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INTRODUCTION |
TIP47 was first identified by a yeast two-hybrid screen using the
cytosolic domain of the cation-dependent mannose
6-phosphate receptor (M6PR)1
as a bait to screen an expression library from human Jurkat cells (1).
A glutathione S-transferase TIP47 fusion protein was
subsequently shown to bind to glutathione S-transferase
fusion proteins of the cytosolic tails of both the
cation-dependent and cation-independent M6PRs in
vitro and thus, this protein was named TIP47
(tail-interacting protein of 47 kDa). Diaz and Pfeffer (1) have proposed that TIP47 directs the
retrieval of M6PRs from a prelysosomal compartment with delivery back
to the trans-Golgi network through the interaction of TIP47
with the cytoplasmic tails of M6PRs. An essentially identical cDNA,
PP17a1 (2), and a truncated version of this cDNA,
PP17a2, have been obtained by screening a human placental
expression library with an antibody raised against a 38-kDa protein
purified from human placenta (3). Interestingly, the amino acid
sequence of TIP47 (PP17a) is highly similar to sequences from the
members of a growing family of lipid droplet-associated proteins that includes perilipins and the adipose differentiation-related protein (ADRP), also called adipophilin. The amino acid sequence of TIP47 (1)
is 43% identical to ADRP throughout the length of the sequence and
bears higher homology (60% identity, 80% similarity to ADRP) in an
amino-terminal region that is shared by the perilipins; hence, these
proteins may all be members of a common family. The perilipins are
encoded by a single gene; alternative splicing yields multiple
mRNAs that are translated into three different isoforms that have
similar amino termini but differ in the amino acid sequences of the
carboxyl termini. The perilipins are specifically associated with lipid
droplets in adipocytes (4, 5) and steroidogenic cells (6) and, to date,
have been found neither in any other cell type nor in any other
intracellular compartment. ADRP (7) is found on lipid droplets in many
cultured cell lines and tissues (8, 9) and is also found on secreted
milk lipid globules (10). The perilipins and ADRP have similar
amino-terminal sequences; 105 consecutive amino acids are 32%
identical, and 65% are similar (11). Detailed subcellular localization
studies are lacking for several additional recently described family
members. S3-12 (12) is a protein of 1403 amino acids that has a
33-amino acid segment that shares sequence similarity with ADRP; this
segment is repeated 29 times. The region of ADRP that is similar to the S3-12 repeats lies outside of the sequence that is conserved between perilipins and ADRP, and hence, it appears that the perilipins lack
strong similarity to the 33-amino acid repeated sequence of S3-12.
Finally, a Blast search (13) of GenBankTM reveals two
predicted sequences from Drosophila that share similar amino
termini with ADRP and perilipins. The function and tissue distribution
of these newly identified Drosophila proteins have not yet
been addressed.
Lipid droplets are largely uncharacterized subcellular organelles found
in the cytosol of most mammalian tissues and cultured cell lines as
well as in the adipose tissue of other chordates, the fat body of
insects, and the seeds of plants. Lipid droplets are spherical
structures composed of a core of triacylglycerols and cholesterol
esters covered by a monolayer of phospholipid. While large lipid
droplets in adipose tissue store the body's major energy supply as
triacylglycerols, histological sections of other nonadipose tissues
including liver, intestine, muscle, kidney, heart, adrenal gland,
testes, ovary, and mammary gland (14-16) also demonstrate the presence
of small lipid droplets. In many of these tissues, the lipid droplets
are primarily composed of cholesterol esters that play an important
role in maintaining cellular cholesterol homeostasis by providing a
source of cholesterol for membrane synthesis when circulating
cholesterol levels are low. Additionally, steroidogenic cells such as
those found in the adrenal cortex, testes, and ovaries use stored
cholesterol esters as a source of substrate for steroid hormone
synthesis. Most nonadipose cultured cell lines package neutral lipids
into small lipid droplets, and the numbers of these lipid droplets are
largely determined by the composition of the culture media (17).
Typical culture medium is very low in lipid content, and the cells
grown in these lipid-starved conditions have few, if any, lipid
droplets. By contrast, the number of cytoplasmic lipid droplets
increases dramatically upon supplementation of culture media with
physiologic levels of free fatty acids or lipoproteins (17, 18). Thus,
the formation of lipid droplets is controlled, at least in part, by the
availability of exogenous or circulating substrates for neutral lipid
synthesis. The release of neutral lipids from lipid droplets is
regulated by extracellular hormones or intracellular mechanisms that
signal the need for energy or building materials for membrane
synthesis. Thus, it is reasonable to expect that the lipid droplet
surface contains proteins involved in controlling this flux, given that
the flux of lipids both to and from lipid droplets are regulated
processes. Nonetheless, few lipid droplet-associated proteins have been
identified, and the study of the proteins associated with lipid
droplets is an emerging area of inquiry.
Due to the striking similarity between the amino acid sequences of
TIP47 (PP17a1), ADRP, and the perilipins, we hypothesized that TIP47 may associate with lipid droplets. Using polyclonal antibodies raised against TIP47 (1), we investigated the subcellular localization of TIP47 and found that it associates with lipid droplets.
These observations raise the question as to whether or not TIP47
functions exclusively as a component of a selective sorting mechanism
that directs proteins to vesicular endosomal compartments and imply a
potential function for TIP47 in the regulation of the metabolism of
neutral lipids.
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EXPERIMENTAL PROCEDURES |
Antibodies--
The anti-human TIP47 polyclonal antiserum was
donated by Dr. Suzanne Pfeffer (1). The anti-mouse ADRP (mADRP)
polyclonal antiserum was donated by Dr. Charles Schultz (National
Institutes of Health, Bethesda, MD). The anti-human ADRP (hADRP)
monoclonal antibody and anti-calnexin antibody were purchased from
Research Diagnostics Inc. (Flanders, NJ) and Transduction Laboratories, Affiniti Research Products Ltd. (Mamhead, UK), respectively. The polyclonal anti-M6PR antibody raised against the cation-independent M6PR was donated by Dr. Thomas Braulke (Georg-August-University, Gottingen, Germany). Tetramethyl rhodamine-conjugated anti-goat and
fluorescein-conjugated anti-rabbit IgGs were obtained from Jackson
Immunoresearch (West Grove, PA). Alexa 594-conjugated anti-rabbit and
Alexa 488-conjugated anti-mouse IgGs were obtained from Molecular
Probes, Inc. (Eugene, OR).
Cell Culture--
Human HeLa cells, human U937 monocytes and
human K562 cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 2 mM glutamine,
100 units/ml penicillin, and 100 µg/ml streptomycin. Murine MA10
Leydig cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 15% horse serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. When cells were
grown in media supplemented with fatty acids, oleic acid was complexed
to bovine serum albumin at a ratio of 6 mol of oleic acid/mol of
albumin; the final concentration is indicated in the figure legends.
All cells were cultured in a 5% CO2 atmosphere at
37 °C.
Northern Blot Analysis--
RNA was extracted using an RNeasy
minikit (Qiagen) following the protocol recommended by the
manufacturer. The extracted RNA was resolved by agarose gel
electrophoresis, transferred to charged nylon membranes (Hybond N+;
Amersham Pharmacia Biotech), and probed following the manufacturer's
protocol for the NorthernMax-Gly kit (Ambion, Austin, TX).
Fluorescence Microscopy--
HeLa cells were cultured on glass
coverslips and were fixed with 2% formaldehyde in phosphate-buffered
saline for 20 min. Primary and secondary antibodies were added
sequentially to the fixed cells in phosphate-buffered saline containing
0.1% saponin and 0.1% bovine serum albumin. The fluorophore Bodipy
493/503 (Molecular Probes) specifically stains neutral lipids (19) and has narrow absorption and emission spectra, thus allowing for the use
and detection of a second fluorophore. Bodipy 493/503 was dissolved in
ethanol at 1 mg/ml and then added to secondary antibody solutions to a
final concentration of 10 µg/ml. Coverslips were mounted on glass
slides using Fluoromount G (Southern Biotechnology Associates Inc.,
Birmingham, AL). Images from the samples were acquired using a Zeiss
LSM 410 confocal microscope (Carl Zeiss Inc., Thornwood, NY).
Metabolic Labeling of Cells and Immunoprecipitation of
TIP47--
For each condition, HeLa cells from confluent monolayers
grown in 150-mm culture dishes were incubated with
[35S]methionine and [35S]cysteine
(EasytagTM EXPRE35S35S; PerkinElmer
Life Sciences) in suspension in Dulbecco's modified Eagle's medium
lacking methionine and cysteine, supplemented with 25 mM
HEPES, pH 7, for 30 min at 37 °C. When incubation was required after
labeling, cells were resuspended in complete Dulbecco's modified
Eagle's medium supplemented with 25 mM HEPES, pH 7. Cells were collected by low speed centrifugation, and pellets were
solubilized in 50 mM Tris, pH 7.4, with 1% Triton X-100,
0.5% sodium deoxycholate, 300 mM NaCl, 1 mg/ml
iodoacetamide, 10 mg/ml leupeptin, 100 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 1 mM benzamidine, and 1 mM EDTA.
Immunoprecipitations of TIP47 were performed, as described previously
(20). The immunoprecipitated proteins were resolved by
SDS-polyacrylamide gel electrophoresis and revealed by fluorography.
Disruption of Cells by Hyperosmotic Shock--
HeLa or MA10
Leydig cells were scraped into phosphate-buffered saline and pelleted
by low speed centrifugation. The cell pellets were dispersed by
vortexing, concurrent with the dropwise addition of 70% (w/w) sucrose
dissolved in lysis buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 10 µg/ml leupeptin, 100 µM
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 1 mM benzamidine, 1 mM EDTA) at room temperature. The cells were then incubated on ice for 10 min, during which time they
were vortexed for 30 s every 2 min. To maximize the osmotic shock,
3 ml of lysis buffer were added rapidly while vortexing the sample. The
homogenate was incubated for an additional 10 min on ice and vortexed
for 30 s every 2 min; the cells were then further disrupted by
passing them through a 27-gauge needle four times.
Fractionation of Cells by Centrifugation of Sucrose
Gradients--
Confluent monolayers of HeLa or MA10 Leydig cells were
disrupted by hyperosmotic shock, as described above, and then
centrifuged for 10 min at 1000 × g at 4 °C. The
supernatant was mixed 1:1 with 70% sucrose (w/w) and layered onto a
cushion of 0.6 ml of 50% sucrose (w/w). A sucrose gradient, as
described in the figure legends, was layered over the sucrose-cell
supernatant. The gradients were centrifuged for 4 h at
154,000 × g in a Beckman SW41Ti rotor at 4 °C. The
buoyant fraction was collected by slicing off the tops of the tubes
with a Beckman tube slicer, and this fraction was brought to 1 ml with
lysis buffer. Eleven additional 1-ml fractions were collected.
Fractionation of HeLa Cells by Differential
Centrifugation--
HeLa cells from a confluent 150-mm dish were
disrupted by hyperosmotic shock, and the homogenate was centrifuged for
10 min at 1000 × g at 4 °C. The supernatant was
collected, adjusted to a volume of 4 ml with lysis buffer, and
centrifuged in a SW60Ti rotor for 1 h at 165,000 × g at 4 °C. The buoyant fraction was collected with a tube
slicer, as described previously.
Analysis of Cellular Fractions--
Immunoblotting was performed
as described previously (8). Neutral lipids were extracted in solvents
and separated by thin layer chromatography and revealed, as described
previously (21). Gradient fractions were assayed for activity of the
cytosolic enzyme lactate dehydrogenase (22) and the integral
endoplasmic reticulum enzyme NADPH- dependent cytochrome c
reductase (23).
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RESULTS |
Characterization of TIP47 mRNA and Protein in HeLa
Cells--
Since the amino acid sequence of TIP47 indicates a close
relationship of TIP47 to a family of lipid droplet-associated proteins (24), we investigated the hypothesis that TIP47 localizes to lipid
droplets. Previously, when a TIP47 (PP17a1) cDNA was
used to probe Northern blots of total HeLa RNA, multiple mRNAs were detected, and when immunoblots were probed with an antibody raised against a 38-kDa protein from placenta that shares sequence identity with TIP47, multiple proteins were detected in most of the tissues that
were tested (2). To characterize the expression of TIP47 in HeLa cells,
we used Northern blotting, immunoblotting, and immunoprecipitation
techniques to show that a single mRNA encodes a single protein
isoform of TIP47 (Fig. 1). A Northern
blot of total RNA from HeLa cells probed with a human TIP47 cDNA
probe showed an mRNA consistent with a size of 1998 base pairs
(Fig. 1A), the predicted size of the TIP47 mRNA (1).
Immunoblots of proteins from HeLa cells probed with anti-TIP47
antiserum revealed a single protein of ~46 kDa (Fig. 1B).
Human K562 cells, which were used in the initial characterizations of
TIP47 (1), also displayed a single protein of ~46 kDa in TIP47
immunoblots (Fig. 1B). Additional immunoblots of proteins
from cultured murine 3T3-L1 adipocytes (not shown) and rat MA10 Leydig
cells (Fig. 5) showed a single TIP47 protein isoform. Furthermore,
anti-TIP47 antiserum immunoprecipitated a single protein of ~46 kDa
from homogenates of HeLa cells and human U937 monocytes (Fig.
1C), and rat MA10 Leydig cells (not shown) radiolabeled with
[35S]methionine and [35S]cysteine. Thus,
several cultured cell lines express a single isoform of TIP47.

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Fig. 1.
HeLa cells have a single mRNA encoding
TIP47; HeLa cells, k562 cells, and U937 monocytes express a single
protein isoform of TIP47. A, Northern blot of total RNA
from HeLa cells hybridized with a radiolabeled cDNA probe for TIP47
(upper panel) and the 18 S ribosomal subunit from
the lanes stained with ethidium bromide (lower
panel). Each lane contains 10 µg of total RNA
extracted from HeLa cells. Fatty Acid + indicates
that the cells were grown with 400 µM oleic acid
complexed to albumin for 19 h before harvest. B,
immunoblots of proteins from HeLa cells (lane 1)
and K562 cells (lane 2) separated by
SDS-polyacrylamide gel electrophoresis and probed with an anti-TIP47
polyclonal antiserum. C, autoradiograph of proteins from
HeLa cells and U937 monocytes that were labeled for 4 h with
[35S]methionine and [35S]cysteine,
immunoprecipitated with anti-TIP47 antiserum, and resolved by
SDS-polyacrylamide gel electrophoresis. D, autoradiograph of
SDS-polyacrylamide gels containing proteins from HeLa cells labeled
with [35S]methionine and [35S]cysteine for
30 min (lanes 1 and 2) and chased
without label for 6 h (lane 2) before
immunoprecipitation with anti-TIP47 antiserum. E, immunoblot
of proteins from lipid-loaded MA10 Leydig cell homogenates probed with
anti-mADRP antiserum (lane 1) or anti-TIP47
antiserum and anti-mADRP antiserum (lane
2).
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Since ADRP and TIP47 share sequence similarity, it is possible that the
anti-TIP47 antiserum recognizes ADRP. To test this, immunoblots of
proteins from MA10 Leydig cell homogenates were probed with anti-mADRP
antiserum (Fig. 1E) and then reprobed with the anti-TIP47
antiserum, without stripping the anti-mADRP antibody from the blots.
The anti-mADRP antiserum recognized a single protein band and the
anti-TIP47 antiserum recognized a single band of a slightly higher
molecular weight, thus demonstrating that the TIP47 antibody and the
ADRP antibody recognize discrete proteins.
The expression of perilipins (21) and ADRP (8, 25), lipid
droplet-associated proteins related to TIP47, are regulated by the
addition of fatty acids to the medium of cultured cells. By contrast,
we observed no change in the level of TIP47 mRNA (Fig.
1A) or protein (Fig. 6) following the addition of oleic acid
to the culture medium. We did, however, observe a dramatic change in
the subcellular compartmentalization of TIP47 following the addition of
fatty acids to the culture medium (Figs. 4-6). Additionally, the
immunoprecipitation of TIP47 under nondenaturing conditions yielded a
single radiolabeled band (Fig. 1, C and D),
thus suggesting that TIP47 may not form stable complexes with other
cellular proteins Finally, when using a pulse-chase protocol, greater
than 50% of radiolabeled TIP47 was stable throughout a 6-h chase
period (Fig. 1D), thus demonstrating that cellular TIP47 has
a relatively long half-life.
Antibody to TIP47 Decorates Neutral Lipid Droplets--
The
subcellular localization of TIP47 was examined by immunofluorescence
microscopy of HeLa cells grown in both the absence and presence of
supplemental lipids. Subcellular lipid droplets were visualized with
Bodipy 493/503, a stain for neutral lipids. Furthermore, since TIP47
has been described as a protein that interacts with the M6PR (1), HeLa
cells were doubly stained with anti-M6PR antiserum and anti-TIP47
antiserum, and the localization of the two proteins was examined by
immunofluorescence microscopy. The staining pattern for M6PR in HeLa
cells was greatest in the juxtanuclear region with some staining of
peripheral vesicles and the plasma membrane (Fig.
2A) and resembled that
previously shown for M6PR in embryonic bovine trachea cells and COS
cells (1); by contrast, the staining for TIP47 was both diffuse through the cytoplasm and specifically localized to punctate structures but was
rarely coincident with the staining for M6PR when cells were doubly
stained for both proteins (Fig. 2A). When the cells were
supplemented with fatty acids to increase neutral lipid storage, the
anti-TIP47 antiserum stained primarily larger, uniformly spherical structures that were not localized to the regions of most intense M6PR
staining (Fig. 2B).

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Fig. 2.
Anti-TIP47 polyclonal antiserum stains lipid
droplets in HeLa cells. HeLa cells were fixed with formaldehyde,
permeabilized with saponin, and stained. A, C,
E, and G show HeLa cells grown under normal low
lipid-containing culture conditions; B, D,
F, and H show HeLa cells grown with 600 µM supplemental oleate for 19 h. A and
B show staining with anti-TIP47 antiserum in green and
staining with anti-M6PR antiserum in red.
C and D show staining with anti-TIP47 antiserum
in green and neutral lipid staining in red;
coincident staining is depicted in yellow. The
inset to D shows a 3.4-fold magnification of the
indicated region of the lower cell. E and F show
staining with anti-TIP47 antiserum in green and anti-hADRP
antiserum in red; coincident staining is depicted in
yellow. Many cells showed staining for TIP47 but not ADRP;
when both stains were observed in a cell, they were usually coincident.
G and H show HeLa cells stained with anti-hADRP
antiserum in green and neutral lipid staining in
red. Inset to H shows a 4.1-fold
magnification of the indicated region of the cell to the
left. Bars, 10 µm.
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Cells grown under normal culture conditions and doubly stained for
neutral lipids and TIP47 revealed that the punctate structures detected
by the anti-TIP47 antiserum are lipid droplets, since the two stains
colocalized (Fig. 2C). Lipid loading of the cells increased
the sizes of the lipid droplets slightly and allowed the detection of a
distinct ring of TIP47 staining surrounding the neutral lipid core
(Fig. 2D). Interestingly, we observed that ~80% of HeLa
cells grown in the standard low lipid-containing culture medium
apparently lacked lipid droplets, while 20% of these cells contained
detectable lipid droplets; in the cells lacking lipid droplets, the
TIP47 staining appeared to be diffuse throughout the cytoplasm (data
not shown). When the cells were grown in culture medium containing
supplemental fatty acids, all of the cells displayed lipid droplets,
most of which were stained with the anti-TIP47 antiserum.
The lipid droplet protein ADRP, which shares 43% identity with TIP47,
covers the surfaces of lipid droplets in a number of types of cells (8,
9); this localization was confirmed in HeLa cells doubly stained for
neutral lipid and ADRP in the current study (Fig. 2, G,
H, and inset of H). To compare the
subcellular distribution of ADRP and TIP47, HeLa cells were doubly
stained with anti-hADRP monoclonal antibody and anti-TIP47 antiserum. In cells grown under normal culture conditions without lipid
supplementation, most of the cells that contained detectable lipid
droplets displayed staining for both TIP47 and ADRP (Fig.
2E). In lipid-loaded cells, the lipid droplets of almost all
of the cells stained strongly with anti-TIP47 antiserum, while the
lipid droplets of approximately half of the cells were simultaneously
stained with anti-hADRP antibody (Fig. 2F); occasional cells
showed lipid droplets that were decorated solely with anti-ADRP
antibodies. The coincident staining of lipid droplets with both
antibodies suggests colocalization of these highly related proteins on
the surfaces of lipid droplets. Additionally, these observations imply
that HeLa cells are a heterogeneous population of cells with regard to
the expression of lipid droplet-associated proteins. Furthermore, this
experiment provides additional proof that the anti-hADRP antibody and
the anti-TIP47 antiserum recognize distinct proteins despite the
similarities of the amino acid sequences between the two proteins,
since the lipid droplets of some cells stain only with anti-TIP47
antiserum, while others stain only with anti-hADRP antibody.
Finally, the association of TIP47 with lipid droplets is not unique to
HeLa cells; lipid droplets in primary mouse fibroblasts, human
melanocytes, and 3T3-L1 adipocytes all show surface immunostaining for
TIP47 (data not shown).
The Subcellular Distribution of TIP47 Is Unchanged When Cells Are
Treated with Brefeldin A--
The treatment of cells with the fungal
metabolite brefeldin A causes extensive rearrangements of membranous
compartments in the secretory pathway, including the recruitment of
endosomal structures into a tubular reticulum (26). As expected, the
treatment of HeLa cells with brefeldin A caused a redistribution of
M6PR staining from a primarily juxtanuclear region to more peripheral structures, while having no obvious effect on TIP47 staining (Fig. 3), thus demonstrating that the bulk of
the TIP47 is located in a brefeldin A-insensitive compartment, while
the bulk of M6PR is in a brefeldin A-sensitive compartment.

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Fig. 3.
Brefeldin A alters the distribution of M6PR
but fails to perturb the localization of TIP47. HeLa cells were
cultured in medium supplemented with 600 µM oleate for
18 h. Cells shown in the lower panels were
then treated for 20 min with 5 µg/ml brefeldin A; cells in the
upper panels were incubated in the absence of
brefeldin A. The cells were immediately fixed and doubly stained with
anti-TIP47 antiserum (green; center
panels) and anti-M6PR antiserum (red;
left panels). The right
panels show superimposed staining for TIP47 and M6PR.
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TIP47 Cofractionates with Neutral Lipids--
To assess the
subcellular localization of TIP47 biochemically, HeLa cells and MA10
Leydig cells were fractionated by flotation on sucrose density
gradients. Lipid droplets have a neutral lipid core comprising most of
their volume and rendering them buoyant in aqueous solution, while
membranous subcellular compartments have a higher protein/lipid ratio,
giving them greater density. In HeLa or MA10 Leydig cells grown in
standard low lipid-containing culture media, TIP47 (Fig.
4A, Fatty
Acid (
), and Fig.
5A, Fatty Acid (
)) fractionated with lactate dehydrogenase (Figs.
4B and 5B), an enzyme used as a marker for
cytosol. Low levels of TIP47 also cofractionated with calnexin, a
marker for endoplasmic reticulum, in HeLa cells (Fig. 4A),
and with cytochrome c reductase activity, an alternative
marker for endoplasmic reticulum, in MA10 Leydig cells (Fig. 5,
A and B). In fractions from HeLa cell
homogenates, the distributions of TIP47 and M6PR overlapped in the
lower portion of the sucrose gradients (Fig. 4A). These
findings are consistent with the subcellular distribution described
previously for TIP47 in COS cells (1). In the current studies, when
HeLa cells and MA10 Leydig cells were grown under standard low
lipid-containing culture conditions, no neutral lipid was detected in
the gradients (Fig. 4A or 5A,
Triacylglycerol panels, Fatty
Acid (
)), thus indicating a lack of detectable lipid
droplets in cellular fractionation experiments. Clearly, some of these
cells contain lipid droplets, as shown in microscopy experiments, as
depicted in Fig. 2, C and G.

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Fig. 4.
Lipid loading of cells increases the
accumulation of triacylglycerols in lipid droplets; TIP47 becomes
buoyant and fractionates with lipid droplets. HeLa cells were
grown in culture medium supplemented with 600 µM oleic
acid, (Fatty Acid (+)), or normal low
lipid-containing culture medium (Fatty Acid
( )), for 19 h before being harvested, homogenized, and
fractionated by centrifugation of sucrose gradients. Each gradient had
a 0.6-ml 50% (w/w) sucrose cushion, 3.4 ml of postnuclear supernatant
corresponding to 7 mg of cellular protein in 40% (w/w) sucrose and 1 ml of 35% sucrose, overlaid with a 7-ml linear 0-30% sucrose
gradient. A shows immunoblots depicting the distributions of
the endosomal marker, M6PR, ER protein, calnexin, and TIP47, and thin
layer chromatography data depicting the distributions of
triacylglycerol (lipid droplets) and cholesterol (membranes) across the
fractions from the gradients. B shows the distribution of
the activity of the cytosolic enzyme lactate dehydrogenase across the
gradients.
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Fig. 5.
When triacylglycerols accumulate, TIP47 is
recruited from the cytosolic fraction onto lipid droplets, while ADRP
is found exclusively on lipid droplets. MA10 Leydig cells were
cultured in 600 µM supplemental oleate (Fatty
Acid (+)) or normal low lipid-containing culture medium
(Fatty Acid ( )) for 19 h before being
harvested, homogenized, and fractionated by the centrifugation of
sucrose gradients. Each gradient had a 0.6-ml 50% (w/w) sucrose
cushion, 5 ml of postnuclear supernatant corresponding to 15 mg of
cellular protein in 40% (w/w) sucrose, 1.5 ml of 35% sucrose, 1.5 ml
of 20% sucrose, and 1.5 ml of 10% sucrose, overlaid with 1.9 ml of
lysis buffer. A shows immunoblots depicting the
distributions of ADRP and TIP47 and thin layer chromatography data
depicting the distributions of triacylglycerol (lipid droplets) and
cholesterol (membranes). B shows the distributions of the
activities of the cytosolic enzyme lactate dehydrogenase ( ) and the
ER membrane marker NADPH-dependent cytochrome C
reductase ( ).
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Most cultured cells store small amounts of triacylglycerols and
cholesterol esters in a few lipid droplets, and when the cells are
incubated with fatty acids, the mass of triacylglycerol stored in the
droplets increases. The lipid droplets of Leydig cells contain
primarily cholesterol esters; this cholesterol serves as a substrate
for the synthesis of steroid hormones (27). In the current studies,
when MA10 Leydig cells were grown in media supplemented with fatty
acids, they accumulated both cholesterol esters (data not shown) and
triacylglycerols (Fig. 5A, Fatty Acid (+), Triacylglycerol). By contrast, HeLa cells accumulated
primarily triacylglycerols. For both cell types, triacylglycerols were
detected predominantly in the most buoyant fractions (Figs.
4A and 5A, Fatty Acid (+),
Triacylglycerol panels, lane
12). Under these culture conditions, the most buoyant
fractions also contained the greatest mass of TIP47, thus demonstrating
that TIP47 localizes to newly synthesized lipid droplets.
Following the incubation of either HeLa cells or MA10 Leydig cells with
fatty acids, ~70 or 25% of the total cellular TIP47 localized to
lipid droplets in subcellular fractions from HeLa cells or MA10 Leydig
cells, respectively; an additional portion of the TIP47 shifted from
the cytosol to fractions containing markers for the endoplasmic
reticulum in each type of cell. In fractions from HeLa cells grown in
the absence of supplemental lipids, TIP47 was found primarily in
fractions 1-5 (Fig. 4A, Fatty Acid
(
)) that contained the highest levels of lactate dehydrogenase (Fig.
4B), a cytosolic marker. When these cells were incubated with fatty acids, 20% of the mass of TIP47 was found in fractions 4 and 5 that contained the majority of calnexin, a marker for endoplasmic
reticulum, and cholesterol, a marker for cellular membranes (Fig.
4A, Fatty Acid (+)), with a
corresponding decrease in the mass of TIP47 found in fractions 1-3.
Likewise, in fractions from MA10 Leydig cells grown in the absence of
supplemental lipids, TIP47 colocalized with lactate dehydrogenase in
fractions 1-6 (Fig. 5, A and B, Fatty
Acid (
)), and lipid loading shifted ~10% of TIP47 to
fractions 6-8, which contained the majority of cholesterol and
activity of NADPH-dependent cytochrome c
reductase, a marker for endoplasmic reticulum (Fig. 5, A and
B, Fatty Acid (+)). Thus, when cells
are incubated under conditions that promote the synthesis of neutral
lipids and the packaging of these lipids into lipid droplets, TIP47
moves into the most buoyant fraction that contains lipid droplets, as
well as into the fractions that contain the bulk of the endoplasmic
reticulum, the site of neutral lipid synthesis (28-30), and,
presumably, the initial site of lipid droplet formation.
To confirm the observation of the colocalization of TIP47 and ADRP at
the surfaces of lipid droplets obtained in the microscopy studies, the
subcellular fractions from the sucrose gradients were immunoblotted for
ADRP. MA10 Leydig cells, which show more homogeneous ADRP expression
than HeLa cells (Fig. 2), were used to compare the subcellular
distribution of TIP47 and ADRP. ADRP was not detected in fractions from
cells grown in standard low lipid-containing culture media (Fig.
5A, Fatty Acid (
), ADRP panel); however, when the cells were supplemented with fatty
acids, ADRP became easily detectable and was found almost exclusively in the most buoyant fractions (Fig. 5A, Fatty
Acid (+), ADRP panel). By contrast,
lipid loading of the cells did not change the total cellular mass of
TIP47 but, rather, dramatically altered the subcellular distribution of
TIP47 from a predominantly cytosolic localization onto both lipid
droplets and membranes.
Lipid Loading Induces the Movement of TIP47 from the Cytosol onto
Newly Synthesized Lipid Droplets--
Culturing cells with
supplemental fatty acids causes TIP47 to associate with lipid droplets,
as characterized by both immunofluorescence microscopy and subcellular
fractionation experiments (Figs. 2, 4, and 5). To characterize the
recruitment of TIP47 from the cytosol onto lipid droplets, HeLa cells
were cultured in media supplemented with fatty acids for 0-8 h before
harvest and subcellular fractionation. During this time, the total
cellular mass of TIP47 was minimally changed (Fig.
6, Total TIP47). By
1 h of culture in the presence of supplemental fatty acids, the
levels of triacylglycerols in the most buoyant fraction increased to
clearly detectable levels concomitant with the appearance of TIP47 in
this fraction (Fig. 6, Triacylglycerol and
Buoyant TIP47). During the 8-h incubation of the
cells with fatty acids, both the mass of triacylglycerol and the mass
of TIP47 in the most buoyant fraction increased proportionally and in
parallel, while the cytosolic pool of TIP47 was depleted (data not
shown); these observations suggest that TIP47 moves onto nascent lipid
droplets.

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Fig. 6.
TIP47 accumulates in parallel with
triacylglycerol in the most buoyant fraction. HeLa cells were
incubated with supplemental 600 µM oleate for the times
indicated before being disrupted by hyperosmotic shock and fractionated
by differential centrifugation. The most buoyant fraction was collected
from each gradient and assayed for triacylglycerol (Buoyant
TG; upper panel) by thin layer
chromatography and TIP47 (Buoyant TIP47;
middle panel) by immunoblotting. The lower panel
(Total TIP47) shows an immunoblot of TIP47 in
0.9% of the starting postnuclear supernatants prior to fractionation;
this panel indicates that the total mass of cellular TIP47
is equivalent at all times. The exposure time for the immunoblot in the
middle panel was different from that of the
lower panel; exposure times were selected to best
compare the TIP47 levels within a given panel.
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DISCUSSION |
The primary finding of this study is that TIP47 is a lipid
droplet-associated protein. In microscopy experiments, TIP47 staining was observed on the surfaces of small, rare lipid droplets in HeLa
cells grown in low lipid-containing media as well as on the somewhat
larger and more numerous lipid droplets of cells grown in media
supplemented with fatty acids to increase neutral lipid storage.
Furthermore, TIP47 colocalized with ADRP, a lipid droplet-specific protein (8, 9, 10), on the surfaces of lipid droplets in HeLa cells
grown under both conditions. Subcellular fractionation of both human
HeLa cells and murine MA10 Leydig cells grown under lipid-enriched
conditions showed that much of the cellular TIP47 localizes to the
buoyant lipid droplet fraction along with the majority of the cellular
triacylglycerol, cholesterol ester, and ADRP.
TIP47 shares sequence homology with at least two other lipid
droplet-associated proteins, ADRP and perilipins. Perilipins localize
exclusively to lipid droplets in adipocytes and steroidogenic cells
(4-6) and have been found in no other subcellular compartment. ADRP
has also been localized to lipid droplets in a wide variety of cells
and tissues (8-10). By contrast, TIP47 is abundant in the cytosol of
HeLa cells and MA10 Leydig cells cultured in typical low
lipid-containing medium lacking supplemental lipids, as well as
associated with the few small lipid droplets that occur under these
culture conditions. Interestingly, the addition of physiological levels
of fatty acids to the culture medium leads to the rapid synthesis of
triacylglycerols, and the accumulation of TIP47 on lipid droplets with
a concurrent loss of TIP47 from the cytosol, thus suggesting that TIP47
is recruited from the cytosol onto nascent lipid droplets.
TIP47 has been proposed to be a cargo selection device for the sorting
of M6PRs into transport vesicles (1). Several observations make it
difficult to reconcile our data with an exclusive role for TIP47 in
determining the specificity of M6PR sorting in the cell. First, in
addition to having a cytosolic localization, TIP47 associates with
lipid droplets in a manner that is responsive to the status of neutral
lipid synthesis and storage in the cell. Second, while the subcellular
localization of M6PR is dramatically altered by the treatment of cells
with brefeldin A, the distribution of TIP47 is unaffected; thus, the
two proteins appear to occupy different subcellular compartments.
Third, TIP47 has 43% sequence identity with ADRP over the entire
length of the proteins; thus, it is likely that these proteins maintain
a similar secondary structure. It is unlikely that ADRP plays a general
role in protein trafficking, since ADRP has been found only in cells
storing neutral lipid, with most or all of the protein on lipid
droplets. Fourth, we have found that strong alkaline solutions of 100 mM sodium carbonate fail to extract both TIP47 and ADRP
from isolated lipid droplets (data not shown); this treatment has been
shown to disrupt electrostatic interactions while leaving hydrophobic
interactions intact (23). Thus, both ADRP and TIP47 display similar
characteristics of proteins integrally associated with lipid droplets.
In conclusion, the current data describing the localization and tight
association of TIP47 with lipid droplets, together with the sequence
similarity between TIP47 and other lipid droplet-associated proteins,
support the hypothesis that TIP47 plays a role in the metabolism of
intracellular neutral lipids and not in the sorting of secretory proteins.
Does a portion of cellular TIP47 associate with endosomes?
Immunofluorescence microscopy experiments conducted in both the presence and absence of brefeldin A failed to support the localization of TIP47 to M6PR-containing endosomes. While subcellular fractionation experiments indicated that a portion of TIP47 colocalized with membrane
fractions that contained endosomal markers, it is important to note
that these fractions also contained markers for other membranous
compartments such as ER; thus, the fractions containing membranes were
not adequately resolved to unambiguously identify which membrane
contains a small portion of the total cellular TIP47. Furthermore, the
observation that the association of TIP47 with membranes is dependent
upon lipid availability is suggestive of a role for TIP47 in lipid
trafficking or lipid droplet assembly. Although these data do not
preclude the possibility that a small amount of TIP47 is on an
endosomal compartment, they clearly show that the bulk of TIP47 is
either soluble or associated with neutral lipid droplets.
Most mammalian cells efficiently sequester and store neutral lipids in
droplets, yet very little is known about this process. The final steps
of neutral lipid synthesis occur in the endoplasmic reticulum (28-30).
It is highly likely that the initial events that lead to the formation
of lipid droplets occur in the endoplasmic reticulum and that the
droplets then dissociate from these membranes, since these droplets are
coated by a unique subset of cellular proteins that are not found in
other compartments (5, 8, 9). In the current study, TIP47 moved from
the cytosol to both membranes and lipid droplets during conditions that
promoted the rapid synthesis of neutral lipids and the formation of
lipid droplets. These observations raise the possibility that TIP47 has
a role in the packaging of neutral lipids into droplets.
We have found TIP47 to be widely expressed and localized to lipid
droplets, solidifying the notion that the perilipin/ADRP/TIP47 family
of proteins may function in regulating the storage of neutral lipids.
Chordates store much of the chemical energy required for their
existence in triacylglycerol-cored lipid droplets. Failure to have or
to mobilize this energy store is perilous when food supplies are
scarce. Conversely, in the human population, food consumption often
exceeds energy requirements, and the resulting obesity is a major
factor in the development of cardiovascular disease and diabetes. Thus,
the appropriate storage and mobilization of neutral lipid is critical
for viability. The lipid droplet proteins, ADRP and perilipins, are
located on the surfaces of the lipid droplets, between the stored
neutral lipids and the soluble lipases that mobilize these lipids; both
ADRP and perilipins have been proposed to play roles in maintaining
lipid homeostasis (31, 32). Given that TIP47 is found on the surfaces
of lipid droplets and that its cellular distribution is dependent upon the storage of neutral lipids, it is likely that TIP47 may also play a
role in lipid metabolism.