From the a Lipoprotein and Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa, Canada K1Y 4W7, the b Department of Biochemistry, Microbiology, and Immunology, the i Department of Pathology and Laboratory Medicine, University of Ottawa, Canada K1H 8M5, the e Cell Biology Program, Memorial Sloan-Kettering Cancer Center and Graduate Program in Biochemistry and Structural Biology, Weill Graduate School of Medical Sciences of Cornell University, New York, New York 10021, and the g Robarts Research Institute, London, Ontario, Canada N6A 5K8
Received for publication, November 25, 2002, and in revised form, January 15, 2003
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ABSTRACT |
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We examined the role of S-linked
palmitoylation of human apolipoprotein (apo) B in the assembly and
secretion of very low density lipoproteins using recombinant human
apoB48. There are four free cysteine residues
(Cys1085, Cys1396, Cys1478,
and Cys1635) within apoB48 that potentially can be
palmitoylated. All four cysteine residues were substituted with serine
by site-specific mutagenesis. The mutant protein was expressed in
transfected rat hepatoma McA-RH7777 cells. Metabolic labeling of the
stably transfected cells with iodopalmitic acid analog showed that the
mutant apoB48 lacked palmitoylation. The lack of palmitoylation had
little impact on the ability of apoB48 to assemble and secrete very low
density lipoproteins or high density lipoproteins. Immunocytochemistry experiments using confocal microscopy failed to reveal any major alterations in the intracellular distribution of the mutant apoB48 at
steady state. Pulse-chase analysis combined with subcellular fractionation showed no apparent deficiency in the movement of the
mutant apoB48 protein from the endoplasmic reticulum to
cis/medial Golgi. However, the mutant apoB48 lacking
palmitoylation showed retarded movement toward the distal Golgi and
increased association (>2-fold) with the membranes of the secretory
compartments. A marginal decrease (by 15-20%) in secretion efficiency
as compared with that of wild type apoB48 was also observed. These
results suggest that lack of palmitoylation may influence the
partitioning of apoB48 between microsomal membranes and microsomal
lumen, but it does not compromise the ability of apoB48 to assemble lipoproteins.
Hepatic apolipoprotein
(apo)1 B100 is synthesized as
a single polypeptide chain consisting of 4536 amino acids and is
secreted as a major protein constituent of very low density
lipoproteins (VLDL). Expression of hepatic apoB100 is regulated
primarily at a post-translational level (1, 2). Proper folding of
apoB100 and its association with lipids are assisted by interaction
with the chaperone-like protein termed microsomal triglyceride
transfer protein (3) and by post-translational modifications of the apoB100 polypeptide (4). Mutations within the microsomal triglyceride transfer protein gene are associated with abetalipoproteinemia, a genetic disorder characterized by the absence of lipoproteins containing apoB in the plasma (5). In vitro mutagenesis
experiments have shown that the assembly and secretion of lipoproteins
containing apoB are markedly affected by abnormal post-translational
modification of apoB, including disulfide bond formation (6, 7),
asparagine-linked glycosylation (8), and cysteine-linked palmitoylation
(9). In most of the in vitro experiments, carboxyl
terminally truncated apoB forms were used as a model to test the
function of apoB post-translational modifications. The mutant apoB
proteins display lowered secretion efficiency, enhanced intracellular
degradation, and impaired ability to assemble into lipid-rich
lipoproteins (6, 7). However, since the ability of apoB to recruit
lipid in forming VLDL is profoundly influenced by apoB length (10),
some of the apoB truncates used in the mutagenesis studies were too
short to assemble VLDL (6, 9). For example, the apoB truncation mutant
used in the palmitoylation experiments was apoB29 (the amino-terminal 29% of the full-length apoB100), which has no ability to form VLDL and
can only form small, dense particles resembling that of high density
lipoproteins (HDL). Therefore, the functional consequence of
palmitoylation of apoB in VLDL assembly and secretion remains unclear.
Palmitoylation of human apoB has been observed in cultured human
hepatoblastoma HepG2 cells (11), in plasma LDL (12), and in transfected
rat hepatoma McA-RH7777 cells (9). In eukaryotic cells, the 16-carbon
fatty acid palmitate is covalently linked to cysteine residues via
thioester bonds (reviewed in Refs. 13, 14). There are 25 cysteine
residues within apoB100, of which 16 are involved in the formation of
eight disulfide bonds (15). One free cysteine residue at the carboxyl
terminus of apoB100 is involved in covalent linkage with apolipoprotein
(a) (16), and the remaining eight cysteine residues can potentially be
modified by palmitic acid. The exact number of palmitic acids that are covalently linked to apoB100 is uncertain (17). Experiments with
apoB100 secreted from HepG2 cells showed that there were three moles of
palmitic acid per mole of apoB100 (11). One study with apoB29 has
mapped a palmitoylation site to Cys1085, which is the only
free cysteine residue within apoB29 (9). Palmitoylation is likely
catalyzed enzymatically by a cytosolic palmitoyl acyl transferase in
cells but can also be achieved non-enzymatically in vitro
(12, 18). The mechanism by which the secretory protein apoB100 gets
palmitoylated by a cytosolic enzyme is unknown.
In rat hepatic cells, both apoB100 and apoB48 can form VLDL that
contains a single copy of apoB100 or apoB48. Kinetic analysis indicates
that the assembly of B48-VLDL (19) and B100-VLDL (20) is achieved
post-translationally in post-endoplasmic reticulum (ER) compartments.
The stepwise assembly of lipids with apoB appears to be initiated when
the apoB polypeptide is membrane-bound in the microsomes. Thus,
association of apoB with membrane represents an important step in the
VLDL assembly dynamics. The mechanisms by which apoB-membrane
interaction is achieved or regulated are currently unclear. One
possibility is covalent conjugation of fatty acyl chains with the apoB
polypeptides. Recent experiments using McA-RH7777 cells transfected
with a carboxyl terminally truncated apoB, apoB29, showed that
abolishing palmitoylation of apoB29 resulted in impaired lipoprotein
assembly and abnormal intracellular distribution of the mutant protein
(9). The non-palmitoylated apoB29 proteins were secreted as dense
lipoprotein particles and were accumulated in the Golgi apparatus (9).
These results are the first indication of functional consequences of
apoB polypeptides lacking palmitoylation. However, since apoB29 can
only assemble HDL-like lipoproteins, this truncated apoB form did not
offer the opportunity to investigate the role of palmitoylation in the assembly and secretion of VLDL. We reasoned that if palmitoylation plays a role in the assembly and secretion of VLDL, then apoB forms
longer than apoB29 and, having the ability to assemble VLDL, would be a
more suitable model. It has been well documented that the naturally
occurring truncated form apoB48 has the ability to form VLDL in the rat
liver. In the current study, we used apoB48 to assess the role of apoB
palmitoylation in VLDL assembly and secretion.
Materials--
Dulbecco's modified Eagle's medium (DMEM) and
fetal bovine serum were obtained from Sigma. ProMixTM (a
mixture of [35S]methionine and
[35S]cysteine, 1000 Ci/mmol), protein
A-SepharoseTM CL-4B beads and horseradish
peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G
antibody were obtained from Amersham Biosciences. Protease
inhibitor mixture and chemiluminescent blotting substrate were obtained
from Roche Molecular Biochemicals. Monoclonal antibody 1D1 raised
against human apoB was a gift of R. Milne and Y. Marcel (University of
Ottawa Heart Institute). Antibody against Preparation of ApoB48 Expression Plasmids Containing Cys-to-Ser
Substitution--
The plasmid encoding wild type apoB48 (pB48wt) was
prepared using the expression vector pCMV5. The coding sequences
encompassing nucleotides 20-6668 of the human apoB cDNA were
excised from the published pB48 (21) by digestion with EcoRI
and MluI. The EcoRI-MluI fragment was
inserted into the polylinker region of pCMV5. The resulting pB48wt
plasmid encodes the signal peptide and the amino-terminal 2152 amino
acids of human apoB100. The Cys-to-Ser substitution was introduced into
pB48wt at Cys1085, Cys1395,
Cys1478, and Cys1635, consecutively, using the
QuikChangeTM site-directed mutagenesis kit (Stratagene
Inc.) (Fig. 1A). Sequences of all primers used for
mutagenesis are shown in Table I. The resulting plasmids pB48C1085,
pB48C1085-1395, pB48C1085-1478, and
pB48C1085-1635 were purified in a CsCl gradient. The
integrity of the coding sequence for each apoB construct was
authenticated through bidirectional sequencing of the plasmid template
using a total of 17 pairs of amplification primers and then in turn
using these to prime direct sequencing reactions that were run on an
Applied Biosystems Prism 377 automated DNA sequencer.
Cell Culture and Transfection--
McA-RH7777 cells were
cultured in DMEM containing 10% fetal bovine serum and 10% horse
serum. Transfection of the cells with expression plasmids encoding wild
type or mutant forms of apoB48 was achieved using the previously
described calcium phosphate precipitation method (22). Expression of
the recombinant proteins was verified by immunoblotting of the
conditioned media using monoclonal antibody 1D1 (23).
Palmitoylation Assay--
Palmitoylation of recombinant wild
type or mutant apoB48 was determined according to the published
protocol (9). Briefly, stable cell lines were pulse-labeled for 4 h with [125I]iodopalmitate (100-250 µCi in 5 ml) and
chased for 16 h in serum-free DMEM. At the end of chase, the
medium was collected and concentrated. The concentrated media were
delipidated using chloroform:methanol, and the delipidated samples were
dissolved in buffer (4% SDS, 40% glycerol, 65 mM
dithiothreitol in 0.5 M Tris-HCl, pH 6.5) prior to PAGE.
Incorporation of [125I]iodopalmitate into secreted apoB48
was visualized using phosphorimaging.
Pulse-chase Analysis of ApoB--
Pulse-chase experiments
(with [35S]methionine/cysteine) for determination of apoB
synthesis and secretion were performed as described previously (7, 20).
The results were expressed as percent of the initial counts of
35S-labeled apoB at the end of pulse labeling.
Analysis of ApoB Particles by Rate Flotation
Ultracentrifugation--
Cells were continuously labeled with
[35S]methionine/cysteine for 2 h in DMEM containing
20% fetal bovine serum and 0.4 mM oleate. The media were
fractionated into VLDL1, VLDL2, and other
lipoproteins by rate flotation ultracentrifugation in a KBr density
gradient as described previously (24). The apoB proteins in each
fraction were immunoprecipitated (using an anti-apoB polyclonal
antibody that recognized both human and rat apoB) and subjected to PAGE and fluorography. In experiments where secreted apoB mass was determined, cells were incubated for 2 h in DMEM containing 20% fetal bovine serum and 0.4 mM oleate. The media were
fractionated in a KBr density gradient as above, and apoB proteins in
each fraction were immunoprecipitated using a polyclonal anti-apoB antibody, resolved by SDS-PAGE, and detected by immunoblot analysis using the monoclonal antibody 1D1.
Pulse-chase and Subcellular Fractionation--
Cells were
pulse-labeled with [35S]methionine/cysteine for 15-30
min and chased for indicated times as described in figure legends. At
the end of chase, cells were homogenized using a ball-bearing homogenizer (25). The postnuclear supernatant was prepared and fractionated into ER, cis/medial Golgi and distal Golgi by
ultracentrifugation using a preformed Nycodenz gradient (37,000 rpm, 90 min, 15 °C, Beckman SW41 rotor). A total of 15 fractions (0.8 ml/fraction) were collected, and an aliquot of each fraction was
subjected to PAGE and immunoblotted to detect Immunocytochemistry--
Cells were plated on coverslips and
fixed with paraformaldehyde in phosphate-buffered saline and
permeabilized with 0.1% Triton X-100 in phosphate-buffered saline as
described previously (20). Monoclonal antibody 1D1 was used to probe
the recombinant human apoB; ER and Golgi were probed with anti-calnexin
and anti- Expression of ApoB48 Mutants That Lacked Palmitoylation--
Of
the nine free cysteine residues in apoB100, four are located within the
amino-terminal 48% of the protein (Fig.
1A, solid ovals).
We used apoB48, a naturally occurring form of apoB that can assemble
VLDL, to study the role of palmitoylation in VLDL assembly. The four
cysteine residues Cys1085, Cys1395,
Cys1479, and Cys1635 within apoB48 were
substituted with serine residues by site-directed mutagenesis (Fig.
1A, opened ovals), and the resulting cDNA
constructs were transfected into McA-RH7777 cells. In stably
transfected cell lines, the mutant apoB48 proteins, namely
B48C1085, B48C1085-1395,
B48C1085-1478, and B48C1085-1635, were
secreted with the expected molecular mass and reacted with monoclonal
antibody 1D1 that was specific for human apoB (Fig. 1B,
bottom panel). Palmitoylation of the apoB
proteins was determined by [125I]iodopalmitate labeling.
Preliminary experiments with parental McA-RH7777 cells showed that the
endogenous rat apoB100 was secreted as palmitoylated proteins (data not
shown), which agrees with the previous report (9).
[125I]Iodopalmitate labeling experiments showed that
B48C1085-1635, in which all free cysteine residues were
mutagenized, was expectedly non-palmitoylated (Fig. 1B,
top panel). In contrast, palmitoylation occurred
in the wild type apoB48 (B48wt) and B48C1085,
B48C1085-1395, and B48C1085-1478 mutants
containing one or more free cysteine residues. Palmitoylation of the
endogenous rat apoB48 was below the limit of detection (Fig.
1B, lane labeled with McA). The intensity of
[125I]iodopalmitate labeling of the recombinant mutant
apoB48 proteins decreased gradually as the number of mutated cysteine
residues increased (Fig. 1B, top
panel), suggesting the presence of multiple palmitoylation
sites within human apoB48.
Lack of Palmitoylation in ApoB48 Did Not Impair VLDL
Secretion--
As shown previously, apoB48 could form VLDL as well as
HDL-like particles in McA-RH7777 cells, and the formation of B48-VLDL was dependent on exogenous oleate (10). The culture media in all
subsequent experiments was therefore supplemented with 20% FBS and 0.4 mM oleate to maximize lipogenesis and VLDL
assembly/secretion. Having ascertained that B48C1085-1635
was non-palmitoylated, we examined the effect of lack of palmitoylation on the assembly and secretion of VLDL. Cell lines expressing comparable levels of B48wt or B48C1085-1635 were metabolically
labeled with [35S]methionine/cysteine, and the
conditioned media were fractionated by rate flotation centrifugation to
separate VLDL1 (Rf > 100) and
VLDL2 (Rf 20-100) from dense
lipoproteins (Fig. 2A). Both B48wt and B48C1085-1635 were secreted predominantly
(>80%) as HDL-like particles and a small proportion as VLDL.
Quantification of 35S-labeled B48wt and
B48C1085-1635 showed that the lack of palmitoylation had
little impact on the density distribution among different lipoprotein
fractions (Fig. 2B). Secretion of VLDL containing endogenous
rat 35S-labeled apoB100 was also similar between B48wt- and
B48C1085-1635-transfected cells. These results were the
first indication that palmitoylation of apoB48 was not essential for
the assembly and secretion of VLDL. To confirm that palmitoylation was
not required for B48-VLDL secretion, we determined secretion of the
apoB48 mass by immunoblotting (Fig. 2C). We also
fractionated the medium lipoproteins by ultracentrifugation in a salt
gradient that was designed to resolve HDL subfractions (9) (Fig.
2D). In either case (Fig. 2, C and D),
there was no apparent difference in the secretion of VLDL or HDL
between B48wt- and B48C1085-1635-transfected cells.
Furthermore, there was no indication of a requirement of palmitoylation
for B48-VLDL secretion from B48C1085-,
B48C1085-1395-, or B48C1085-1478-transfected
cells (Fig. 2E). Thus, abolishing palmitoylation of B48 was
not associated with impairment in B48-VLDL secretion.
Lack of Palmitoylation in ApoB48 Resulted in Decreased Secretion
Efficiency--
We next contrasted secretion efficiency between B48wt
and B48C1085-1635 by pulse-chase experiments (Fig.
3A). In two independent
experiments, the secretion efficiency (i.e. the percent of
total newly synthesized apoB48 that was secreted during chase) for
B48C1085-1635 was reproducibly lower (by 15-25%) than
that of B48wt (top panel). Concomitantly, a
slightly increased B48C1085-1635 (by 20%) compared with
B48wt was retained within the cells during chase (bottom
panel). The recovery of total apoB48 (medium + cell) was
similar between B48wt and B48C1085-1635, suggesting that
the decreased B48C1085-1635 secretion was not accompanied
with enhanced intracellular degradation. Similar pulse-chase
experiments were also performed with cells expressing
B48C1085, B48C1085-1395 or
B48C1085-1478 (Fig. 3B). The secretion
efficiency of the mutant apoB48 proteins decreased gradually as the
number of mutagenized free cysteine residues increased (Fig.
3B, bottom). Once again, the decreased secretion
of apoB48 mutants was accompanied with prolonged intracellular retention (Fig. 3B, top). These data indicate
that although the ability of apoB48 to assemble VLDL is unimpaired by
the lack of palmitoylation, the secretion efficiency of the mutant
proteins is somewhat slightly reduced.
It was reported that mutant apoB29 lacking palmitoylation exhibited an
abnormal accumulation within the Golgi apparatus (9). The prolonged
intracellular retention of B48C1085-1635 shown by
pulse-chase experiments (Fig. 3A) prompted us to examine intracellular distribution of this mutant protein. To this end, we
performed indirect double immunofluorescent co-localization experiments
(Fig. 4). Confocal images showed that
both B48wt and B48C1085-1635 appeared as punctate staining
throughout the cells with high intensity near the perinuclear area
(Fig. 4, A and B, left panels, labeled
with apoB). Merging of fluorescence derived from apoB with
that from calnexin (middle panels, labeled with
CNX) and Lack of Palmitoylation in ApoB48 Resulted in Enhanced Association
with Microsomal Membranes--
To gain kinetic insights into
intracellular trafficking of apoB48, we performed pulse (30 min) and
chase (45 min) experiments combined with subcellular fractionation
(Fig. 6). Preliminary experiments showed
that the majority of microsome-associated 35S-labeled
apoB48 was present in the lumen (see below). Thus, in this experiment,
we monitored trafficking of the lumenal
35S-labeled apoB48 through different subcellular
compartments. At the end of the 30-min pulse, the ER lumenal
35S-labeled B48wt and 35S-labeled
B48C1085-1635 were predominately associated with HDL-like
particles (Fig. 6, A and B, top
panels, labeled with ER). Because protein
synthesis was not synchronized under the current experimental
conditions, some apoB48-HDL particles were found in the lumen of the
Golgi apparatus (panels labeled with cis/medial Golgi and
distal Golgi) and were released into the media at the end of
the 30-min pulse. However, no 35S-labeled apoB48 was
associated with VLDL at this time. After the 45-min chase
(bottom panels), the 35S-labeled
apoB48 exited the ER, became prominent in the distal Golgi, and was
secreted into the media. A small proportion of 35S-labeled
B48 was associated with VLDL within the distal Golgi lumen, but
35S-labeled B48-VLDL was never observed in the ER lumen.
The presence of B48-VLDL, particularly B48-VLDL1, in the
distal Golgi lumen, at the end of the 45-min chase is consistent with a
post-translational, post-ER process for B48-VLDL assembly (19). Similar
to what was observed in continuous labeling experiments (Fig.
2A), secretion of 35S-labeled
B48C1085-1635 as VLDL and HDL was also comparable to that
of 35S-labeled B48wt at the end of chase (Fig. 6, compare
panels labeled with medium), again indicting that the lack
of palmitoylation has no effect on the ability of apoB48 to assemble
lipoproteins. Notably, the lumenal 35S-labeled
B48C1085-1635 presented in different subcellular
compartments was markedly lower than 35S-labeled B48wt at
the end of both pulse and chase time (Fig. 6, compare panel
A with B).
The reduced lumenal B48C1085-1635 during the pulse-chase
period (Fig. 6) raised the possibility that the mutant protein might
bind to microsomal membranes with high affinity. We
hypothesized that the decreased secretion efficiency and
prolonged intracellular retention of B48C1085-1635 was
attributable to altered membrane association. In the next pulse-chase
experiment, we compared membrane-association property together with the
trafficking rate between B48wt and B48C1085-1635 (Fig.
7). The pulse time was reduced to 15 min (from the previous 30 min); thus at the beginning of chase
the 35S-labeled apoB48 was mainly confined to the ER (see
insets of panels labeled with time 0). Moreover,
two extra time points, 15-min and 30-min, were added to the chase (in
addition to the previous single 45-min point) to monitor the rate of
intracellular trafficking of apoB48. As the chase proceeded from 0 to
45 min, the radioactivity associated with apoB48 moved out from the ER lumen and appeared in the distal Golgi lumen, and no apoB48 was accumulated in the cis/medial Golgi (Fig. 7, A
and B, left panels). These results,
together with the data shown in Fig. 6, indicate that apoB48 was
transported mainly in a lumenal HDL-associated form from the
ER to distal Golgi, and it passed through cis/medial Golgi
rather rapidly.
When association of apoB48 with membranes was determined, it became
clear that B48C1085-1635 had increased (by at least
2-fold) membrane binding. At the end of the 15-min pulse, 26% of the
total ER-associated 35S-labeled B48C1085-1635
was membrane-bound, whereas only 11% of the total
35S-labeled B48wt was membrane-bound. The elevated membrane
association of 35S-labeled B48C1085-1635 was
observed throughout the entire chase (Fig. 7, A and
B, right panels). Thus, the average
values of membrane-associated apoB48 in the combined ER,
cis/medial Golgi, and distal Golgi fractions from four chase
time points (i.e. 0, 15, 30, and 45 min) were 29.8 ± 3.0% for B48C1085-1635 and 10.5 ± 1.3% for B48wt
(p < 0.001).
The increased association of B48C1085-1635 with the
membranes was accompanied with reduced lumenal form in all subcellular
compartments (Fig. 7B, left panel).
The percent of total 35S-labeled B48C1085-1635
presented in the lumen of all subcellular compartments was less than
that of 35S-labeled B48wt at every chase time point. The
average values of lumenal apoB48 in the combined ER,
cis/medial Golgi, and distal Golgi fractions from four chase
time points were 70.3 ± 3.0% for B48C1085-1635 and
89.5 ± 1.3% for B48wt (p < 0.001). Notably, the
proportion of total B48C1085-1635 as lumenal form in the
distal Golgi was lower than that of B48wt (compare the left
panels between A and B, inverted
triangles), whereas the proportion of total
B48C1085-1635 as membrane-associated form in the
cis/medial Golgi was more pronounced than that of B48wt
(compare the right panels between A
and B, opened circles). Thus, while
B48C1085-1635 exited the ER at a rate similar to that of
B48wt, B48C1085-1635 (particularly the membrane-bound
form) might have a slightly reduced transit rate between
cis/medial Golgi and distal Golgi.
2-Bromopalmitate Inhibited VLDL Secretion--
Consideration was
given to the possibility that multiple Cys-to-Ser substitution within
apoB48 could have effects that extended beyond loss of palmitoylation.
Hence, it was desirable to confirm the mutagenesis data using other
approaches. It has been reported that 2-bromopalmitate inhibits
palmitoylation (26). We attempted to block apoB palmitoylation using
2-bromopalmitate and found that it indiscriminately inhibited secretion
of both B48wt (Fig. 8A) and
B48C1085-1635 as VLDL (Fig. 8B) in a
dose-dependent manner. Secretion of B48wt or
B48C1085-1635 as HDL-like particles was less affected by
the treatment (Fig. 8). Secretion of endogenous rat
35S-labeled apoB100 was also inhibited by 2-bromopalmitate
treatment (data not shown). The treatment with 2-bromopalmitate had no
effect on apoB synthesis as judged by the equal incorporation of
35S-labeled amino acids into B48wt and
B48C1085-1635 at different doses of the inhibitor (Fig.
8C). Apparently, 2-bromopalmitate exerts an inhibitory
effect on VLDL assembly and secretion independent of apoB
palmitoylation.
Multiple functional roles have been assigned to post-translational
protein palmitoylation. Palmitoylation results in anchoring and
targeting otherwise cytosolic proteins to the inner surface of specific
membrane domains (27-29). Palmitoylation also governs protein-protein
interactions within membranes (12, 18, 30). The reversibility of
protein palmitoylation suggests that it may regulate membrane
activities. While extensive protein palmitoylation studies have focused
on proteins involved in signal transduction, the role of palmitoylation
of apoB, one of the rare secretory proteins that can be palmitoylated,
is not entirely clear. To date, only one report has suggested that
substituting the single free cysteine (Cys1085) with serine
in apoB29 results in impaired lipid recruitment and abnormal
accumulation of the mutant protein within the Golgi apparatus (9). In
the current study, we used the naturally occurring apoB48 as a model to
assess the role of apoB palmitoylation in VLDL assembly and secretion.
Unexpectedly, results obtained from our biochemical (Fig. 2) and
immunocytochemical experiments (Fig. 4) using both transiently and
stably transfected cells do not support the notion that lack of
palmitoylation in apoB48 results in impaired lipoprotein assembly or
secretion. In an attempt to determine the effect of palmitoylation
using a chemical inhibitor 2-bromopalmitate, we observed that this
compound inhibited VLDL secretion regardless of the status of apoB
palmitoylation (Fig. 8). Thus, the use of apoB48 as a model leads us to
conclude that apoB palmitoylation within this truncated apoB form does
not play a role in VLDL assembly and secretion.
The lack of a gross effect of apoB palmitoylation on VLDL
assembly/secretion does not rule out a possible temporal and spatial role that this post-translational modification plays during
intracellular apoB trafficking and VLDL assembly. Recent studies with
the full-length apoB100 have shown that the VLDL assembly proceeds
through membrane-associated apoB100 in the ER and cis/medial
Golgi to eventual acquisition of bulk neutral lipids in a compartment
close to distal Golgi (20). The physical nature of the apoB100-membrane
interaction during VLDL assembly is unclear. One hypothesis is that
covalently linked fatty acid may facilitate the interaction between
apoB100 and membranes (6). Thus, we performed detailed pulse-chase experiments in conjunction with subcellular fractionation to determine the rate of intracellular trafficking and membrane association of the
mutant B48C1085-1635 and contrasted these parameters with
those of B48wt. The following two observations are noteworthy.
First, although immunocytochemistry (Fig. 4) and immunoblot analysis
(Fig. 5) of the fractionated microsomes failed to detect gross changes
in intracellular distribution of the mutant apoB48, two independent
pulse-chase experiments reproducibly revealed slightly lowered
secretion efficiency and prolonged intracellular retention of
B48C1085-1635 (Fig. 3A). The effect of
palmitoylation on apoB48 secretion appeared to be a function of the
extent of this S-acylation. Thus, the more free cysteine
residues are mutagenized, the lower the secretion efficiency and the
greater intracellular retention of the mutant apoB48 proteins (Fig.
3B). Furthermore, the low secretion efficiency of
B48C1085-1635 was associated with slightly reduced transit
rate between cis/medial Golgi and distal Golgi (Fig. 7).
Thus, palmitoylation of apoB may exert a subtle effect in fine-tuning
the kinetics of intracellular trafficking.
Second, the lack of palmitoylation did not diminish association of
apoB48 with the microsomal membranes. Rather, the mutant B48C1085-1635 exhibited enhanced binding to the membranes
of the secretory compartments (Fig. 7). These results suggest that the
normally observed apoB-membrane interaction is probably mediated by
factors other than apoB palmitoylation. As mentioned above, the
physical nature of apoB-membrane interaction is currently unclear.
However, the present studies with apoB48 revealed a marked distinction between apoB100 and apoB48 in their membrane association during lipoprotein assembly. In the case of apoB100, the newly synthesized proteins were almost exclusively membrane-associated within the ER and
only became lumen-borne when they reached distal Golgi as VLDL (20). In
contrast, the majority of newly synthesized apoB48 was present in the
lumen, while only a small proportion was membrane-associated throughout
the secretory compartments (Fig. 7). The difference in membrane
association between apoB100 and apoB48 is striking, which may indicate
that the carboxyl-terminal half of the apoB100 amino acid sequences
governs apoB membrane binding. An important unsolved issue in studies
of VLDL assembly is how the membrane-associated lipoprotein precursor
particles recruit lipid and eventually "fall-off" the membrane as
mature, soluble, lipid-loaded lipoprotein particles that are
secretion-competent. Since there are an additional five free cysteine
residues located within the carboxyl-terminal half of apoB100 and at
least four of which can potentially be palmitoylated, it is of
importance to determine further, using the full-length apoB100 as a
model, whether or not the fatty acids conjugated onto apoB can act as the link that regulates apoB association/dissociation with the membrane
during apoB100-VLDL assembly.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosidase II was a gift
of M. Farquhar (University of California, San Diego, CA). The
anti-calnexin and anti-TGN38 antibodies were obtained from StressGen
and Affinity BioReagents, respectively.
Oligonucleotides used for Cys-to-Ser mutagenesis
-mannosidase II,
calnexin, TGN38, and apoB, respectively (20). In some experiments, the membranes and lumenal contents were isolated from the pooled ER, cis/medial Golgi, and distal Golgi fractions by sodium
carbonate treatment followed by ultracentrifugation as described
previously (20, 25). The lumenal and membrane-associated apoB proteins were analyzed by PAGE and fluorography as described above.
-mannosidase II antibodies, respectively (20). The
experiments were performed using both transiently transfected
McA-RH7777 cells and individually selected G418-resistant colonies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
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Fig. 1.
Construction and expression of apoB48
mutants. A, the top line represents human
apoB100 with free cysteine residues (closed ovals)
indicated. The bottom lines depict wild type apoB48
(B48wt) and four mutant forms of apoB48 containing
Cys-to-Ser substitutions (open ovals) at the potential
palmitoylation sites Cys1085, Cys1395,
Cys1479, and Cys1635. B,
palmitoylation assay of apoB48. Cells were labeled with
[125I]iodopalmitate, and the 125I-labeled
apoB48 were concentrated from the conditioned medium and resolved by
SDS-PAGE. The labeled apoB proteins were visualized using
phosphorimaging (top). An aliquot of the conditioned medium
was subjected to immunoblot analysis using monoclonal antibody 1D1
(bottom).
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Fig. 2.
Buoyant density of lipoproteins containing
apoB48 secreted from transfected cells. A, cells
expressing B48wt or B48C1085-1635 were labeled with
[35S]methionine/cysteine for 2 h in the presence of
20% serum and 0.4 mM oleate. The medium was collected at
the end of labeling and fractionated into VLDL1,
VLDL2, and other lipoproteins by ultracentrifugation.
Endogenous rat apoB100 (rB100) and recombinant apoB48 were
immunoprecipitated from each fraction, and analyzed by SDS-PAGE and
fluorography. B, quantification of 35S-labeled
apoB48. The bands corresponding to apoB48 (shown in A) were
excised, and the associated radioactivity was quantified by
scintillation counting. The values of radioactivity associated with
35S-labeled apoB48 in different lipoprotein fractions are
expressed as percent of total secreted apoB48. C,
immunoblots of B48wt or B48C1085-1635 associated with
lipoproteins. Cells expressing B48wt or B48C1085-1635 were
cultured in media containing 20% serum and 0.4 mM oleate
for 4 h. The conditioned media were fractionated by
ultracentrifugation, and the apoB48 proteins in each fraction were
detected by immunoblotting using monoclonal antibody 1D1. D,
immunoblot of B48wt and B48C1085-1635 associated with
secreted lipoproteins in the media that were fractionated by
ultracentrifugation in a salt gradient (9). E, the
experiments were performed the same as in C, except the that
cells used were transfected with mutants containing incomplete
Cys-to-Ser substitutions. Data presented in C and
E are representatives of five independent experiments using
different stable transformants. IDL, intermediate density
lipoproteins; LDL, low density lipoproteins.
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Fig. 3.
Pulse-chase analysis of apoB48 secretion
efficiency and post-translational stability. A, stably
transfected cells (60-mm dish) expressing B48wt or
B48C1085-1635 were incubated in methionine- and
cysteine-free media for 30 min and pulse-labeled with
[35S]methionine/cysteine (100 µCi/dish) for 30 min.
After pulse labeling, the medium was changed to normal media and
incubated for up to 3 h (chase). Both pulse and chase media
contained 20% serum and 0.4 mM oleate. The media were
collected, cells were lysed at the indicated chase times, and the
35S-labeled apoB48 proteins were recovered by
immunoprecipitation and subjected to SDS-PAGE and fluorography.
Radioactivity associated with secreted apoB48 (top) or
cellular apoB48 (bottom) was quantified. Data are expressed
as % of initial counts at the end of 30-min pulse labeling and are the
average of two independent experiments. The representative fluorograms
of 35S-labeled apoB48 in media and cell during chase are
shown as insets. B, comparison of cell and medium
35S-labeled apoB48 at the end of the 2-h chase between
B48wt- and various mutant-transfected cells. The experiments were
performed as described in A, except that they were done only
once.
-mannosidase II (middle panels,
labeled with ManII), respectively, showed substantial
co-localization of apoB48 (wild type and mutant) with the ER and the
Golgi markers (Fig. 4, right panels, labeled with
overlay). There was no discernible difference that could suggest the lack of palmitoylation resulting in abnormal intracellular distribution of B48C1085-1635 at steady state. The
immunocytochemistry experiments were performed in both stably and
transiently transfected cells. Under no circumstances had different
intracellular distribution between B48wt and B48C1085-1635
been observed (data from transiently transfected cells are not shown).
The immunocytochemistry data were corroborated by subcellular fractionation experiments in which the microsomes were separated using
a Nycodenz gradient. Immunoblot analysis with anti-calnexin, anti-
-mannosidase II, and anti-TGN38 antibody was performed to detect ER, cis/medial Golgi, and distal Golgi markers,
respectively (Fig. 5A), and
antibody 1D1 was used to probe apoB48 (Fig. 5B). Quantification of the intracellular distribution of apoB48 among the
Nycodenz fractions showed almost identical profiles between B48C1085-1635 and B48wt (Fig. 5C). These
results together indicate that the decreased secretion efficiency of
mutant B48C1085-1635 was not associated with abnormal
intracellular distribution at steady state.
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Fig. 4.
Immunocytochemistry of apoB48.
Cells expressing B48wt or B48C1085-1635 were plated on
cover slips and kept in cultured medium containing 20% FBS and 0.4 mM oleate for 2 h. The cells were then fixed and
permeabilized. Monoclonal antibody 1D1 was used to probe the
recombinant human apoB with goat anti-mouse IgG Alexa Fluro-488
conjugate (green) as secondary antibody. The ER and Golgi
were probed with anti-calnexin (CNX) and
anti- -mannosidase II (ManII) antibodies, respectively,
with Alexa Fluro-594-conjugated anti-rabbit IgG (red) as
secondary antibody. The images were captured using the MRC-1024 laser
scanning confocal imaging system. Data are representative of two
experiments with similar results.
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Fig. 5.
Distribution of apoB48 along the secretory
pathway. Cells (100-mm dish, 80% confluent) expressing B48 or
B48C1085-1635 were incubated in DMEM containing 20% FBS
and 0.4 mM oleate for 2 h. The cells were homogenized,
and the postnuclear supernatants were fractionated by Nycodenz gradient
ultracentrifugation. Aliquots of each fraction were resolved by
SDS-PAGE, transferred to nitrocellulose membranes, and subjected to
immunoblotting. A, distribution of marker proteins. The
blots were probed with antibodies against TGN-38, calnexin
(CNX), or -mannosidase II (ManII),
respectively. The intensity of these marker proteins was semiquantified
by scanning densitometry. Data are presented as "% of maximum
value" in which 100% corresponds to the highest value.
B, immunoblots of apoB48 probed with monoclonal antibody
1D1. Bottom, semiquantification of density distribution of
B48wt and B48C1085-1635.
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[in a new window]
Fig. 6.
Pulse-chase analysis of intracellular
distribution and secretion of apoB48. Cells expressing B48wt
(A) or B48C1085-1635 (B) were
pulse-labeled with [35S]methionine/cysteine for 30 min as
described in Fig 3. The chase was carried on for 45 min. Exogenous
oleate (0.4 mM) was present in both pulse and chase. At the
end of pulse (top panels) and chase
(bottom panels), the cells were homogenized and
the postnuclear supernatants were fractioned by Nycodenz gradient
ultracentrifugation. The fractions corresponding to ER,
cis/medial Golgi, and distal Golgi were pooled, and the
lumenal contents of each pooled fraction were released by the sodium
carbonate treatment. Lipoproteins in both the lumenal content and media
were fractionated into VLDL1, VLDL2, and other
lipoproteins as described in Fig. 2. The recombinant apoB48 and
endogenous rat apoB100 (rB100) were recovered by
immunoprecipitation, subjected to SDS-PAGE, and visualized by
fluorography. Repetition of the experiments yielded identical
results.
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Fig. 7.
Comparison of intracellular trafficking
between B48wt and B48C1085-1635. Cells expressing
B48wt (A) or B48C1085-1635 (B) were
pulse-labeled with [35S]methionine/cysteine for 15 min,
and were chased for up to 45 min. Exogenous oleate (0.4 mM)
was present in both pulse and chase. At each chase time, the cells were
homogenized and the postnuclear supernatants were fractionated by
Nycodenz gradient ultracentrifugation. The lumenal contents and
membranes of the pooled ER, cis/medial Golgi, and distal
Golgi fractions were separated by sodium carbonate treatment followed
by ultracentrifugation. The 35S-labeled apoB48 proteins
present in the respective membranes (insets of
right panels) and lumenal contents
(insets of left panels) were recovered
by immunoprecipitation and subjected to SDS-PAGE/fluorography.
Radioactivity associated with 35S-labeled B48wt and
35S-labeled B48C1085-1635 was quantified by
scintillation counting. The values were expressed as % of total
35S-labeled B48 (lumen + membrane) at each chase
time.
View larger version (39K):
[in a new window]
Fig. 8.
Effect of 2-bromopalmitate treatment on
secretion of apoB48. Cells were preincubated with serum-free
medium containing 0.1% BSA and indicated concentrations of
2-bromopalmitate for 7 h and then labeled with
[35S]methionine/cysteine for 2 h in the presence of
20% serum, 0.4 mM oleate, and different concentrations of
2-bromopalmitate. At the end of labeling, the 35S-labeled
B48wt (A) and 35S-labeled
B48C1085-1635 (B) secreted as lipoproteins were
fractionated and analyzed by SDS-PAGE and fluorography as described in
Fig. 2. C, the cell-associated 35S-labeled B48wt
and 35S-labeled B48C1085-1635 at the end of
2-h labeling.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank J. Vance and R. Milne for a critical reading of the manuscript, M. Farquhar, Y. Marcel, and R. Milne for antibodies used in this work, and H. McBride and J. Ngsee for the guidance and assistance in fluorescence microscopy.
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FOOTNOTES |
---|
* This work was supported by Heart and Stroke Foundation of Ontario Grant T-4643 and Canadian Institutes of Health Research Grant MT-15486 (to Z. Y.) and National Institutes of Health Grant GM 57966 (to M. D. R.).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.
c Recipient of the Doctoral Research Award from Canadian Institutes of Health Research and Heart & Stroke Foundation of Canada.
d Both authors contributed equally to this work.
f Recipient of the Student Award from Canadian Institutes of Health Research and Heart & Stroke Foundation of Canada.
h Canada Research Chair (Tier 1) in Human Genetics and a Heart and Stroke Foundation of Ontario Career Investigator.
j Scientist of Canadian Institutes of Health Research. To whom correspondence should be addressed. Tel.: 613-798-5555 (ext. 18711); Fax: 613-761-5281; E-mail: zyao@ottawaheart.ca.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M211995200
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ABBREVIATIONS |
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The abbreviations used are: apo, apolipoprotein; VLDL, very low density lipoproteins; HDL, high density lipoproteins; ER, endoplasmic reticulum; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum.
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