From the Department of Nutritional Sciences, Rutgers, State University of New Jersey, New Brunswick, New Jersey 08901
Received for publication, July 3, 2002, and in revised form, October 25, 2002
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ABSTRACT |
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The perilipins are the most abundant proteins
coating the surfaces of lipid droplets in adipocytes and are found at
lower levels surrounding lipid droplets in steroidogenic cells.
Perilipins drive triacylglycerol storage in adipocytes by regulating
the rate of basal lipolysis and are also required to maximize
hormonally stimulated lipolysis. To map the domains that target and
anchor perilipin A to lipid droplets, we stably expressed fragments of perilipin A in 3T3-L1 fibroblasts. Immunofluorescence microscopy and
immunoblotting of proteins from isolated lipid droplets revealed that
neither the amino nor the carboxyl terminus is required to target
perilipin A to lipid droplets; however, there are multiple, partially
redundant targeting signals within a central domain including 25% of
the primary amino acid sequence. A peptide composed of the central
domain of perilipin A directed a fused green fluorescent protein to the
surfaces of lipid droplets. Full-length perilipin A associates with
lipid droplets via hydrophobic interactions, as shown by the
persistence of perilipins on lipid droplets after centrifugation
through an alkaline carbonate solution. Results of the mutagenesis
studies indicate that the sequences responsible for anchoring perilipin
A to lipid droplets are most likely domains of moderately hydrophobic
amino acids located within the central 25% of the protein. Thus, we
conclude that the central 25% of the perilipin A sequence
contains all of the amino acids necessary to target and anchor
the protein to lipid droplets.
All proteins contain sequences of amino acids that specify their
ultimate subcellular localization. These molecular zip codes may
consist of a short motif such as the KDEL sequence that serves as a
retention signal to hold proteins in the endoplasmic reticulum (1, 2)
or may be quite complex like mitochondrial targeting signals that lack
amino acid identity, but are enriched in hydrophobic, hydroxylated, and
positively charged amino acids, and have the potential to form
amphipathic Lipid droplets are spherical organelles found in many types of
eukaryotic cells that are composed of a core of neutral lipids covered
by a monolayer of phospholipids, free cholesterol, and proteins.
Depending on the cell type, the number of lipid droplets, the relative
mass of stored triacylglycerol and cholesterol esters, and the protein
composition of the droplet vary. Adipocytes store almost exclusively
triacylglycerol in enormous lipid droplets that may exceed 100 µm and
constitute the major energy storage depot of the body. Many other cells
store cholesterol ester in tiny droplets; this cholesterol is used to
maintain cellular cholesterol levels for membrane synthesis, and, in
specialized cells in the adrenal cortex, testes, and ovaries, it serves
as a source of substrate for steroid hormone synthesis (4). To date,
few lipid droplet-associated proteins have been identified in mammals,
yet recent functional studies show that these proteins serve essential roles in regulating neutral lipid storage and release (5-9).
The perilipins are a family of three protein isoforms (10, 11) encoded
by a single gene that are localized exclusively to lipid droplets in
adipocytes (12, 13) and steroidogenic cells (14). Perilipin A is the
most abundant isoform in both cell types, whereas perilipin C is unique
to steroidogenic cells; low levels of perilipin B can be found in both
types of cells. The perilipins share a common amino-terminal region,
and each isoform has a unique carboxyl-terminal end (10, 11, 14). Perilipin A is a relatively abundant protein on adipocyte lipid droplets and functions to increase cellular triacylglycerol storage by
decreasing the rate of triacylglycerol hydrolysis (5-8); thus, is
required to maximize the storage of triacylglycerols in adipose tissue
(6, 7). Furthermore, perilipin A is multiply phosphorylated by
cAMP-dependent protein kinase
(PKA)1 following the
stimulation of lipolysis in adipocytes (15), and serves an additional
role in controlling the release of triacylglycerol at times of
need (6, 9).
Current models for lipid droplet assembly favor the nucleation of lipid
droplets as a lens of neutral lipid within the membrane bilayer of the
endoplasmic reticulum that pinches off and enters the cytosol following
the accumulation of sufficient neutral lipid (4). Perilipins are
synthesized on free ribosomes rather than on endoplasmic
reticulum-bound ribosomes (16, 17); thus, nascent perilipins must
travel to and assemble onto lipid droplets post-translationally. The
processes that control the directing of nascent proteins to lipid
droplets and the assembly of these proteins onto the droplet have not
been elucidated. The purpose of this study is to identify the
structural domains that mediate the targeting and anchoring of
perilipin A to lipid droplets. Deletion mutations of perilipin A were
stably expressed in 3T3-L1 fibroblasts, a cell line that does not
express endogenous perilipins. Targeting of mutated perilipins to lipid
droplets was assessed by immunofluorescence microscopy and
immunoblotting of subcellular fractions; effective anchoring of the
mutated proteins into the droplet was assayed by subjecting isolated
lipid droplets to alkaline carbonate solutions, in a classic test for
hydrophobic interactions. The studies show that the central 25% of
perilipin A contains all of the amino acid sequences required to target
and anchor the protein to lipid droplets.
Materials--
Pfu DNA polymerase was purchased from
Stratagene. Alexa Fluor 488-conjugated goat anti-rabbit IgG and Bodipy
493/503 were obtained from Molecular Probes, Inc. (Eugene, OR).
Rhodamine Red X-conjugated goat anti-guinea pig IgGs, fluorescein
isothiocyanate-conjugated goat anti-guinea pig IgGs, and lissamine
rhodamine-conjugated goat anti-rabbit IgGs were obtained from Jackson
ImmunoResearch Laboratories, Inc. (West Grove, PA). RNase inhibitor was
from 5 Prime Cell Culture--
3T3-L1 preadipocytes and 293T cells were
cultured as previously described (5).
Expression of Perilipin A in 3T3-L1 Fibroblasts--
The coding
sequence of the cDNA for mouse perilipin A was amplified by
polymerase chain reaction (PCR) using Pfu DNA polymerase and
oligonucleotide primers corresponding to the ends of the regions of the
perilipin A cDNA sequence that needed to be amplified, with added
HindIII sites. When the perilipin A cDNA was amplified to truncate the 5' end, an ATG codon was inserted into the 5' primer;
when perilipin A cDNA was amplified to truncate the 3' end, a TGA
stop codon was inserted into the 3' primer. Internal deletions were
created by replacing the cDNA sequence to be deleted by a
BglII or XbaI site, introduced by PCR
oligonucleotide primers. The amplified cDNA sequences were ligated
into the unique HindIII site of the pSR Expression of Green Fluorescent Protein (GFP)-Perilipin Fusion
Proteins--
The cDNA sequence for a mutated GFP engineered to
fluoresce more efficiently at the wavelength for fluorescein was used
(19) (mutation 2). The GFP mutation 2 cDNA was amplified from the
pGAL-GFP plasmid (gift of Dr. Joseph Nickels, MCP Hahnemann School of
Medicine) by PCR, using primers matching the 5' and 3' ends of the
coding sequence of the GFP cDNA with added HindIII sites
at both ends, and an added XbaI site proximal to the 3' end
to aid in subsequent subcloning steps; the amplified cDNA was then
ligated into the HindIII site of the pSR Fluorescence Microscopy--
Cells were grown on glass
coverslips and fixed with 3% paraformaldehyde in phosphate-buffered
saline (PBS). 3T3-L1 fibroblasts expressing GFP constructs were
observed under the microscope without fixation. Cells expressing
amino-terminally truncated perilipin A were probed with polyclonal
antibodies raised against the carboxyl-terminal domain of perilipin A
( Subcellular Fractionation--
Confluent monolayers of 3T3-L1
fibroblasts stably expressing full-length or mutated perilipin A were
incubated with either 400 µM oleic acid complexed to
fatty acid-free bovine serum albumin (BSA) at a 6:1 molar ratio, when
the BSA was purchased from Sigma, or with 600 µM oleic
acid complexed to fatty acid-free BSA (6:1) when the BSA was purchased
from Biocell Laboratories, Inc. (Rancho Dominguez, CA) for 24 h to
increase triacylglycerol synthesis and storage (5); the two different
levels of fatty acid-albumin complexes were necessary to obtain
approximately the same level of lipid loading of the cells. Cells were
harvested by scraping into cold PBS and pelleted by low speed
centrifugation. Pelleted cells were resuspended and lysed in a
hypotonic solution containing 10 mM Tris, pH 7.4, 1 mM EDTA, 10 mM sodium fluoride, 10 µg/ml leupeptin, 1 mM benzamidine, and 100 µM
[4-(2-aminoethyl)benzenesulfonylfluoride] hydrochloride, for 10 min
at 4 °C, followed by 10 strokes in a Teflon/glass Dounce
homogenizer. The homogenate was centrifuged for 30 min at 26,000 × g at 4 °C, and the floating lipid layer was recovered
after slicing off the tops of the tubes with a Beckman tube-slicer. The
infranatant and pellet fractions were also collected.
Analysis of Cellular Fractions--
The lipid droplet-containing
fractions were concentrated and delipidated by overnight precipitation
with cold acetone at Polysome Profile Sucrose Gradients--
Confluent monolayers of
3T3-L1 fibroblasts were harvested on ice by scraping the cells into
cold PBS and were pelleted by low speed centrifugation at 4 °C.
Cells were resuspended in lysis buffer (10 mM Tris, pH 7.4, 15 mM KCl, 10 mM MgCl2, 0.1%
Triton X-100, 20 mM dithiothreitol, 53 mM
cycloheximide, 0.025% heparin, and 2 units/ml RNase inhibitor),
incubated for 10 min on ice, and homogenized with 10 strokes of a
Teflon/glass homogenizer. The homogenate was centrifuged at 12,000 × g at 4 °C for 15 min, and the resulting supernatant
was layered over a 10-50% sucrose gradient placed on a 1-ml 60%
sucrose cushion. The sucrose solutions contained 20 mM
Hepes, pH 7.2, 0.25 M KCl, 10 mM
MgCl2, 20 mM dithiothreitol, 53 mM
cycloheximide, 0.5 mg/ml heparin, and 3.3 units/ml RNase inhibitor. As
a control for the fractionation of untranslated mRNAs, half of each
post-mitochondrial supernatant fraction was adjusted to 20 mM EDTA and layered onto a gradient containing 20 mM EDTA. Gradients were centrifuged for 3 h at
180,000 × g at 4 °C in a Beckman SW40Ti rotor. 12 1-ml fractions were harvested on ice and stored at Northern Blot Analysis--
Total RNA was extracted from
cultured cells using RNeasy minicolumns (Qiagen), and from polysome
profile fractions with TRIzol LQ (Invitrogen) according to the
protocols of the manufacturers. RNA was separated by electrophoresis in
1% agarose gels using NorthernMaxTM-Gly reagents (Ambion,
Inc.). RNA was transferred electrophoretically to MagnaCharge nylon
membranes (Osmonics), and the membranes were probed with
32P-labeled cDNA probes corresponding to the
full-length coding sequence of perilipin A using the ExpressHyb
hybridization solution from Clontech.
Predicted Structural Motifs of Murine Perilipin A--
The
predicted amino acid sequence of murine perilipin A includes 517 amino
acids containing several notable domains (Fig. 1; see also Ref. 17). Sequences from
amino acids 17 to 121 of both rat and murine perilipin A are 32%
identical and 65% similar to the amino-terminal region of adipophilin
(10) and 38% identical and 60% similar to the amino-terminal region
of TIP47; adipophilin (20, 23) and TIP47 (21) are ubiquitously
distributed lipid droplet-associated proteins. Between amino acids 111 and 182, perilipin A contains five sequences of 10-11 amino acids that are predicted by the LOCATE program to form amphipathic Ectopically Expressed Perilipin A Targets to Lipid Droplets in
Cultured Fibroblasts--
To elucidate the targeting motifs
responsible for directing nascent perilipin A to lipid droplets, we
used cultured cell lines that lack endogenous perilipins, but form
lipid droplets when incubated with exogenous fatty acids. Previous
studies indicated that perilipin A expressed in 3T3-L1 fibroblasts (5)
or CHO-K1 fibroblasts (not shown) targets to lipid droplets and tethers to the droplets in a manner indistinguishable from the association of
endogenous perilipins with droplets in differentiated adipocytes; furthermore, the ectopic perilipin is found only on lipid droplets in
subcellular fractionation experiments, and in no other cellular compartment (5). Cultured 3T3-L1 fibroblasts ectopically expressing perilipin A were stained simultaneously for neutral lipids and for
perilipins and were compared with cells expressing the retroviral expression vector lacking an inserted cDNA (referred to as control cells) (Fig. 2). Although both cell types
contained lipid droplets in the presence (Fig. 2, B and
D) or absence (Fig. 2, A and C) of
excess oleic acid in the culture medium, control cells did not express
perilipin A (Fig. 2, A and B). Expression of
perilipin A caused an increase in the numbers and sizes of
intracellular lipid droplets, and altered the localization of the
droplets from a dispersed to a clustered arrangement (Fig. 2,
C and D), when compared with control cells (Fig.
2 and Ref. 5). Furthermore, perilipin A was only found on the surfaces
of lipid droplets (Fig. 2 and Ref. 5), as in adipocytes (13), although
the droplets were much smaller in the fibroblasts.
Identification of the Domain(s) of Perilipin A Required for Its
Targeting to Lipid Droplets--
To identify the regions of perilipin
A that are necessary for its targeting to lipid droplets, truncation
and internal deletion mutations of the murine perilipin A cDNA were
ligated into a retroviral expression vector and used to stably
transfect 3T3-L1 fibroblasts that lack endogenous perilipins. A summary
of data collected from cells stably expressing selected mutations of
perilipin A is shown in Table I. Sites
for truncation were designed to test potential requirements for the
various notable domains (Fig. 1) in mediating the targeting of
perilipin A to lipid droplets. Targeting of the mutated perilipins was
assessed by immunofluorescence microscopy (Figs.
3-5)
using antibodies directed against either amino- or carboxyl-terminal peptides of perilipin A. Three or more microscopy experiments were
conducted for each mutated perilipin construct; each experiment involved the observation of all of the densely subconfluent cells on
three or more coverslips per construct. When the results indicate that
a mutated perilipin has targeted to lipid droplets, then greater than
95% of the stably selected cells displayed a perilipin signal on lipid
droplets. Furthermore, cells on additional coverslips in each
experiment were lipid-loaded to increase the storage of triacylglycerol
in lipid droplets, thus facilitating the observation of perilipin
targeting; under these conditions, the targeting of the indicated
mutated perilipins to lipid droplets was observed in 100% of the
cells. In all cases, mutated perilipins that were scored as failing to
target to lipid droplets were not observed on lipid droplets in any
cells, whether or not the cells were lipid-loaded.
To confirm the localization of the mutated perilipins, lipid droplets
were isolated from stably transfected cells and the proteins contained
in the lipid droplet fractions were delipidated, separated by SDS-PAGE,
and immunoblotted for perilipins (Fig. 6); additionally, supernatant fractions
and the membrane pellets were probed for the presence of perilipins
(data not shown). For the immunoblotting experiments, the cells were
grown in the presence of 400 µM oleic acid to increase
the number of lipid droplets per cell, and hence the amount of
expressed perilipin (16); we observed an increase in the protein mass
of the expressed mutated perilipins that targeted to lipid droplets
under lipid-loading conditions (data not shown), suggesting that these
truncated perilipins are stabilized by binding to lipid droplets
similarly to full-length perilipin A (16). By contrast, no protein mass
was observed for non-targeting mutated perilipins in any subcellular
fraction (data not shown).
Identification of Potential Amino-terminal Targeting Sequences of
Perilipin A--
To identify potential targeting motifs within the
amino terminus of perilipin A, a series of amino-terminal truncation
mutations of perilipin A were stably expressed in 3T3-L1 fibroblasts
(Table I, Figs. 3 and 6). Removal of the first 81 amino acids from the amino terminus of perilipin A (mutation N1) does not impair the ability
of the mutated protein to target to lipid droplets in 3T3-L1
fibroblasts (Figs. 3 and 6, Table I); hence, the consensus site for
phosphorylation by PKA (aa 78-81) does not play an essential role in
perilipin A localization. The amino-terminal domain is shared by
perilipins, adipophilin, and TIP47, making it a logical sequence to
contain information required for the targeting of lipid
droplet-associated proteins; however, truncation of this entire
amino-terminal sequence (removal of the first 121 aa; mutation N2) does
not prevent the targeting of perilipin A to lipid droplets (Table I,
Figs. 3 and 6). Therefore, this conserved amino-terminal domain does
not appear to contain essential targeting information. The amino acids
immediately following this conserved domain contain five short
sequences that are predicted to form amphipathic Identification of Potential Carboxyl-terminal Targeting Sequences
of Perilipin A--
Cells stably expressing mutated perilipins
containing successive carboxyl-terminal truncations were studied to map
potential carboxyl-terminal targeting motifs. The extreme
carboxyl-terminal region of perilipin A contains three consensus sites
for PKA phosphorylation. To test the importance of these sites in the
targeting of perilipin to lipid droplets, mutated perilipins C1
(expressing aa 1-489) and C2 (expressing aa 1-429) were studied and
shown to target to lipid droplets (Figs. 4 and 6, Table I), thus
indicating that the consensus sites for phosphorylation by PKA are
dispensable. Mutated perilipin expressing only the sequences common to
perilipins A and B (mutated perilipin C3, expressing aa 1-405) also
targeted to lipid droplets (Figs. 4 and 6, Table I), thus showing that the unique carboxyl-terminal domains of perilipins A and B are not
necessary for the targeting of perilipins to lipid droplets. Furthermore, truncation of the entire carboxyl terminus following H3
(mutation C4; aa 1-364) yielded a mutated perilipin that targeted to
lipid droplets. Finally, mutated perilipin C7 (expressing aa 1-302),
which includes the amino terminus of perilipin A through the first
third of the acidic domain, fails to target to lipid droplets (Fig. 4,
Table I) and cannot be found in the lipid droplet (Fig. 6), soluble, or
membrane-containing fractions of lysed cells (data not shown). In
summary, truncation of the final 193 carboxyl-terminal amino acids (or
38% of the sequence) does not alter the intracellular localization of
perilipin A, whereas the additional disruption of the acidic domain
eliminates the targeting of perilipin to lipid droplets. These
experiments implicate the central acidic domain as a potential
determinant for the targeting of perilipin A to lipid droplets.
Interestingly, lipid droplets in cells expressing mutated perilipins
C2-C6 (Fig. 4) are arranged differently than those found in cells
expressing full-length perilipin A (Fig. 2); as more of the carboxyl
terminus is successively removed, the lipid droplets gradually lose the
tightly clustered appearance and become dispersed throughout the cytoplasm.
Identification of Core Targeting Motifs Using Internal Deletion
Mutations of Perilipin A--
Because both the amino and carboxyl
termini appear to be dispensable in targeting perilipin A to lipid
droplets, the role of the central domain, comprising ~25% of the
protein, was investigated. Mutated perilipins containing internal
deletions, in which a defined central portion of the protein was
deleted from otherwise intact perilipin A, were stably expressed in
cells. Because the central acidic domain was implicated in the
targeting of perilipin A to lipid droplets in studies of cells
expressing carboxyl-terminal truncated perilipins, cells were
transfected with a mutated perilipin lacking only the acidic domain
(mutated perilipin Cells Stably Transfected with Non-targeting Mutated Perilipins
Express mRNA for the Mutated Constructs--
The non-targeting
mutated perilipins were undetectable by immunofluorescence microscopy
of stably transfected cells, and by immunoblotting of proteins from
subcellular fractions isolated from these cells. These results implied
that either the mutated perilipin constructs were not expressed, or the
nascent proteins were unstable and rapidly degraded. To test the
possibility that the constructs that yielded non-targeting mutated
proteins were not expressed, total RNA was isolated from cells stably
expressing constructs from both targeting and non-targeting perilipin
mutations; the RNA was separated electrophoretically on agarose gels,
transferred to charged nylon membranes, and probed with radiolabeled
perilipin cDNA probes. Cells transfected with either targeting or
non-targeting mutated perilipin cDNAs generally had comparable
levels of mRNA for the mutated perilipin constructs (Fig.
7), thus implying similar levels of
transcription of all constructs.
mRNAs from Mutated Perilipins That Fail to Target to Lipid
Droplets Are Translated--
Because the cDNAs for the
non-targeting mutated perilipin constructs were transcribed (Fig. 7),
the failure to observe protein encoded by these constructs was the
result either of the failure of the cells to translate the
corresponding mRNAs or of instability and rapid degradation of the
nascent mutated perilipins. To address the possibility that the
mRNAs for these mutated perilipins were not translated, we
fractionated post-mitochondrial supernatants from cells stably
transfected with the non-targeting C7, N6, or The Central Domain of Perilipin A Is Sufficient to Redirect the
Soluble GFP to Lipid Droplets--
The analysis of cells expressing
deletion mutations of perilipin A indicated that the essential domains
that direct the targeting of nascent perilipin to lipid droplets are
potentially redundant sequences found within the central 25% of the
protein that is limited by H1 and H3. To determine whether portions of
this domain are sufficient to target proteins to lipid droplets, we
created a fusion construct of the cDNA sequence of the soluble GFP
ligated 5' to the cDNA sequence encoding the amino acids from the
start of H1 to the end of H2 (aa 233-345; mutated perilipin GP2) of perilipin A. Additional fusion constructs of the GFP cDNA ligated 5' to 1) the cDNA encoding full-length perilipin A (perilipin construct GP1), 2) the cDNA encoding mutated perilipin A lacking the first 248 amino acids (mutated perilipin GP3), 3) the cDNA encoding the first 232 amino acids of perilipin A (mutated perilipin GP4), 4) the cDNA encoding the first 302 amino acids of perilipin A
(mutated perilipin GP5; data not shown), and 5) the cDNA encoding the last 153 amino acids (mutated perilipin GP6; data not shown) were
also prepared. Data collected for these perilipin-GFP fusion proteins
are shown, in part, in Fig. 9 and are
summarized in Table II. Fluorescence
microscopy demonstrated that the full-length perilipin A-GFP fusion
protein was efficiently targeted to lipid droplets (Fig. 9,
GP1). Furthermore, lipid droplets coated with the
GFP-perilipin A fusion protein were arranged in clusters (Fig. 9,
GP1) comparable with the pattern observed in cells
expressing full-length perilipin A (Fig. 2C, and Ref. 5).
The fusion protein containing a portion of the central region of
perilipin A fused to GFP also targeted to lipid droplets (Fig. 9,
GP2), thus suggesting that portions of the central domain
are sufficient to redirect a soluble protein to the surfaces of lipid
droplets; however, the lipid droplets coated with the truncated
perilipin-GFP fusion protein labeled GP2 retained a dispersed
arrangement (Fig. 9, GP2) comparable with that of control
cells lacking perilipins (Fig. 2, A and B). A GFP
fusion protein of perilipin lacking the amino-terminal 248 amino acids
also targeted to lipid droplets (Fig. 9, GP3), but lipid
droplets retained a mostly dispersed arrangement in the cytoplasm. The
targeting of GP3 to lipid droplets is interesting, because a comparable
mutated perilipin lacking the fused GFP (mutated perilipin N6; Fig. 3
and Table I) fails to target to lipid droplets. These findings support
the concept that a short sequence of amino acids (e.g. aa
234-248) is not required to direct perilipin to lipid droplets, but
instead, that potentially redundant sequences within the central
domain, which may be stabilized by the additional GFP sequence, are
critical. The remaining mutated perilipin-GFP fusion proteins including those containing short amino-terminal (Fig. 9, GP4) and
carboxyl-terminal (GP6; data not shown) perilipin peptides, and a
longer perilipin peptide lacking the carboxyl terminus (GP5; data not
shown) failed to target, and cells displayed only a diffuse faint
fluorescent signal. Although the GP4 and GP6 mutated perilipins lacked
all sequences within the central putative targeting domain and, hence, gave the expected results of failing to target to lipid droplets, the
GP5 construct contained approximately half of the central domain, but
was unable to support the targeting of the mutated perilipin-GFP fusion
protein to lipid droplets. Control cells expressing soluble GFP
displayed diffuse green fluorescence throughout the cytoplasm, and no
specific staining of lipid droplets (Fig. 9 G).
The Central Hydrophobic Domains Are Essential to Anchor Perilipin A
to Lipid Droplets--
Previous observations have suggested that
perilipins are very tightly associated with lipid droplets, probably
via hydrophobic interactions; the release of perilipins from isolated
lipid droplets requires detergents such as sodium dodecyl sulfate (12).
Furthermore, the flotation of lipid droplets in 100 mM
carbonate, pH 11.5, fails to remove perilipins; this classic technique
was used initially by Fujiki et al. (24) to remove
peripherally associated proteins from microsomes, thus leaving integral
membrane proteins associated with the phospholipid bilayers. Although
lipid droplets lack a membrane bilayer, the resistance of full-length
perilipin A to removal by alkaline carbonate treatments suggests that
perilipin A associates with lipid droplets through hydrophobic
interactions. We used this classic protocol to study the nature of the
association between the mutated perilipins and lipid droplets. Lipid
droplets were isolated by centrifugation from 3T3-L1 cells stably
transfected with the different mutated perilipin constructs. The
isolated lipid droplets were further centrifuged through an alkaline
carbonate solution, and the presence of mutated perilipins was assessed by immunoblotting of delipidated proteins from the buoyant lipid droplet fractions that floated in the second centrifugation step. Perilipin mutations N1-N4 were detected on lipid droplets that had
been centrifuged through carbonate, thus indicating that amino acids
1-222 are not required to embed perilipin A into lipid droplets (Fig.
10A). The further removal of
10 amino acids (mutation N5) eliminated the association of the mutated
perilipin with carbonate-washed lipid droplets. Perilipin mutations
C1-C5 were detected on lipid droplets that had been centrifuged
through carbonate (Fig. 10B), but the further removal of H2
(mutation C6) impaired the ability of the mutated perilipin to embed
into lipid droplets. These observations imply that the central portion
of perilipin with its hydrophobic domains is required for the anchoring
of the protein to lipid droplets. Interestingly, although the The major finding of this study is that the central domain of
perilipin A, representing 25% of the amino acid sequence, contains all
of the sequences that are required to target the protein to lipid
droplets. A portion of this region, comprising H1 through H2, is
sufficient to redirect soluble GFP to lipid droplets. There appear to
be redundancies among a combination of targeting signals within this
central region. The removal of various small portions of the central
region fails to eliminate the targeting of perilipin to lipid droplets,
but removal of the entire sequence prevents targeting. Hence, we
suggest that the signals required to target perilipin A to lipid
droplets are a combination of hydrophobic domains in conjunction with
the acidic domain. When these targeting domains are present within the
entire protein, they are dispensable individually or in some
combinations, but the presence of at least the sequence from H1 through
H2 is sufficient to redirect GFP to lipid droplets, and the absence of
the entire sequence from H1 through H3 is necessary to eliminate the
targeting of perilipin A to lipid droplets. Thus, we conclude that
perilipin A does not contain a single short amino acid "zip code"
responsible for directing its targeting to lipid droplets.
The domains mediating the anchoring of perilipin A to lipid droplets
are similar to those responsible for targeting perilipins to lipid
droplets. Results obtained from subjecting lipid droplets to alkaline
carbonate solutions suggest that perilipin A associates with lipid
droplets via hydrophobic interactions; this observation implies that
the hydrophobic domains of perilipin may embed into the core of the
lipid droplet to anchor the protein. Our results indicate that H3, in
the absence of the sequence comprising H1 through H2, but in the
context of the remaining amino- and carboxyl-terminal sequences
(mutation We hypothesize that perilipin A is anchored onto lipid droplets through
the three hydrophobic domains, which embed into the neutral
lipid-filled core, whereas the central highly charged acidic domain
loops away from the surface of the lipid droplet. The sequences with
the characteristics of amphipathic TIP47 and adipophilin share a high degree of sequence similarity with
the perilipins and, furthermore, like perilipins, target to and
associate with lipid droplets, most likely through hydrophobic interactions, because the proteins resist removal by the extraction of
lipid droplets with alkaline carbonate solutions. Although a recent
study has questioned the association of TIP47 with lipid droplets (25),
further studies by Miura and co-workers (26), and in our
laboratory,2 have confirmed
the localization of TIP47 to lipid droplets. To date, no studies have
addressed the molecular basis of the targeting of TIP47 or adipophilin
to lipid droplets. Surprisingly, the amino-terminal domain of 105 amino
acids that is highly conserved within this family of proteins is not
required to mediate the targeting of perilipin A to lipid droplets,
thus suggesting that this domain may play another, as yet
uncharacterized role in the function of these lipid droplet-associated
proteins. Furthermore, the dispensability of the amino terminus of
perilipin is remarkable in light of the observation that the earliest
synthesized sequences in the amino termini of many different proteins
contain essential targeting information. TIP47 and adipophilin are less
highly conserved in the central sequences that target and anchor
perilipins to lipid droplets. None of the members of this family of
proteins contain extensive stretches of hydrophobic amino acids,
although hydropathy plots of the proteins show short moderately
hydrophobic sequences for all members. An alternative mechanism of
anchoring these proteins to lipid droplets may be through the acylation
of cysteine residues; Heid and co-workers (23) have shown that
adipophilin is likely acylated. Future studies are needed to determine
the mechanism required to direct these additional family members to
lipid droplets. A comparison of the sequences of TIP47 and adipophilin
may also be instructive to explain some significant differences in the targeting behavior of TIP47 relative to the other family members; in
addition to localizing to lipid droplets, TIP47 can be found in the
cytosol of most cells (21, 27), and has been proposed to play a role in
endosomal trafficking of the mannose 6-phosphate receptor as part of a
complex with Rab 9 (28-30). Finally, additional members of this
protein family have been identified in Drosophila, Bombyx, and Dictyostelium (11); both
Drosophila proteins, as well as the Dictyostelium
protein, target to lipid droplets when expressed in Chinese hamster
ovary cells in culture (26).
While perilipins, adipophilin, and TIP47 are naturally occurring lipid
droplet-associated proteins, several additional proteins have recently
been demonstrated to localize to lipid droplets under some conditions
in laboratory experiments. Although caveolins normally associate with
small invaginations along the cytoplasmic face of the plasma membrane,
ectopically expressed caveolins have been shown to localize to lipid
droplets in cultured cells when plasma membrane targeting signals are
deleted, endoplasmic reticulum retrieval signals are added, or when
cells are treated with oleic acid or brefeldin A (31-33). Deletion
mutagenesis experiments described in these studies suggest that
caveolins associate with lipid droplets via a central hydrophobic
domain. Additionally, when expressed in cultured cell lines, the core
proteins of both the hepatitis C virus and the GB virus-B
associate with lipid droplets via a central highly hydrophobic domain
(34), and localize to the cytoplasm and several other membranes within
the cell. Thus, in all lipid droplet-associated proteins studied to
date, hydrophobic domains have been implicated in targeting of the
proteins to lipid droplets.
Mutated perilipins that are not found on lipid droplets cannot be found
in any other subcellular compartments and appear to be synthesized but
rapidly degraded. Because perilipins are stabilized upon association
with lipid droplets (16), we hypothesize that the removal of essential
targeting sequences produces mutated perilipins in the cytoplasm that
fail to become stabilized by anchoring to lipid droplets and, hence,
are rapidly degraded. Alternatively, the removal of specific sequences
may either prohibit the proper folding of the mutated perilipins,
leading to failure to target and subsequent degradation, or promote
much slower synthesis or more rapid degradation of the mutated protein,
thus eliminating subsequent targeting to droplets. Interestingly,
although the removal of a few extra amino acids from an already
truncated perilipin construct prevented targeting of the translated
product and promoted degradation (as seen for mutated perilipins N6 and
C7), the deletion of these few amino acids from otherwise intact
perilipin had no effect on targeting or on the stability of the protein
whatsoever (perilipin mutations
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices (3). Proteins specifically associated with
lipid droplets have only recently been described, and the amino acid
sequences required to target nascent proteins to lipid droplets are
uncharacterized. The purpose of this study is to identify the
structural motifs responsible for directing the targeting of
perilipins, the major proteins coating the prominent lipid droplets of
adipocytes, to lipid droplets.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 Prime, Inc. (Boulder, CO). Geneticin was purchased from Mediatech, Inc. (Herndon, VA).
MSVtkneo
retroviral expression vector (18). The procedure used to assemble the
retrovirus, transduce 3T3-L1 fibroblasts, and select cells stably
expressing the cDNAs was described previously (5). Cells used for
control conditions were selected to stably express the retroviral
vector lacking perilipin A cDNA. Stably selected cells from
multiple transduction experiments were used for each of the mutated
perilipin constructs in all of the experiments.
MSVtkneo
retroviral expression vector. The resulting expression vector was named
pSR
MSVtkneo-GFP. After amplification by PCR using 5' and 3' primers
containing added XbaI sites, full-length or truncated
perilipin A cDNA sequences were ligated in frame into the
XbaI site of pSR
MSVtkneo-GFP. GFP-perilipin fusion
constructs in the retroviral expression vector were stably or
transiently expressed in 3T3-L1 fibroblasts following the previously
described procedure (5). Cells for control conditions stably expressed
GFP. The GFP fusion proteins consist of GFP separated at its carboxyl
terminus from the amino terminus of perilipin A by a hinge sequence of
5 prolines.
-COT) (20). Cells expressing carboxyl-terminally truncated, or
perilipin A mutated by internal deletions, were probed with antibodies
directed against the amino terminus of perilipin A (
-PAT) (20). The
-PAT antibodies do not detect the structurally similar protein,
adipophilin (20). When cells were simultaneously stained for perilipins
and neutral lipids, Bodipy 493/503 was added to the solutions
containing the fluorescently labeled secondary antibodies at a final
concentration of 10 µg/ml (21). Cells were viewed with a Nikon
Eclipse E800 fluorescence microscope equipped with a Hamamatsu Orca
digital camera interfaced with a Power Macintosh G4. Images were
acquired and processed using Improvision Openlab software.
20 °C, followed by solubilization in 2×
concentrated Laemmli's sample buffer (22); the proteins were separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred electrophoretically to nitrocellulose membranes.
Immunoblots were probed with the primary antibodies described above,
with the inclusion of polyclonal antibodies raised against full-length
rat perilipin A (12) for some samples, and horseradish
peroxidase-conjugated secondary antibodies (Sigma), and developed using
enhanced chemiluminescence reagents from Amersham Biosciences.
80 °C for
subsequent RNA extraction and analysis.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-pleated sheets (17). Three domains of moderate hydrophobicity of 18 (aa
243-260; H1), 23 (aa 320-342; H2), and 16 (aa 349-364; H3) amino
acids are located in the center of the sequence surrounding a highly
acidic domain (aa 291-318) where 19 of 28 consecutive amino acids are
glutamic or aspartic acid. Six consensus phosphorylation sites for PKA
(consensus: R(R/K)XS) are located throughout the predicted
protein sequence; the PKA consensus site serines are in positions 81, 222, 276, 433, 492, and 517. It is not known whether all of these
serines are phosphorylated when adipocytes are lipolytically
stimulated, thus activating PKA. The first 405 amino acids are common
to perilipins A and B; the unique carboxyl terminus of 112 amino acids
of perilipin A is depicted, whereas perilipin B contains a different
sequence of 16 amino acids (10, 11). The aim of this study is to
elucidate the roles that the depicted motifs may play in targeting
perilipin A to lipid droplets and in mediating the anchoring of
perilipin A to lipid droplets.
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Fig. 1.
Schematic diagram of mouse perilipin A
structural domains. Perilipin A contains 517 amino acids. Starting
from the amino terminus, perilipin A contains a sequence of 105 amino
acids similar to adipophilin and TIP47 (amino acids 17-121), five
10-amino acid domains with amphipathic -pleated sheet character
(between amino acids 111 and 182), three sequences of moderate
hydrophobicity (amino acids 243-260, 320-342, and 349-364), a
highly acidic region (amino acids 291-318), and six consensus sites
for phosphorylation by PKA at the indicated positions. The
vertical line indicates the limit of the
amino-terminal region common to perilipins A and B; the carboxyl
terminus following amino acid 405 is unique to perilipin A.
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Fig. 2.
Ectopic perilipin A localizes to lipid
droplets in 3T3-L1 fibroblasts. Control 3T3-L1 fibroblasts
(A and B) and cells stably expressing perilipin A
(C and D) were lipid-loaded (B and
D) for 24 h with 400 µM oleic acid
complexed to albumin. Cells were prepared for immunofluorescence
microscopy and probed for perilipin (green) using
-PAT. Neutral lipids (red) were simultaneously stained
with Bodipy 493/503. Each panel depicts a single cell. The
background fluorescence of the cells has been enhanced to make the
cells more visible. Bar = 10 µm.
Summary of experiments
series, deleted amino acids are indicated. A "+" in the Targets?
column indicates the detection of the mutated perilipins on lipid
droplets by immunofluorescence microscopy and immunoblotting; a
"
" indicates the failure to detect the protein on lipid droplets.
A "+" in the Anchors? column indicates the association of the
mutated perilipins with lipid droplets following an alkaline carbonate
wash. A "+" in the Clusters? column indicates the appearance of
clustered lipid droplets in cells following the stable expression of
the mutated perilipin; a "+/
" indicates the appearance of limited
clusters of lipid droplets. NA, not applicable.
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Fig. 3.
Localization of amino-terminal truncation
mutations of perilipin A in 3T3-L1 fibroblasts. A,
3T3-L1 fibroblasts stably expressing full-length perilipin A
(FL), vector control (C), and amino-terminal
truncation mutations of perilipin A (N1-N6) were prepared
for microscopy and probed for perilipin using the -PAT antibody
(FL and C) or
-COT (N1-N6).
Bar = 10 µm. Differences in the relative intensity of
lipid droplet staining for the FL panel relative
to the other panels reflects the use of different
antibodies, and not the relative levels of expression of the N1-N6
mutated perilipins when compared with full-length perilipin.
B, schematic diagrams of the expressed portions of
full-length and amino-terminal truncation mutations of perilipin A
labeled N1-N6.
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Fig. 4.
Localization of carboxyl-terminal truncation
mutations of perilipin A in 3T3-L1 fibroblasts. A,
3T3-L1 fibroblasts stably expressing carboxyl-terminal truncation
mutations of perilipin A (C1-C7) were prepared for
microscopy and probed for perilipin using the -PAT antibody.
B, schematic diagrams of the expressed portions of
carboxyl-terminal truncation mutations of perilipin A labeled
C1-C7.
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Fig. 5.
Localization of internal deletion mutations
of perilipin A in 3T3-L1 fibroblasts. A, 3T3-L1
fibroblasts stably expressing internal deletion mutations of perilipin
A ( 1-
5) were prepared for microscopy and probed for
perilipin using the
-PAT antibody. Bar = 10 µm.
B, schematic diagrams of internal deletion mutations of
perilipin A labeled
1-
5. The deleted portion of perilipin A is
represented by a dotted line.
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Fig. 6.
Immunoblots depicting the localization of
mutated perilipins on isolated lipid droplets. Confluent 3T3-L1
fibroblasts ectopically expressing mutated perilipin A were cultured in
the presence of oleic acid for 24 h. Cells were harvested and
fractionated; the lipid droplet fraction was delipidated with acetone,
and the component proteins were solubilized in Laemmli's sample
buffer, separated by SDS-PAGE, transferred electrophoretically to
nitrocellulose membranes, and probed for perilipin using either the
-PAT antibody (panels B and C) or a
polyclonal antibody raised against full-length rat perilipin A
(panel A). A, lane
1, full-length perilipin A; lane 2,
N1; lane 3, N2; lane 4, N3;
lane 5, N4; lane 6, N5;
lane 7, N6. Asterisks identify the
major perilipin bands for full-length and mutated perilipins. Lower
molecular weight bands likely represent either degradation products or
nonspecific proteins bound by the antibodies; higher molecular weight
bands are nonspecific proteins. B, lane
1, full-length perilipin A; lane 2,
C1; lane 3, C2; lane 4, C3;
lane 5, C4; lane 6, C5;
lane 7, C6; lane 8, C7;
lane 9, control cells (no perilipin). Full-length
perilipin A generally migrates as a doublet; C1 and C2 also show this
pattern. C, lane 1,
1;
lane 2,
2; lane 3,
3;
lane 4,
4; lane 5,
5.
Each lane contains lipid droplet proteins from an equivalent mass of
cells.
-pleated sheets
(17); these sequences were likely candidates for motifs that could be
involved in directing or anchoring perilipins to lipid droplets.
Surprisingly, mutation N3, which further deletes these five sequences
(removal of the first 182 aa), targets to lipid droplets. Removal of an
additional 40 amino acids (to aa 222; mutation N4), which includes a
second PKA consensus phosphorylation site, yielded a mutated perilipin
that also targeted to lipid droplets (Table I, Figs. 3 and 6); however,
removal of the following 26 amino acids (to aa 248; mutation N6),
including a portion of H1, eliminated the localization of the mutated
perilipin to lipid droplets. The expression of mutated perilipin N6 in
cells was not detected by immunofluorescence microscopy (Fig. 3) or by
the immunoblotting of proteins from lipid droplets (Fig. 6), or other subcellular fractions including pelleted membranes and supernatants containing microsomes and soluble proteins (data not shown); hence, the
non-targeting mutated perilipin was likely rapidly degraded within the
cells. Mutated perilipin N5 (protein expressed from aa 233 to 517) was
the shortest amino-terminally truncated perilipin that targeted to
lipid droplets and was visualized by immunofluorescence microscopy
(Fig. 3) and by the immunoblotting of proteins from lipid droplets
(Fig. 6, Table I). Thus, removal of the amino acid sequences preceding
H1 (to aa 233) failed to prevent targeting of mutated perilipin to
lipid droplets, whereas removal of the following 15 amino acids
including the disruption of H1 yielded a mutated perilipin that failed
to target to lipid droplets and was not detected anywhere else in the cell.
1). Surprisingly, mutated perilipin
1 targeted
to lipid droplets in 3T3-L1 fibroblasts (Figs. 5 and 6, Table I), thus
indicating that the acidic domain is dispensable for targeting nascent
perilipins to lipid droplets when the remainder of the carboxyl
terminus is intact. Additionally, the mutated perilipin created by
removing both the central acidic domain and the adjacent H2 (mutated
perilipin
2, lacking aa 291-342) targeted to lipid droplets (Figs.
5 and 6, Table I). Amino-terminal truncation studies suggested a role
for H1 in directing the targeting of perilipin A to lipid droplets.
Hence, a mutated perilipin was constructed that contained a deletion
from H1 up to the acidic domain (lacking aa 233-290, mutated perilipin
3); this mutated perilipin targeted to lipid droplets (Figs. 5 and
6, Table I), as did the mutated perilipin lacking this entire sequence
and H2 (lacking aa 233-342, mutated perilipin
4; Figs. 5 and 6,
Table I). We reasoned that the targeting of the
4 mutated perilipin to lipid droplets may be directed by the remaining H3, so we expressed a mutated perilipin lacking the entire central region (lacking aa
233-364, mutation
5) in 3T3-L1 fibroblasts; the
5 mutated perilipin failed to target to lipid droplets (Figs. 5 and 6, Table I).
Like other non-targeting perilipin mutations, the
5 mutated perilipin was not detected in cells by immunofluorescence microscopy (Fig. 5) or by the immunoblotting of proteins from lipid droplets (Fig.
6) or other subcellular fractions (data not shown). These results
suggest that there are multiple and somewhat redundant targeting
signals in the central domain comprising 25% of the amino acid
sequence; motifs such as the hydrophobic domains may be individually
dispensable in the presence of the remaining sequences, and to remove
all targeting signals requires the deletion of the entire central
domain. Furthermore, the deletion of some of the central sequences of
perilipin A led to dispersion of the lipid droplets throughout the cells.
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Fig. 7.
Cells transfected with both targeting and
non-targeting mutated perilipins express mRNAs for perilipin.
Total RNA isolated from confluent 3T3-L1 fibroblasts ectopically
expressing mutated perilipins was separated electrophoretically on
agarose gels and transferred to charged nylon membranes. Membranes were
probed with radiolabeled full-length perilipin A cDNA probes
(top panels of A-C); blots were
stripped and re-probed for -actin (lower panels of
A-C). A, lane 1,
full-length perilipin A; lane 2, N1;
lane 3, N2; lane 4, N3;
lane 5, N4; lane 6, N5;
lane 7, N6; lane 8, control
cells (no perilipin). B, lane 1,
full-length perilipin A; lane 2, C1;
lane 3, C2; lane 4, C3;
lane 5, C4; lane 6, C5;
lane 7, C6; lane 8, C7;
lane 9, control cells. C,
lane 1, full-length perilipin A; lane
2,
1; lane 3,
2; lane
4,
3; lane 5,
4; lane
6,
5; lane 7, control cells.
5 mutated perilipin
constructs on sucrose gradients. Northern blot analysis of RNA
extracted from the gradient fractions showed that mRNAs for the
three mutated perilipins were efficiently translated (Fig.
8), because the majority of perilipin
mRNA was isolated from dense fractions, thus indicating the
association of multiple ribosomes. Although these data are
qualitatively comparable with results obtained for the translation of
unmodified perilipin A (data not shown, and in Ref. 16), they provide
no information regarding the relative rates of protein synthesis of the
non-targeting mutated perilipins relative to the rates of synthesis of
either unmodified perilipin A or the targeting mutated perilipins. It is possible that differences in the rates of translational extension and termination for the non-targeting mutated perilipins are slower, resulting in lower levels of protein expression.
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Fig. 8.
mRNAs from mutated perilipins that fail
to target to lipid droplets are translated. Confluent 3T3-L1
fibroblasts expressing non-targeting mutated perilipins were harvested,
and post-mitochondrial supernatants were fractionated on 10-50%
sucrose gradients in the presence (B, D, and
F) or absence (A, C, and E)
of EDTA; EDTA releases mRNAs from ribosomes and serves as a control
to identify fractions containing non-translated mRNA. Twelve
fractions were collected from each gradient following centrifugation.
Total RNA was extracted from these fractions and was analyzed by
Northern blotting; blots were probed for perilipins using a full-length
perilipin A cDNA probe. A and B are samples
from cells expressing mutated perilipin N6; C and
D are samples from cells expressing mutated perilipin C7;
E and F are samples from cells expressing mutated
perilipin 5. The high density of fractions containing abundant
mRNA for the mutated perilipins (A, C, and
E) indicates efficient translation of the mRNAs.
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Fig. 9.
The central domain of perilipin A is
sufficient to redirect the soluble green fluorescent protein to lipid
droplets. A, 3T3-L1 fibroblasts stably or transiently
expressing GFP fused to full-length perilipin A (GP1), GFP
fused to the central region of perilipin including amino acids 233-345
(GP2), GFP fused to mutated perilipin A lacking the
amino-terminal 248 amino acids (GP3), GFP fused to a
perilipin peptide containing amino acids 2-232 (GP4), and
GFP alone (G) and growing on coverslips were mounted on
glass slides in PBS and visualized with a light microscope. Full-length
perilipin A (GP1), the central domain of perilipin
(GP2), and mutated perilipin lacking the amino terminus
(GP3) altered the localization of GFP from a diffuse
cytoplasmic staining pattern (G) to localization at the
surfaces of lipid droplets; lipid droplets coated with full-length
perilipin A fused to GFP were clustered, whereas droplets coated with
the mutated perilipins fused to GFP (GP2 and GP3)
were dispersed in the cytoplasm. By contrast, the amino-terminal
peptide of perilipin failed to direct GFP to lipid droplets
(GP4). The background fluorescence of the cells was enhanced
to make the cells more visible. Bar = 10 µm.
Summary of experiments with GFP-perilipin A fusion proteins
" indicates the
failure to detect the fusion protein on lipid droplets. A "+" in
the Clusters? column indicates the appearance of clustered lipid
droplets in cells following the expression of the mutated perilipin-GFP
fusion protein in cells. NA, not applicable.
2,
3, and
4 mutated perilipin containing two, two, or one
hydrophobic domains, respectively, adhere to carbonate-washed lipid
droplets (Fig. 10C), the C6 and N5 mutated perilipins do
not, suggesting that the individual hydrophobic domains may not be
equally effective in anchoring perilipins to lipid droplets in the
absence of the remaining amino- and carboxyl-terminal sequences. We
cannot exclude the possibility that sequences in addition to the
hydrophobic domains are essential in positioning and anchoring
perilipins to lipid droplets.
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Fig. 10.
The central hydrophobic domains are
essential to anchor perilipin A to lipid droplets. Confluent
3T3-L1 fibroblasts ectopically expressing mutated perilipin A were
cultured in the presence of oleic acid for 24 h. Cells were
harvested and fractionated; lipid droplets were centrifuged through an
alkaline carbonate solution, the floating lipid droplet fractions were
delipidated, and the solubilized proteins were separated by SDS-PAGE,
electrophoretically transferred to nitrocellulose membranes, and
immunoblotted with either -PAT antibodies (panels
B and C) or antibodies raised against full-length
rat perilipin A (panel A). A,
lane 1, full-length perilipin A; lane
2, N1; lane 3, N2; lane
4, N3; lane 5, N4; lane
6, N5; lane 7, N6. B,
lane 1, full-length perilipin A; lane
2, C1; lane 3, C2; lane
4, C3; lane 5, C4; lane
6, C5; lane 7, C6; lane
8, C7. C, lane 1,
1;
lane 2,
2; lane 3,
3;
lane 4,
4; lane 5,
5.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4), is sufficient to mediate the targeting and
anchoring of perilipin to lipid droplets, but results obtained with
other mutations indicate that H3 is dispensable. However, if H3 and the
remainder of the carboxyl terminus are deleted, then both H1 and H2 are
necessary to tether the protein to the lipid droplets. It is likely
that the context of the amino acids surrounding the hydrophobic domains
plays a role in positioning the protein with respect to the droplet,
and that the individual hydrophobic domains may not play equivalent
roles in embedding the protein into the droplet. This idea gains
support from the observation that perilipin mutation N5 targets to
lipid droplets but fails to withstand the carbonate wash conditions
despite the presence of three hydrophobic domains; in the absence of
the adjacent amino acids, the positioning of this mutated protein at
the surface of the droplets may be insufficient to support firm
anchoring into the droplet.
-pleated sheets may be shallowly
embedded into the surface phospholipids of the droplets; this
positioning of the amino terminus may help perilipin A to serve its
function of stabilizing the lipid droplet. Alternatively, these regions
could be folded in upon themselves with the hydrophobic faces in close
proximity, in which case the "globular" arrangement would limit the
contact of the protein with the surface of the lipid droplet and may
lead to a less stabilizing configuration. In our hypothetical model of
perilipin association with lipid droplets, the extreme amino and
carboxyl termini may be positioned near the surface of the lipid
droplet, without being embedded. These sequences contain dispersed
charged amino acids that might engage in interactions with other
cellular proteins. Deletion of the carboxyl terminus or portions of the
central domain leads to the dispersion of lipid droplets (Figs. 4 and
5) that appear in a clustered arrangement when full-length perilipin A or amino-terminal deletion mutations (Fig. 3) are expressed; thus, residues within the carboxyl terminus and central domain may
participate in interactions that promote the clustering of lipid
droplets in one or two regions of the cytoplasm.
1 and
3); these observations
suggest that the perilipin sequence lacks short segments that are
required to prevent rapid degradation. The only mutated perilipins that failed to target lacked large portions of the protein sequence. Furthermore, the addition of oleic acid to the culture medium, which
has been demonstrated to stabilize overexpressed perilipin A (16),
failed to stabilize the non-targeting mutated perilipins.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Susan Fried, Nathan Wolins, and Judith Storch for helpful advice and critical review of the manuscript. We thank Dr. Constantine Londos for sharing reagents and for useful discussions. We also thank Dr. Joseph Nickels for the GFP expression vector and advice. We are also grateful to Boris Rubin, Maryellen Sepelya, Deanna DiDonato, Amy Marcinkiewicz, Jennifer Lin, Justin P. Hart, and Lawrence Park for technical support.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK54797 and by a research award from the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom all correspondence should be addressed: Dept. of
Nutritional Sciences, 96 Lipman Dr., Rutgers, State University of New
Jersey, New Brunswick, NJ 08901. Tel.: 732-932-6524; Fax: 732-932-6837; E-mail: brasaemle@aesop.rutgers.edu.
Published, JBC Papers in Press, October 28, 2002, DOI 10.1074/jbc.M206602200
2 D. L. Brasaemle, N. E. Wolins, and B. Rubin, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
PKA, cAMP-dependent protein kinase;
-COT, polyclonal
antibodies raised against the carboxyl terminus of perilipin A;
-PAT, polyclonal antibodies raised against the amino terminus of
perilipin A;
BSA, bovine serum albumin;
GFP, green fluorescent protein;
H1, H2, and H3, first, second, and third hydrophobic domains of
perilipin A, respectively;
PBS, phosphate-buffered saline;
aa, amino acid(s).
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REFERENCES |
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