top fraction. We therefore
combined the radioactive TG in the d 1.055 g/ml fractions in all
calculations. The bottom fraction was excluded because its proximity to the
membrane pellet increased the risk of contamination with microsomes. In the
absence of the activator, TG and caveolin were not released
(Fig. 3B).
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Electron Microscopy of the d 1.055 g/ml
FractionThe d
1.055 g/ml fraction consisted of
rounded filled structures (Fig.
4A). Most were 100400 nm in diameter and were
similar in size and appearance to the lipid droplets recovered from the
d
1.018 g/ml fraction of homogenized 3T3 L1 cells and the
smaller lipid droplets isolated from rat adipocytes lysed with hypotonic
buffer (Fig. 4A).
Immunoelectron microscopy showed that caveolin was present on filled
structures of the same size as those isolated by gradient ultracentrifugation
(Fig. 4B). Control
experiments demonstrated that lipid droplets from the cell-free system did not
derive from the partially purified cytosolic activator.
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Protein Composition of the d 1.055 g/ml
FractionAs shown by immunoblotting, caveolin 1 and 2, but not 3,
were present on the lipid droplets (d
1.055 g/ml fraction)
isolated from the cell-free system (Fig.
5A). Virtually all of the caveolin was in the denser
fractions obtained by gradient ultracentrifugation after treatment with 1%
Triton (Fig. 5B),
indicating that the caveolin was detergent-soluble
(14). This was also the case
for the caveolin in lipid droplets from 3T3-L1 cells that had been induced for
10 days (Fig. 5C). The
lipid droplets from the cell-free system also contained ADRP
(Fig. 5A) but not
perilipins (Fig. 5A)
or Tip 47 (not shown).
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To analyze further the protein composition of the formed lipid droplets, we
incubated the cell-free system in the presence or absence of the partially
purified cytosolic activator and analyzed the d 1.055 g/ml
fraction by SDS-PAGE (Fig. 6).
In the presence of the activator (Fig.
6I), seven major silver-staining bands were present. In
its absence, no proteins were detected
(Fig. 6II). Incubation
of the purified activator with the DGAT substrate alone showed that none of
the silver-stained bands in the d
1.055 g/ml fraction derived
from the interaction between proteins in the activator and this substrate
(Fig. 6III).
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Bands 6 and 7 (Fig.
6I) reacted with antibodies to caveolin 1 and 2 on
immunoblot. Band 5 migrated on the gel with an estimated molecular mass of
50 kDa and reacted with ADRP antibody. Band 4 (
54 kDa) was cut out,
trypsinized, and analyzed by MALDI-TOF. Identified trypsin fragments (with no
or only one missed cleavage) were used to search the complete non-redundant
data base at NCBI. Vimentin (mouse) got a probability score (Mascot) of 104
(scores above 74 are significant at the 95% level). Band 3 (
70 kDa) was
identified by tandem MS/MS (quadrupole time-of-flight). In a search of the
NCBI data base, GRP-78 (mouse) got a score of 172. No spectra were obtained
from bands 1 and 2.
In summary, the lipid droplets formed in the cell-free system contained caveolin 1 and 2, ADRP, vimentin, and GRP-78. Immunoblot demonstrated that all these proteins were present on the high salt-washed microsomes but not in the partially purified cytosolic activator. MALDI-TOF analysis after SDS-PAGE confirmed that the activator did not contain caveolin, ADRP, vimentin, or GRP-78. Thus, the proteins on the lipid droplets generated in the cell-free system derived from the microsomes and not from the fraction containing the cytosolic activator.
Lipid Composition of the d 1.055 g/ml
FractionAs shown by TLC, the lipid droplets from 3T3-L1 cells
(Fig. 7A) and those
from the cell-free system (Fig.
7B) contained TG, diacylglycerol, phosphatidylcholine,
phosphatidylethanolamine, and a small amount of phosphatidylserine.
Diacylglycerol was relatively more abundant in the d
1.055 g/ml
fraction from the cell-free system than in lipid droplets from 3T3-L1 cells.
By using antibodies to ADRP, we were able to immunoprecipitate 6070% of
the d
1.055 g/ml fraction (measured as radioactive TG). TLC of
the bound fraction demonstrated TG, diacylglycerol, phosphatidylcholine,
phosphatidylethanolamine, and phosphatidylserine
(Fig. 7C).
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Lipid Droplet Formation Requires DGAT Substrates and TG
BiosynthesisIn the absence of diacylglycerol and palmitoyl-CoA, TG
biosynthesis in the cell-free system was virtually abolished, and no caveolin
accumulated in the d 1.055 g/ml fraction
(Fig. 8A). Removing
these DGAT substrates also abolished the accumulation of TG in the d
1.055 g/ml fraction, even when expressed as the proportion of the very
small amount of TG synthesized. Thus, the formation of lipid droplets from the
microsomes is dependent on the DGAT substrate and on the rate of TG
biosynthesis. To address this further, we removed either the cold
palmitoyl-CoA or the cold diacylglycerol from the DGAT substrate
(Fig. 8B). In the
absence of palmitoyl-CoA, TG formation in the cell-free system and the
accumulation of caveolin and TG in the lipid droplets (d
1.055
g/ml fraction) were significantly reduced. In the absence of diacylglycerol,
only a small amount of TG was formed, and no caveolin and virtually no TG
accumulated in the droplets.
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Lipid Droplet Formation in the Cell-free System Requires
PLD1-Butanol, but not 2-butanol, inhibited lipid droplet formation
in the cell-free system, as measured by the appearance of caveolin and TG in
the d 1.055 g/ml fraction
(Fig. 9A).
Because1-butanol acts as a nucleophile during PLD-catalyzed hydrolysis of
phosphatidylcholine, phosphatidylbutanol is formed instead of PA. 2-Butanol,
which is less nucleophilic, had no effect. Thus, the assembly of lipid
droplets in the cell-free system involves the formation of PA and PLD. Another
PLD inhibitor, 2,3-diphosphoglycerate, also inhibited droplet formation
(Fig. 9B).
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Next we investigated the effects of the cytosolic activator on PLD
activity. The partially purified activator increased both PLD activity and
lipid droplet formation in the cell-free system
(Table I). Moreover, droplet
formation and PLD activity increased after the partial purification
(Table I). If the cytosolic
activator activates a PLD involved in the formation of the lipid droplets, it
should be possible to replace the activator with constitutively active PLD or
PA. This was the case. After addition of active PLD or exogenous PA in the
absence of the activator, newly formed TG and caveolin accumulated in the
d 1.055 g/ml fraction (Fig.
10). Thus, PLD activity in the microsomes is essential for lipid
droplet formation in the cell-free system.
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DISCUSSION |
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The formation of lipid droplets required the DGAT substrate and ongoing biosynthesis of TG. However, the release of the lipid droplets was dependent on the formation of PA and on PLD activity. This conclusion is based on several observations. First, release of the droplets was inhibited by 1-butanol but not by 2-butanol, an indication that a PLD and the formation of PA are essential to the process (21, 32). Moreover, 2,3-diphosphoglycerate inhibited the release of the TG-containing structures. Second, lipid droplet formation required an activator present in the 160,000 x g supernatant of rat adipocytes. This activator also induced PLD activity in the microsomes of the cell-free system. Third, the activator could be replaced by a constitutively active PLD or by PA. PLDs are highly regulated by ADP-ribosylation factor 1 (44, 50, 51), protein kinase C (5153), and phosphatidylinositol 4,5-bisphosphate (5456), for example, and may participate in intracellular membrane "budding" processes (1922). However, the role of PLD in the formation of the lipid droplets within microsomes remains to be elucidated.
All proteins present on the lipid droplets formed in the cell-free system have been identified on lipid droplets in intact cell.
Caveolin participates in the transport of lipids, particularly cholesterol
(15), and binds fatty acids
(17). Its presence on
cytosolic lipid droplets
(35)
and its induction by hepatic overexpression of PPAR1, which causes
adipogenic steatosis (57),
suggest that caveolin is involved in TG storage. Consistent with this notion,
caveolin-1-deficient mice are lean and resistant to diet-induced obesity
(58). It has been proposed,
based on its intracellular sorting
(18,
59) and its lipid binding
capacity, that caveolin serves as a shuttle for intracellular lipids
(18). In support of this
model, our findings in this study that caveolin is released from microsomes
with lipid droplets suggest that caveolin may help sort newly formed TG from
microsomes into a cytosolic form. Our results also support the suggestion
(2,
6) that caveolin "buds
off" with lipid droplets from the microsomal membranes
(35).
Although vimentin is a cytosolic protein, the vimentin on the lipid droplets was derived from the microsomal membrane. Vimentin-type intermediate filaments interact with lipid bilayers (60), and this interaction is mediated by the highly positively charged N-terminal domain (61). Vimentin has also been shown to be associated with both ER (62) and the Golgi apparatus (6264). Vimentin forms a cage of intermediate filaments around cytosolic lipid droplets (65). In 3T3-L1 cells, expression of a dominant-negative vimentin reduces the formation of lipid droplets (66), suggesting that this protein is essential for their assembly. Our observations are compatible with such a conclusion. However, vimentin-deficient mice do not have any obvious phenotype (67). Thus, vimentin may promote lipid droplet formation but not be essential for this process. Alternatively, lipid droplets may be formed through more than one mechanism.
The ADRP on the lipid droplets was also derived from a membrane-associated pool, indicating that it can be targeted to the microsomal membranes. ADRP can be acylated (1, 68), and these fatty acids may take part in the interaction between ADRP and the membrane, because covalently linked fatty acids participate in the membrane targeting of other proteins (e.g. ADP-ribosylation factor 1) (6971). The cell-free system was based on microsomes from cells induced for 2 days. Such cells assemble mostly small lipid droplets, like those formed in the cell-free system. ADRP is mostly present on smaller lipid droplets and, when overexpressed, seems to stimulate lipid droplet formation (7). Together our observations and the results from the literature may suggest that ADRP binds to the microsomal membrane and participate in the budding of the small lipid droplets.
Perilipins were not expressed in the 3T3-L1 cells used to prepare the microsomes and were not present in the partially purified cytosolic activator. Therefore perilipins were not present on the lipid droplets assembled in the cell-free system. Thus, small lipid droplets can be assembled in the absence of perilipins. However, perilipins are dominant proteins on cytosolic lipid droplets (1, 2). Because these proteins interact with hormone-sensitive lipase (810), they may protect the droplets against lipolysis (11) and perhaps thereby promote the formation of the large droplets seen in the adipocytes.
GRP-78 is a chaperon protein that contains a KDEL sequence and is therefore restricted to the ER/Golgi region of the secretory pathway. Because GRP-78 is a major protein in the ER, its presence on the lipid droplets from the cell-free system may reflect leakage of the ER-derived microsomes. The released protein may adhere to the assembled particles. However, in one study, GRP-78 appeared to be present on the cytosolic side of the surface layer of lipid droplets (72), suggesting that the surface layer is derived from the membrane of the ER (72). This is in agreement with the model proposed for the assembly of lipid droplets (2, 9, 73). According to this model the triglycerides will during the biosynthesis "oil-out" between the leaflets of the bilayers, forming lens-shaped structures in the membrane. The lipid droplet, covered by a monolayer, will be formed by the budding of such a lens from the membrane. The structure will be stabilized by proteins bound to both the luminal and cytosolic surface of the membrane.
The lipid droplets that were assembled in the cell-free system differed
somewhat in density from those isolated from 3T3-L1 cells (1.0551.018
versus 1.018 g/ml, respectively). This may be due to a difference
in the load of TG, perhaps reflecting a difference in the efficiency of TG
biosynthesis in the two systems. If so, we would expect to find at least a
small amount of denser droplets in 3T3-L1 cells. Such droplets were found in
the 1.055 and 1.034 g/ml fractions isolated from the 3T3-L1 cells (not shown).
A difference in the triglyceride load of the droplets is supported by the
qualitative analysis which suggested that the lipid droplets in the d
1.018 g/ml fraction of 3T3-L1 cells have a higher TG:phospholipid ratio
than the d
1.055 g/ml fraction from the cell-free system.
Another possibility is that the lipid droplets formed in the cell-free system
had a higher diacylglycerol:TG ratio than the droplets isolated from the
cells; however, a portion this diacylglycerol could represent contamination
from the DGAT substrate.
In conclusion, we propose that lipid droplets are formed from regions of the microsomal membrane that contains caveolin, vimentin, and ADRP. The process is dependent on DGAT activity and is driven by the activity of a PLD and the formation of PA.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Wallenberg Laboratory, Sahlgrenska University Hospital/S, SE-413 45 Göteborg, Sweden. Tel.: 46-31-342 1956; Fax: 46-31-82 37 62; E-mail: Sven-Olof.Olofsson{at}medkem.gu.se.
1 The abbreviations used are: TG, triglyceride(s); ADRP, adipocyte
differentiation-related protein; DGAT, diacylglycerol acyltransferase; ER,
endoplasmic reticulum; GRP-78, 78-kDa glucose regulatory protein; MALDI-TOF,
matrix-assisted laser desorption/ionization time-of-flight; MS, mass
spectrometry; PA, phosphatidic acid; PLD, phospholipase D.
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ACKNOWLEDGMENTS |
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REFERENCES |
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