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INTRODUCTION |
The correct folding of apolipoprotein B
(apoB)1 into its mature,
secretion-competent form is a complex process that leads to the
formation and secretion of triacylglycerol (TAG)-rich lipoproteins, chylomicrons, and the atherogenic lipoproteins, very low density lipoproteins (VLDL) (1, 2). A two-step model for the formation of VLDL
was originally proposed on the basis of electron microscopic studies in
rat liver (3). This model was further supported by latter studies in
rat liver (4), in rat hepatoma cells (McA-RH7777; Refs. 5-8), and in
transgenic mice lacking the gene for apoB in the intestine (9).
According to this model, the first step involves partial lipidation of
apoB to form a primordial intermediate with HDL/LDL-like density. In
the second step this intermediate fuses with a large apoB-free lipid
droplet composed primarily of TAG to form nascent VLDL or chylomicrons.
In HepG2 cells, however, these distinct steps were not clearly
demonstrated. Nonetheless, the initial steps of lipidation are thought
to be similar to other systems in that they are mediated by microsomal
triglyceride transfer protein (MTP) (reviewed in Refs. 10-12) and
occur co-translationally, while apoB is bound to the ER membrane (13).
Partial lipidation of apoB leads to formation of an intermediate with
HDL/LDL-like densities. This intermediate is then released into the ER
lumen. Subsequent recruitment of lipids by this intermediate seems to occur continuously (13) to ultimately form and secrete larger TAG-rich
particles with primarily LDL-like density and, to a lesser extent,
particles with VLDL and IDL-like densities.
In the absence of sufficient lipids, primarily TAG and cholesterol
esters (1, 14, 15), or when MTP activity is diminished, either as a
result of mutations (11, 16) or as a result of specific inhibitors (7,
17), apoB is unable to form lipoproteins and is targeted to
proteasome-dependent degradation both co- and post-translationally (18, 19) in hsp70- and hsp90-mediated mechanisms
(20-22).
In addition to MTP, the folding of apoB, like that of other secretory
proteins, seems to require the assistance of ER-resident molecular
chaperones including BiP, calnexin, calreticulin (CRT), ERp72, and
GRP94 (18, 23-28). Molecular chaperones bind transiently to nascent
polypeptides to prevent their aggregation, and thereby maintain them in
conformations competent for efficient folding. Once correct folding is
attained, chaperones dissociate and folded proteins exit the ER.
However, if nascent polypeptides fail to attain their native form, they
are retained in the ER bound to chaperones, which ultimately target
them for proteasome-dependent degradation (29) to prevent
their transport to their target destination (30-34).
Given the complexity of apoB folding, which involves lipidated
membrane-bound and luminal intermediates, it was of interest to
characterize in great detail the involvement of chaperones in various
stages of its biogenesis.
We hypothesized that the membrane-bound pool of apoB will interact with
molecular chaperones. However, it was unclear whether partially
lipidated apoB that is translocated into the ER lumen as a primordial
intermediate is still dependent on the assistance of molecular
chaperones for its subsequent folding steps and, if so, whether these
chaperones dissociate from apoB-containing particles before their
transport to the Golgi. This study was designed to address these questions.
We demonstrate that the same array of molecular chaperones interact
with both the membrane-bound and the luminal pools of apoB. However,
the relative level of BiP, GRP94, ERp72, and cyclophilin B (CyPB)
associated with the luminal pool of apoB is severalfold higher than
that associated with the membrane-bound pool, suggesting a role for
these chaperones in facilitating the release of apoB from the
microsomal membrane. We further demonstrate that GRP94, ERp72, BiP,
calreticulin (CRT), and CyPB remain bound to apoB that is transported
to the Golgi, albeit the chaperone/apoB ratio was lower than that in
the ER. This is consistent with the idea that these chaperones play
multiple roles both in early and in late folding events occurring in
the ER and the Golgi, respectively. Thus, apoB is recognized as an
incompletely folded protein throughout most of the secretory pathway.
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EXPERIMENTAL PROCEDURES |
Materials
Antibodies--
Goat polyclonal antibodies to apoB were from
Biodesign and Calbiochem. Mouse monoclonal antibodies to apoB 2D8 were
kindly provided by Dr. E. Marcel and Dr. Milne (University of Ottawa, Canada). B4 was a generous gift from Dr. J. Fruchard (Université de Lille, Lille, France) with permission from Dr. H. Bazin (University of Louvain, Louvain, Belgium). Polyclonal antibodies to GRP94 were
kindly provided by Dr. S. Cala (Wayne State University, Detroit, MI).
Rat monoclonal antibodies to GRP94, mouse monoclonal antibodies to BiP,
rabbit antibodies to ERp72 and to calreticulin were from Stressgen
Biotechnologies Corp. (Victoria, British Columbia, Canada); rabbit
antibodies to CyPB and mouse monoclonal antibodies to Golgin 97 were
from Molecular Probes. Monoclonal antibodies to GPP130 were kindly
provided by Dr. H. Hauri (University of Basel, Basel, Switzerland).
Mouse monoclonal antibodies to p58 and horseradish peroxidase
(HRP)-conjugated secondary antibodies were from Sigma.
Other Materials--
Dulbecco's modified Eagle's medium and
fetal calf serum were from Invitrogen. Cycloheximide, leupeptin,
aprotinin, and essentially fatty acid-free albumin were from Sigma.
N-Acetyl-L-leucinyl-L-leucinyl-L-norleucinal (ALLN) and dithiobis(succinimidyl propionate) (DSP) were from Roche
Molecular Biochemicals, and 4-[2-aminoethyl]benzyl sulfonyl fluoride (AEBSF) was from Calbiochem. Oleic acid (OA) was from Neucheck
Inc. Gelatin- and protein G-Sepharose were from Amersham Biosciences. PVDF membranes were from Millipore, and Centricon microconcentrators were from Amicon. Reagents for the ECL system and
[35S]methionine/cysteine (specific activity > 1,000 Ci/mmol) were purchased from NEN Life Science Products.
Biotinylated lectin Sambucus nigra
agglutinin (SNA-I) was from EY Labs. HRP-conjugated extravidin was from
Sigma. BMS-197636 was a generous gift from Dr. D. Gordon (Bristol Myers Squibb).
Methods
Cell Cultures, Pulse-chase Experiments, and KBr Density Gradient
Fractionation--
HepG2 cells were pulse-labeled with
[35S]methionine/cysteine and chased as described (26).
Cell monolayers were subjected to cross-linking, then solubilized and
immunoprecipitated with antibodies to apoB (26). Media were collected
and spun at 800 × g for 5 min, concentrated using
microconcentrators, adjusted to 1.25 g/ml with solid KBr, overlaid by
1.006 g/ml solution, and spun in an SW41 rotor at 40,000 rpm for 20-24
h. Density fractions were collected from the top and their density
determined by an Abbe Refractometer (American Optical Corp.). Fractions
were washed in microconcentrators, adjusted to 2% CHAPS, 50 mM HEPES, and subjected to immunoprecipitation.
Preparation and Extraction of Microsomes--
HepG2 cells were
incubated for 2 h in media supplemented with 0.8 mM
oleic acid complexed to 0.2 mM albumin and 25 µg/ml ALLN to increase lipid availability and inhibit degradation, respectively. Cells were either subjected to cross-linking and then scraped with a
rubber policeman (26) or were scraped without prior cross-linking. Washed cells were suspended in homogenization buffer (0.25 M sucrose, 10 mM HEPES, pH 7.5, and the
following protease inhibitors: 1 mM AEBSF, 5 µg/ml ALLN,
and 10 µg/ml each aprotinin and leupeptin), then homogenized with a
tight-fitting Dounce homogenizer with 20 strokes. In some experiments,
homogenates were also passed through a 25-gauge needle. Homogenates
were spun for 5 min at 6,000 × g, and post-nuclear
supernatants were spun for 40 min at 200,000 × g at
3 °C to pellet the microsomes.
Microsomes were extracted either with carbonate alone (0.1 M Na2CO3, pH 7.4) (35) or with
carbonate supplemented with 0.025% sodium deoxycholate, with or
without 1.2 M KCl (carbonate/DOC or carbonate/DOC/K,
respectively) (36). Samples were then spun at 100,000 × g
for 30 min to separate the soluble contents (luminal) from the
membranes. Soluble contents were neutralized, and salts removed by
ultrafiltration. Membranes and luminal contents were then adjusted to
2% CHAPS, 50 mM HEPES containing protease inhibitors as
described above, and apoB immunoprecipitated. In some experiments luminal contents and membranes were adjusted to 1% SDS in PBS, heated
to 65 °C for 15 min, diluted to 0.1% SDS with 1% Triton X-100 in
PBS, and subjected to immunoprecipitation.
Subcellular Fractionation on Sucrose Gradients--
HepG2 cells
were incubated for 2 h in media supplemented with oleic acid and
ALLN as described before. Cells were either subjected to cross-linking
and then scraped with a rubber policeman (26) or were scraped without
cross-linking as indicated in the figures. Washed cells were suspended
in homogenization buffer, homogenized, and centrifuged as described
above. Postnuclear supernatants were adjusted to 1.3 M
sucrose and overlaid with 1.2, 1.15, 0.86, and 0.25 M
sucrose, 10 mM HEPES (37). Gradients were spun for 18 h at 24,000 rpm at 4 °C in SW41 rotor and eight fractions collected from the top as follows: top 1 ml, light Golgi (interface
0.25/0.86 M sucrose), heavy Golgi (interface 0.86/1.15
M sucrose), 1.15 M sucrose, 1.2 M
sucrose, interface (1.2/1.3 M), and heavy microsomes (1.3 M and pellet). The concentration of sucrose in each
fraction was determined by measuring their refractive index as
described before. Fractions were then washed in 0.25 M
sucrose by centrifugation in a Ti 50.3 rotor (Beckman) at 40,000 rpm
for 1 h at 4 °C. Pellets were extracted either with carbonate
or carbonate/DOC/K as described above. Fractions were then centrifuged
for 30 min at 100,000 × g to separate membranes from
luminal contents. Membranes were washed in PBS and then solubilized in
2% CHAPS. Luminal contents were adjusted to pH 7.4, concentrated using
microconcentrators, and adjusted either to PBS, pH 7.4, or to 2%
CHAPS, 50 mM HEPES, pH 7.4, both containing protease
inhibitors as described above. Membranes and luminal contents were then
processed for immunoprecipitation. In some experiments, luminal
contents were subjected to KBr density gradient centrifugation as
described above and apoB in density fractions immunoprecipitated.
Immunoprecipitation and Western Blotting--
Samples
were pre-cleared by incubation with gelatin- and protein G-Sepharose
and nonimmune goat or rabbit serum, mouse, or rat IgGs for 1 h at
4 °C. Beads were removed by centrifugation, and supernatants were
incubated at 4 °C for 18-20 h with antibodies to apoB, GRP94, or
ERp72 along with appropriate controls as indicated in the figures.
Immunocomplexes were captured with protein G-Sepharose as described
(26). For native immunoprecipitation, beads were washed in PBS followed
by phosphate buffer containing 0.5 M NaCl and, finally, in
PBS.
Immunocomplexes were solubilized by heating the beads to 95 °C in
Laemmli sample buffer (38) containing 10%
-mercaptoethanol and 6 M urea, then resolved by 4-15% SDS-PAGE followed by
electrotransfer onto PVDF membranes (26). ApoB, chaperones, ER, and
Golgi marker proteins were probed with appropriate antibodies,
visualized by the ECL system (NEN Life Science Products), and
quantified by densitometry, using Duoscan T1200 Agfa Densitometer and
Zero-Dscan image analysis system.
Lectin Blotting--
Microsomes were prepared from
un-cross-linked HepG2 cells as described above, then extracted with
carbonate and luminal contents subjected to native immunoprecipitation
with either rabbit antiserum to GRP94 or nonimmune rabbit serum.
Immunocomplexes were resolved on 4-15% SDS-PAGE and transferred onto
PVDF membranes. Nonspecific sites were blocked by incubating the
membranes in 5% albumin, PBS for 1 h. Membranes were then
incubated in either buffer alone (phosphate buffer, pH 6.8, 1 mM CaCl2, 0.01% albumin) or buffer containing
20 milliunits/ml neuraminidase for 20-24 h at 37 °C. Membranes were
then washed and incubated with biotinylated SNA-I, which was visualized
using the ECL system following incubation with HRP-extravidin.
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RESULTS AND DISCUSSION |
Chaperones Interact Both with the Membrane-associated Pool of ApoB
and with the Pool Released into the Microsomal Lumen--
Unlike other
secretory proteins, the newly translated pool of apoB remains
associated with the ER membranes during early stages of folding, which
involve MTP-dependent lipidation to form a primordial lipoprotein intermediate that is ultimately released into the ER lumen
(39). We hypothesized that it is during these early stages of folding
that molecular chaperones would be most critical for its successful
folding and maturation. The partially lipidated intermediate that is
translocated into the lumen is conceivably more stable than the
membrane-bound pool and, therefore, may not require the assistance of
molecular chaperones, so that it could proceed to acquire the bulk of
lipids to form VLDL/IDL independently of chaperones. Alternatively,
this intermediate may still depend on chaperone assistance for its
subsequent folding steps. In that case, two scenarios may be
envisioned: (a) it remains associated with the same array of
molecular chaperone as those interacting with the membrane-bound pool,
or (b) it remains associated with a subset of chaperones
that may be necessary to keep it in a conformation competent for
binding the bulk lipids. To test these possibilities, we determined
first whether chaperones are associated with the luminal pool of apoB.
To that end, crude microsomes were prepared from HepG2 cells that were
pre-incubated with 0.8 mM oleate and the calpain inhibitor
ALLN, which also effectively inhibits proteasomes (40) to increase
folding efficiency. Microsomes were subjected to three extraction
protocols to release the luminal contents: carbonate (0.1 M, pH 11.5) alone or carbonate supplemented either with
deoxycholate (carbonate/DOC) or deoxycholate and KCl (carbonate/DOC/K). The latter two protocols were demonstrated to increase the efficiency of extraction of lipidated apoB from microsomal membranes prepared from
McA-RH7777 cells (36). Immunoblotting showed that the inclusion of
0.025% DOC, which is well below its critical micellar concentration, did not solubilize the microsomal membranes as evidenced by the fact
that the integral membrane protein, calnexin, was not extracted (data
not shown). Therefore, these protocols can be applied to HepG2-derived
microsomes. Quantification of apoB in membranes and lumen showed that
the amount of apoB extracted by carbonate-supplemented DOC with or
without KCl was similar (data not shown), and both protocols typically
led to higher levels of extracted apoB compared with carbonate alone
albeit the difference between these protocols was not statistically
significant. Thus, an average of approximately 48 ± 12 (range,
30-60%) and 62 ± 10% (range, 47-72%) (n = 5)
of the total pool of apoB was extracted by carbonate and carbonate/DOC, respectively. Therefore, either carbonate alone or carbonate/DOC with
or without KCl can be used interchangeably with sufficient recovery of
the luminal apoB pool for further characterization.
Fig. 1A shows that apoB
released either by carbonate or carbonate/DOC (lanes 1 and
3, respectively) was associated with the major ER-resident
molecular chaperones including GRP94, ERp72, calreticulin, CyPB, and
BiP (data not shown). Importantly, the level of co-immunoprecipitated
chaperones was substantially higher than the corresponding controls
(compare lanes 1 and 3 to
lanes 2 and 4, respectively).
Therefore, co-immunoprecipitated chaperones reflect apoB-chaperone
interactions existing in living cells. Furthermore, following
quantification of apoB and chaperone bands, we calculated
the chaperone/apoB ratio and found that it was similar whether
microsomes were extracted with carbonate or carbonate/DOC/K.

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Fig. 1.
Molecular chaperones interact both with the
membrane-bound and the luminal pools of apoB. A, HepG2 cells
were incubated for 2 h with 0.8 mM oleic acid and 25 µg/ml ALLN. Cell monolayers were then incubated with DSP to
cross-link cellular proteins. Crude microsomes were prepared and then
extracted with either carbonate (lanes 1 and
2) or carbonate/DOC/K (lanes 3 and
4). Luminal contents were separated from membranes, then
adjusted to 2% CHAPS and subjected to immunoprecipitation with goat
antiserum to human apoB (lanes 1 and
3) or nonimmune goat serum (lanes 2 and 4) followed by SDS-PAGE and Western blotting.
B, cells were incubated as above and microsomes extracted
with carbonate/DOC. Membranes (lanes 5 and
6) and luminal contents (lanes 7 and
8) were adjusted to 1% SDS in PBS and heated to 65 °C
for 15 min. The samples were then adjusted to 0.1% SDS with 1% Triton
and subjected to immunoprecipitation either with antiserum to apoB
(lanes 5 and 7) or goat nonimmune
serum (lanes 6 and 8) followed by
Western blotting. ApoB and chaperones were visualized by the ECL
system, as described under "Experimental Procedures." M,
membranes; L, lumen; NIS, nonimmune serum.
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Next, it was of interest to determine the chaperone/apoB ratio in the
lumen compared with the membrane-bound pool. In these series of
experiments, however, we could not determine with certainty whether the
spectrum and the level of molecular chaperones associated with these
pools of apoB are comparable, because the efficiency of
immunoprecipitated membrane-bound apoB was very low following solubilization of microsomal membranes by CHAPS. To substantially increase the efficiency of solubilization, membranes and luminal contents were heated to 65 °C in the presence of 1% SDS prior to
immunoprecipitation with antibodies to apoB. Fig. 1B shows that the same array of chaperones is associated both with the membrane-bound and the luminal pools of apoB (lanes
5 and 7, respectively), namely GRP94, ERp72,
calreticulin, CyPB and BiP (data not shown). When antiserum to apoB was
replaced with nonimmune serum, chaperone protein bands were diminished
(lanes 6 and 8), strongly suggesting that the observed interactions of these chaperones with apoB exist in
living cells. Notably, the presence of SDS in the immunoprecipitation buffer led to a substantial reduction in background, consistent with
reduction of nonspecific ionic interactions between chaperone proteins
and the protein G-Sepharose beads (lanes 6 and
8). Quantification of apoB and chaperone bands revealed that
chaperone/apoB ratio in the lumen was at least 3-4-fold higher than
that in the membranes. This was consistently observed for GRP94, ERp72,
and CyPB, although the -fold increase varied between experiments. In
contrast, calreticulin/apoB ratio in the lumen was essentially similar
to that in the membranes. These findings therefore clearly demonstrate
different roles for these chaperones in the folding and maturation of
apoB. Thus, GRP94, ERp72, BiP, and CyPB presumably play a role not only
in early folding events occurring cotranslationally while apoB is membrane-bound, but they also seem to facilitate the release of the
lipidated intermediate from the membrane. One possibility is that they
replace hydrophobic interactions between apoB and the ER membrane,
thereby promoting its detachment from the membrane and providing
stability to this intermediate once translocated into the lumen.
Calreticulin on the other hand, being a lectin-like chaperone, may be
involved in other aspects of apoB folding, so that no additional
molecules are recruited to interact with the primordial intermediate
prior to or after its release into the lumen. Nonetheless, calreticulin
seems to play a role both during early and late folding events, as it
also remains associated with the primordial intermediate after its
translocation into the lumen.
What is the nature of the membrane-bound pool of apparently full-length
apoB? Because these experiments were carried out under conditions that
support optimal lipoprotein assembly (e.g. by providing
oleic acid and inhibiting degradation by ALLN), this pool of apoB does
not represent misfolded molecules. Rather, this pool is likely to
represent the precursor of the primordial intermediate, which
ultimately matures into secretion-competent lipoproteins with VLDL,
IDL, and LDL-like densities. Being membrane-bound and having an
apparent size of full-length apoB, this pool is presumably not
fully-translated, although very close to it (41). It follows that these
molecules are almost fully lipidated as translation is closely
coupled to lipidation in HepG2 cells (41). This pool, being
in the process of translation, interacts both with the translocon and
the ribosome (41). This could be partly responsible for a fraction of
the pool to be nonextractable by either carbonate or carbonate/DOC in
protocols involving cross-linking of proteins. In addition, it is
conceivable that domains enriched in
-strands predicted to form
amphipathic
-sheets (42-44) interact with the luminal leaflet of
the ER membrane until sufficient core lipids are recruited to form the
primordial intermediate.
These experiments also demonstrated for the first time a direct
interaction between CyPB and apoB. This suggests that CyPB is involved
in the folding of apoB and may even be critical for its successful
folding. This is based on studies showing that the immunosuppressant,
cyclosporine A, prevents the secretion of apoB (45), concurrently with
diminished interaction of CyPB with
apoB.2 Although cyclosporine
A also binds to cyclophilin A, its cytosolic residence makes it an
unlikely candidate chaperone to promote folding of apoB, which takes
place in the ER. In fact, it was demonstrated that, unlike the CFTR
receptor (46), interaction of apoB with the cytosolic chaperones Hsp70
and Hsp90 leads to its targeting for degradation (20-22). CyPB is both
a folding catalyst belonging to peptidyl prolyl
cis-trans-isomerases and a chaperone (47-49).
Either activity or both may be important for the correct folding of
apoB, because (a) apoB has a number of prolines that may be
dependent on the peptidyl prolyl
cis-trans-isomerase activity of CyPB for
efficient folding, and (b) CyPB, like GRP94 and ERp72, seems
to play a role in mediating the detachment of the primordial intermediate from the membrane. Further studies will be necessary to
characterize the role of CyPB in apoB maturation in more detail.
Chaperones Interact with Lipidated ApoB with HDL-like Density
Released into the Microsomal Lumen--
The previous series of
experiments (Fig. 1) demonstrated that molecular chaperones are
associated with the luminal pool of apoB. However, these experiments
did not provide direct evidence for the interaction between these
chaperones and the lipidated pool of apoB. To directly demonstrate such
interactions, microsomes prepared from cells following a 30-min
pulse-labeling period were extracted with carbonate, and the soluble
contents were subjected to KBr density gradient fractionation. This
extraction protocol was chosen for two reasons: first, to allow for a
direct comparison with previous studies involving careful
characterization of apoB-100-containing particles in HepG2 cells (13);
second, we determined that there was no qualitative difference in the
density distribution of particles released by carbonate or
carbonate/DOC (data not shown) consistent with other studies (36). Fig.
2A shows that approximately
80% of the newly synthesized pool of apoB was found in fractions
having HDL-like density with a peak at 1.12 g/ml. This pool of apoB was associated with GRP94, ERp72 (panel C), as well
as BiP, calreticulin, and CyPB (data not shown).

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Fig. 2.
Lipidated apoB present in the microsomal
lumen is associated with molecular chaperones and has a peak density of
HDL-like lipoproteins, whereas secreted apoB fractionates with VLDL/IDL
and LDL-like lipoproteins. HepG2 cells were incubated for 2 h
with 0.8 mM OA, then pulse-labeled with
[35S]methionine/cysteine for 30 min and chased for 2h.
Cells harvested after the pulse were subjected to cross-linking, and
crude microsomes prepared and extracted with carbonate. Luminal
contents and conditioned media collected after a 2-h chase were
adjusted to 1.25 g/ml with solid KBr and subjected to density gradient
centrifugation. ApoB in density fractions was immunoprecipitated with
antibodies to apoB, resolved by SDS-PAGE, visualized by
autoradiography, and quantified using Imagequant and expressed as
percentage of total in lumen (A) and media (B).
Chaperones associated with apoB in lumen were identified by Western
blotting (C).
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Because the association of chaperones with lipidated apoB is likely to
increase the overall density of apoB-containing particles, it was
necessary to ensure that the observed density distribution of apoB does
not result from excessive cross-linking of proteins that are not
otherwise associated with apoB in living cells. To that end, we
performed another series of experiments in which cells were not
subjected to cross-linking prior to microsomal preparation. These
experiments showed that the density distribution of apoB present in the
lumen was very similar to that obtained following cross-linking of
cellular proteins (data not shown).
The size distribution of intracellular apoB-containing particles in
HepG2 cells was analyzed by Bostrom et al. (13), who demonstrated that particles isolated from HDL-like densities
(e.g. 1.065-1.170 g/ml) had a mean diameter of
approximately 25 and 20 nm following pre-incubation of cells with or
without oleate, respectively (13). This size is typical of plasma LDL
particles, which contain apoB-100 as their only protein constituent.
The fact that the mean density of these particles is in the HDL range is consistent with these particles having higher protein to lipid ratio
compared with plasma LDL that is contributed by the associated chaperones. Indeed, these investigators suggested, among other possibilities, that perhaps the discrepancy between the size and the
density may be due to the presence of proteins (unknown at the time),
other than apoB on the lipoproteins isolated from the microsomal lumen
(13). However, because molecular chaperones have a slow turn-over rate
(e.g. several days), no additional proteins were detected
following a short term radiolabeling protocol (13).
These series of experiments clearly demonstrated that apoB is released
into the lumen as a lipidated intermediate with an HDL-like density
that is still dependent on the assistance of molecular chaperones for
its subsequent folding steps.
The density distribution of secreted apoB-containing particles is very
different from that of the intracellular pool, as a significant
fraction, approximately 44 and 34% is found in lower densities,
e.g. d < 1.02 and
1.02 < d
1.03 g/ml, respectively (Fig.
2B), clearly reflecting a higher lipid to protein ratio presumably because of both higher lipid content and lower protein content (caused by absence of chaperones). None is found in HDL-like densities, consistent with the finding that the particles are secreted
free of chaperones (data not shown) and with the hypothesis that
apoB-100 cannot attain a stable conformation when associated with
particles having a lipid-poor core. This can be attributed to those
domains starting at apoB-21 enriched in
-sheets (42, 43), which
presumably "prefer" to be bound to TAG because this is a more
energetically favorable state (43, 44). Therefore, it is conceivable
that these domains are also most likely to be engaged in interaction
with molecular chaperones, which stabilize them until enough lipids are recruited.
Chaperones Are Associated with Advanced Folding Intermediates of
ApoB That Are Transported to the Golgi--
The major folding steps of
newly translated proteins that enter the secretory pathway take place
in the ER, where resident chaperones and folding catalysts assist in
their folding and assembly by direct physical interaction. Such
interactions persist until proper folding is attained, whereby the
affinity between chaperones and folding intermediates declines,
ultimately leading to the release of chaperones (31, 32). Correctly
folded proteins are then transported to the Golgi for further
modifications and for sorting to their target destination.
Because the folding of apoB is much more complex than most other
secretory proteins and we have demonstrated that molecular chaperones
remain associated with apoB following its translocation into the lumen
as a lipidated intermediate (Fig. 2), it was of interest to determine
at what stage of maturation chaperones dissociate from apoB. To that
end, cell homogenates were fractionated on sucrose density gradients,
apoB in subcellular fractions immunoprecipitated, and associated
chaperones identified by Western blotting. Fig. 3A shows the distribution of
marker proteins. Both Golgi markers, e.g. Golgin 97 (50),
GPP130 (51), and p58 (52), and the ER marker calnexin (53) are shown.
As evidenced from this figure, all Golgi markers are present in
fraction 3. GPP130 demonstrated to reside in the cis/medial Golgi (51)
is also present to a certain extent in fraction 2. On the basis of this
analysis, fraction 3 represents cis/medial Golgi. This fraction also
contains the trans-Golgi network (TGN) as it also contains the entire
pool of sialylated apoB (data not shown).

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Fig. 3.
Molecular chaperones are associated with apoB
localized to the ER and the Golgi. HepG2 cells were incubated for
2 h with oleic acid and ALLN, subjected to cross-linking, and
homogenized as described under "Experimental Procedures."
Postnuclear supernatants were adjusted to 1.3 M sucrose,
overlaid with 1.2, 1.15, 0.8, and 0.25 M sucrose, and
centrifuged for 16 h at 86,000 × g. Fractions
were collected, washed, and extracted with carbonate/DOC/K. ApoB was
immunoprecipitated with polyclonal antibodies to apoB. Marker proteins
(A), luminal apoB and associated chaperones (C)
were analyzed by immunoblotting and quantified by densitometry. ApoB in
each fraction was expressed as percentage of total apoB recovered
(B). Chaperone/apoB ratio in the Golgi was expressed as
percentage of chaperone/apoB in the ER (D). Values for
GRP94, ERp72, and CRT represent the average of at least three
experiments. Error bars indicate ± standard
error. BiP and CyPB represent the average of two experiments.
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The distribution of calnexin, an integral membrane protein of the ER,
is quite different, as it is found predominantly in heavy microsomes.
Thus, the ER fractionates with heavy microsomes. From here on, heavy
microsomes and ER will be used interchangeably.
The distribution of luminal apoB is shown in panel
B. As expected, a substantial fraction, approximately 40%
is found in heavy microsomes, which include the ER. A smaller but
sizable fraction, approximately 20%, was found in cis/medial Golgi
(fraction 3). Such distribution suggests that, during its folding, apoB
resides primarily in the ER demonstrated to be the site where its major folding steps take place (54). The presence of a sizable fraction of
the intracellular pool of apoB in the Golgi is consistent with other
reports demonstrating the presence of apoB in Golgi from HepG2 cells
(55). Similarly, VLDL particles were also detected in Golgi from rat
(56) and mouse liver (57). The presence of apoB in this compartment
under steady-state conditions is consistent with modifications of apoB
such as glycosylation and phosphorylation (58-62), as well as possible
remodeling by exchange of phospholipids between the Golgi membranes and
the nascent particles (63, 64).
Most interesting is the finding that apoB present in the Golgi
compartment is associated with the same spectrum of molecular chaperones as those bound to apoB in the ER. These include
GRP94, ERp72, calreticulin, BiP, and CyPB (panel
C). These findings therefore demonstrate that apoB exits the
ER in complexes containing the same chaperones assisting in its early
folding stages. It is possible that other molecular chaperones such as
protein disulfide isomerase (PDI) are also present in these complexes.
Chaperone/apoB ratio within the subcellular compartments is shown in
Fig. 3D. It appears that within the early secretory pathway
(e.g. ER) the chaperone/apoB ratio is substantially higher.
Thus, chaperones dissociate from the particles before transport to the
Golgi. The relative amount is dependent on the specific chaperones.
Thus, calreticulin/apoB ratio in Golgi is approximately half that in
the ER, GRP94/, and ERp72/apoB ratios are approximately 30 and 20%,
respectively, and CyPB/ and BiP/apoB decline to less than 10% (Fig.
3D). Thus, it appears that a substantial amount of chaperone
molecules dissociate from apoB-containing lipoproteins prior to their
arrival at the Golgi compartment consistent with apoB being in a more
advanced folding stage. Nevertheless, the fact that a sizable fraction remains bound to apoB during and after its transport to the Golgi is
consistent with different roles for these chaperones in late folding
events occurring in post-ER compartments.
To ensure that the chaperones co-precipitated with the pool of apoB
that is present in the Golgi interact with apoB in living cells, we
performed two series of experiments.
In one series, cells were exposed to BMS-197636, a specific inhibitor
of MTP that blocks the lipid transfer activity of MTP in
vitro (65). As a result, apoB is unable to form lipoproteins and
is rapidly degraded in a proteasome-dependent mechanism
(17). Under these conditions it is expected that the major pool of apoB would be largely unfolded/misfolded and therefore primarily localized to the ER. Therefore, only very low levels of chaperones are expected to co-precipitate with antibodies to apoB in the Golgi. We found that
this inhibitor reduced the amount of secreted apoB by greater than 80%
(data not shown) concomitantly with a substantial reduction in the
level of apoB in the Golgi compared with control levels (compare
lane 1 with lane 3).
Consistent with the diminished level of apoB in the Golgi, the level of
GRP94 and ERp72 co-immunoprecipitated with apoB is also greatly reduced
(compare lane 1 to lane 3). The reduced level of apoB in the ER lumen of cells treated with the
inhibitor (compare lane 4 to lane 2)
is consistent with diminished lipidation of apoB, thus preventing its
translocation into the lumen.
Together, these findings demonstrate that co-precipitation of
ER-resident molecular chaperones with apoB in the Golgi is dependent on
the presence of apoB.
In the next series of experiments, cross-linking of cellular proteins
was eliminated to minimize manipulations prior to fractionation and
immunoprecipitation. To that end, un-cross-linked cells were homogenized and subjected to subcellular fractionation.
Immunoprecipitation of luminal contents was carried out under native
conditions, so that apoB-chaperone interactions were minimally
disrupted. We used polyclonal antibodies to GRP94 and two monoclonal
antibodies to apoB, 2D8 and B4, which bind to a domain encompassing
residues 1438-1480 (66) and 1854-1878 (67), respectively. As shown in
Fig. 5, antibodies to GRP94 co-immunoprecipitated apoB both in the
Golgi (lane 9) and in the ER (lane
11), whereas control samples that were incubated with
nonimmune serum co-precipitated substantially less apoB
(lanes 10 and 12, respectively).
Similarly, antibodies to apoB, B4, specifically co-immunoprecipitated
GRP94 from Golgi and ER (lanes 5 and
7, respectively), whereas control samples precipitated only
low levels of GRP94 (lanes 6 and 8, respectively). In a similar series of experiments, 2D8 and B4 antibodies co-immunoprecipitated calreticulin (data not shown), GRP94
and ERp72 from the Golgi (lanes 1 and
3, respectively) and the ER (lanes 2 and 4, respectively). Thus, the observed apoB-chaperone interactions following cross-linking of cellular proteins does not
result from excessive cross-linking of cellular proteins, which are not
otherwise in close contact with apoB, but rather reflect interactions
that exist in living cells, and as long as the immunoprecipitation
protocol does not require detergents, these interactions are not disrupted.
Thus, from the experiments shown in Figs.
4 and 5, we
conclude that ER-resident molecular
chaperones interact with apoB both in the ER and in the Golgi. We
calculated the relative amount of apoB associated with GRP94 in the ER
and Golgi from the amount of apoB immunoprecipitated with antibodies to
GRP94 and the efficiency of the immunoprecipitation of GRP94. We found
that 40-60 and 25-35% of the total pool of apoB in the ER and the
Golgi, respectively, is associated with GRP94 under steady-state
conditions.

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Fig. 4.
MTP inhibitor changes the subcellular
distribution of apoB and associated chaperones. HepG2 cells were
incubated for 2 h with OA and ALLN without (lanes
1 and 2) or with 100 nM MTP
inhibitor, BMS-197636 (lanes 3 and 4).
Cells were then processed as in Fig. 3. ApoB and associated chaperones
were identified by immunoblotting. G, Golgi.
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Fig. 5.
Molecular chaperones co-immunoprecipitate
with apoB in Golgi and ER without prior cross-linking of cellular
proteins. Cells were incubated and processed as in Fig. 3, except
that cellular proteins were not cross-linked prior to subcellular
fractionation. Luminal contents of Golgi (lanes
1, 3, 5, 6, 9,
and 10) and ER (lanes 2, 4,
7, 8, 11, and 12) fractions
obtained by carbonate extraction were neutralized and subjected to
immunoprecipitation with monoclonal antibodies to apoB, 2D8
(lanes 1 and 2) or B4
(lanes 3-5 and 7), or with rabbit
antiserum to GRP94 (lanes 9 and 11).
Control samples were incubated with nonimmune mouse IgGs
(lanes 6 and 8) or nonimmune rabbit
serum (lanes 10 and 12) under native
conditions. Immunocomplexes were resolved on SDS-PAGE and apoB and
molecular chaperones identified by immunoblotting. Three representative
experiments are shown: experiment 1 (lanes 1-4), experiment
2 (lanes 5-8), and experiment 3 (lanes 9-12).
In experiment 1, all lanes are from the same exposure time. In
experiments 2 and 3, Golgi and ER should not be compared directly as
they are from different exposure times. Samples immunoprecipitated with
antibodies and the corresponding controls within the same compartment
are from the same exposure time and can be compared with each other.
G, Golgi; E, ER; NI, nonimmune (IgG or
serum).
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Interestingly, the pool of apoB that was immunoprecipitated from the
Golgi (Fig. 5) (lanes 1 and 3)
migrated slightly slower than its ER-derived counterpart
(lanes 2 and 4) consistent with modifications of apoB occurring in a post-ER compartment, conceivably, the Golgi itself. These include modifications of the glycan residues, phosphorylation (62) or both. These differences between the pools of
apoB were observed in a number of experiments and provide further
support for efficient separation between the early secretory compartment, the ER, and the Golgi, and for the presence of distinct pools of apoB within these compartments. These findings, therefore, strongly suggest that apoB-molecular chaperone complexes exist within
the Golgi compartment in living cells, excluding the possibility that
such complexes are derived from vesicles originating in the ER.
Overall, these studies clearly demonstrate that apoB is transported to
the Golgi as part of complexes containing ER-resident molecular
chaperones. The presence of different levels of chaperones compared
with the ER suggests distinct roles for these chaperones in late
folding events occurring in the Golgi.
The Major Pool of ApoB Present in the Golgi and in Heavy Microsomes
Is Lipidated and Is Associated with the Same Array of Molecular
Chaperones--
The next series of experiments was designed to
determine whether the chaperone-associated pool of apoB present in the
Golgi is lipidated. Fig. 6A
shows that the majority of the apoB pool in the ER is found in
1.08 < d < 1.2 g/ml, whereas that in the Golgi
is found at lower densities, 1.02 < d < 1.08 g/ml. Nevertheless, apoB present in this lower density range is still
associated with the same array of chaperones associated with the pool
of apoB recovered from the ER, including GRP94, ERp72, BiP,
calreticulin, and CyPB (Fig. 6B). The lower density of apoB
in the Golgi reflects a higher lipid to protein ratio. This could be
caused by either an increase in the relative content of lipid, decrease
in relative content of protein without change in lipid content, or
both. Increase in lipid content can occur by recruitment of neutral
lipids in post ER pre-Golgi compartments, or in the Golgi itself. Such
a model is consistent with continuous recruitment of lipids in these cells rather than a two-step model demonstrated in liver and McA-RH7777 cells (3-8). The release of a substantial fraction of chaperones from
apoB-containing particles prior to exit of particles from the ER (Fig.
3D) may partially account for the shift in the density of
the particles in the Golgi with no substantial addition of lipid. To
test this possibility, we estimated the content of lipid in the ER and
Golgi.

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Fig. 6.
Density distribution of apoB in Golgi and
ER. Cells were incubated, and processed as in Fig. 3. Golgi
and ER were extracted with carbonate/DOC/K and luminal contents
adjusted to 1.25 g/ml with solid KBr and subjected to density gradient
centrifugation. Twelve fractions were collected from the top and
density measured as described under "Experimental Procedures."
Fractions were combined as follows: fraction 1, d 1.02 g/ml; fraction 2, 1.02 < d < 1.08 g/ml;
fraction 3, 1.08 < d < 1.21 g/ml; and fraction
4, d > 1.21 g/ml. ApoB in combined fractions was
immunoprecipitated and quantified by densitometry as described in Fig.
3. A, distribution of apoB in density fractions expressed as
percentage of total apoB in Golgi (squares) and ER
(circles). B, an immunoblot showing apoB and
associated molecular chaperones in Golgi (left) and ER
(right).
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The relative amount of lipid and protein was calculated from the
density of the particle, which equals to the sum of protein and lipid
density using densities of 1.33 and 0.94 g/ml for protein and lipid,
respectively.
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(Eq. 1)
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Using average densities of 1.16 and 1.05 g/ml for particles in the
ER and Golgi, respectively, we calculated that the fraction of protein
and lipid in particles in the ER is 0.56 and 0.44, and in the Golgi
0.28 and 0.72, respectively.
The molecular mass of protein is the sum of the mass of apoB and
associated chaperones. To calculate that, we made two alternative assumptions. Particles in the Golgi have at least one molecule of each
of the identified chaperones (e.g. GRP94 as a homodimer, ERp72, BiP, calreticulin, and CyPB) per particle (or per apoB molecule). Alternatively, there are two chaperone molecules per particle. According to the first assumption, the mass of protein in
Golgi-derived particles is approximately 0.95 × 106
daltons. The mass of protein in ER-derived particles where the chaperone/apoB ratio is severalfold higher, was calculated from the
data presented in Fig. 3D and found to be approximately
2.52 × 106 daltons. From the lipid to protein ratio,
the molecular mass of lipid in particles derived from Golgi and ER is
2.44 and 1.98 × 106 daltons, respectively. Thus,
according to the first assumption, lipid content in Golgi-derived
particles is approximately 23% higher than that in ER-derived particles.
If calculations are made assuming two chaperones per particle, the mass
of protein in Golgi and ER-derived particles is 1.36 and 4.52 × 106 daltons, respectively, and that of lipid is 3.50 and
3.55 × 106 daltons, respectively. According to the
second assumption, the content of lipid in Golgi-derived particles is
similar to that of ER-derived particles. Thus, it is possible that
changes in the amount of chaperones per particle can have a noticeable
effect on the density of the particle. Although it is conceivable that the number of individual chaperone molecules per particle varies, the
calculations shown above give a reasonable estimate of the actual number.
The size of the particles in the ER and the Golgi was calculated from
the anhydrous volume (V) of the particle shown in Equation 2.
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(Eq. 2)
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Assuming a spherical particle, the radius (r) can be
calculated from the following equation.
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(Eq. 3)
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The calculated size of particles in the ER and Golgi is 230 and
210 Å diameter, respectively, assuming one chaperone molecule/apoB in
Golgi or 280 and 244 Å, respectively, assuming two chaperone molecules/apoB in Golgi. Because these calculations are based on the
volume of an anhydrous sphere, the actual size of these particles is
approximately 20% larger.
Clearly, the size and the density of the particles reflect the fact
that there are proteins other than apoB on these particles. According
to these calculations, particles in the Golgi seem slightly smaller
than those in the ER. This may be a result of the fact that the shape
of the particles is not a perfect sphere; therefore, the calculations
based on a spherical shape may deviate to a certain extent from the
actual size. This is probably more pronounced for particles in the ER,
which appear to contain severalfold more chaperone proteins compared
with Golgi-derived particles. Nonetheless, these calculations give a
good estimate of the true size of the hydrated particles, particularly
as they are in agreement with those measured by negative-stain electron
microscopy (13). Thus, under steady-state conditions, particles of at
least LDL or IDL/VLDL size are present both in the ER and in the Golgi.
This may indicate that in HepG2 cells the major recruitment of lipids
occurs in the ER.
Notably, the density of apoB-containing particles in the Golgi is still
substantially higher than that of the secreted particles, 40% of which
are found in d < 1.02 g/ml (Fig. 2B). This
is presumably because of associated chaperones in the Golgi. In the
search for proteins that might be involved in the second step of VLDL
assembly, Stillmark et al. (28) found that PDI,
GRP94, ERp72, BiP, and calreticulin co-fractionated with apoB-48-VLDL
from rat liver. However, the subcellular localization of these
particles was not determined.
GRP94 Is Associated with ApoB That Had Been Transported to the
TGN--
The previous series of experiments clearly demonstrated that
ER-resident molecular chaperones are associated with apoB that had been
transported to the Golgi complex. However, it was unclear in which
compartment within the Golgi these chaperones dissociate from
apoB-containing particles. We were interested in determining whether
molecular chaperones interact with apoB that had been transported to
distal compartments of the secretory pathway, such as the TGN. To do
that, we took advantage of the fact that the enzyme sialyltransferase
resides in the TGN, and used lectin blotting to identify sialylated
apoB. In the experiment depicted in Fig. 7, luminal contents were subjected to
immunoprecipitation with antibodies to GRP94, followed by lectin and
immunoblotting. Membranes were incubated either in buffer alone
(lanes 1 and 2) or buffer containing
neuraminidase (lanes 3 and 4).
Membranes were then probed with the lectin SNA-I that preferentially
binds to sialic acid attached to terminal galactose in
-2,6 linkage.
Bound SNA-I was visualized following binding of HRP-avidin. A clear
band of SNA-I was observed in the sample that was immunoprecipitated
with antibodies to GRP94 and was incubated in buffer alone
(lane 1), whereas only a faint band was seen in
the control sample in which antibodies were replaced by nonimmune serum
(lane 2). As expected, incubation of samples with
neuraminidase prevented the binding of SNA-I (lane
3), consistent with removal of terminal sialic acid residues
by this enzyme. Thus, the binding of SNA-I reflects the presence of
sialic acids. Probing of these membranes with antibodies to apoB
confirmed that the bands identified with SNA-I represent apoB-100.
These findings, therefore, are consistent with the notion that at least
a fraction of the pool of apoB that was modified by enzymes residing in
the TGN was associated with GRP94. Similar results were obtained using
antibodies to ERp72 (data not shown). From the relative amount of the
sialylated pool of apoB that was co-precipitated with antibodies to
GRP94 and from the efficiency of GRP94 immunoprecipitated with these
antibodies, we estimated that approximately 15-25% of the sialylated
pool of apoB is associated with GRP94. Thus, at least GRP94 and
possibly ERp72 remain associated with apoB even after it had been
transported to the TGN.

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Fig. 7.
GRP94 is associated with apoB that has gone
through TGN. Cells were incubated with OA and ALLN and then
homogenized without prior cross-linking of proteins, and microsomes
were prepared and extracted with carbonate. Luminal contents were then
subjected to immunoprecipitation with rabbit antibodies to GRP94
(lanes 1 and 3) or nonimmune serum
(lanes 2 and 4) and immunocomplexes in
duplicates resolved by SDS-PAGE and electrotransferred onto a membrane.
Membrane was blocked with albumin, and then one half was incubated with
buffer alone (lanes 1 and 2) and the
other half with buffer containing neuraminidase (lanes
3 and 4) for 20 h at 37 °C. Both halves
were then probed sequentially with the lectin SNA-I followed by
antibodies to apoB. Bound lectin was visualized by extravidin-HRP.
NIS, nonimmune serum.
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Given the role of ER-resident molecular chaperones in primary
quality control in the ER (31, 34), the question remains as to what is
the role of their prolonged interaction with apoB well beyond the ER?
It is generally thought that these chaperones assist in the folding of
nascent polypeptides in the ER. The progression of the folding process
is very closely monitored by these chaperones so that successful
folding leads to the release of the properly folded nascent proteins,
which then exit the ER and move to the Golgi free of chaperones.
However, if correct folding is not achieved, misfolded proteins are
retained in the ER bound to these chaperones, which prevent their
transport to their target destination. This mechanism is very efficient
in mammalian cells, so that only a very small fraction of misfolded
proteins escapes this primary quality control (31, 34). Backup
mechanisms beyond the ER (provided by a different set of proteins; Ref.
31) ensure that those that escaped the ER are prevented from delivery
to their destination. Consistent with such a tight quality control
mechanism, it was demonstrated that apoB can be targeted for
degradation at different stages of its maturation beyond the ER (15,
40, 55, 68, 69). This, however, is an unlikely scenario occurring in
HepG2 cells under the conditions used in this study, because the cells
were incubated under conditions that support optimal folding of apoB
resulting in a very efficient secretion. A more likely explanation for
the prolonged interaction between ER-resident chaperones and apoB is
that the maturation of apoB to form VLDL/IDL is a multistep process
that begins in the ER and continues throughout the secretory pathway
including distal compartments of the Golgi, as demonstrated by a number
of investigators (56, 57, 63, 64, 70). These include modifications of
apoB such as glycosylation and phosphorylation, remodeling of the
phospholipid on the surface of the particles, and possibly continued
lipidation (71, 72). All of these may tag apoB as an incompletely
folded (e.g. it is on a path for successful folding), rather
than a misfolded protein, therefore requiring assistance of ER-resident
chaperones for its stabilization. Molecular chaperones could also
facilitate the transport of apoB-containing particles through the
secretory pathway, consistent with a proposal that chaperones are also
involved in protein trafficking, as demonstrated for
Drosophila whereby the cyclophilin homologue, ninaA, appears
to be required for rhodopsin Rh1 as it travels through the distal
compartments of the secretory pathway (73), whereas CyPB and HSP47 were
suggested to play a role in export of procollagen from the ER as they
were found associated with procollagen in the intermediate
compartment (74).
ApoB-associated chaperones could dissociate just before apoB VLDL/IDL
particles are packaged into secretory vesicles, consistent with
the finding that the pool of apoB recovered in the light Golgi
fraction (Fig. 3, fraction 2) is not associated
with chaperones. This fraction presumably contains secretory vesicles
(37).
If, however, apoB fails to undergo complete maturation either in the
early or in distal compartments of the Golgi, these chaperones may then
target it for degradation.
In summary, we demonstrated the following. (a) CyPB directly
interacts with apoB and therefore plays a role in folding and maturation of apoB. (b) all five major ER-resident molecular
chaperones and folding catalysts (GRP94, ERp72, BiP, CRT, and CyPB)
interact with the membrane-bound pool of apoB conceivably to keep it in a conformation competent for MTP-mediate lipidation. Except for CRT,
these chaperones seem to facilitate the translocation of lipidated apoB
into the microsomal lumen and to provide stability to the primordial
intermediate in the lumen. (c) ApoB is transported to the
Golgi in complexes containing the same molecular chaperones as those
interacting with the luminal pool of apoB in the ER except that
chaperone/apoB ratio is lower. Thus, apoB leaves the ER as an
incompletely folded intermediate. (d) Because GRP94
interacts with apoB that has gone through the TGN it is conceivable
that this chaperone (and perhaps others) plays a role in late folding events in the maturation of apoB occurring in distal compartments of
the secretory pathway.
On the basis of the data presented in this report, we propose a model
(Fig. 8) showing that the maturation of
apoB is dependent not only on efficient MTP-mediated lipidation, but
also on other ER-resident molecular chaperones that seem to play
multiple roles in the biogenesis of apoB to form TAG-rich lipoproteins
occurring throughout the secretory pathway, including the ER and distal Golgi compartments, contrary to current ideas assigning a role for
molecular chaperones primarily in folding events occurring in the
ER.

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Fig. 8.
A proposed model for chaperone-assisted
folding of apoB to form lipoproteins. Molecular chaperones
interact with apoB as it emerges through the translocon (shown in
red) into the ER lumen. Following elongation, domains which
are presumably enriched in amphipathic -sheets interact with the
membrane (43, 44) cotranslationally, where initiation of MTP-mediated
lipidation leads to the formation of a small lipid core (shown in
yellow) (step I). Ongoing
translation-coupled lipidation leads to elongation, enlargement of the
core, and recruitment of additional molecular chaperones
(step II). Recruitment of additional neutral
lipid molecules leads to completion of translation (41), followed by
dissociation of the ribosome (step III). At this
stage enough lipids had been recruited and the lipidated intermediate
begins to detach from the ER membrane. It appears, however, that for it
to completely detach, additional chaperone molecules, GRP94, ERp72, and
CyPB, but not CRT, are recruited to facilitate the release presumably
by replacing the hydrophobic interactions with the membrane and to
stabilize the primordial intermediate following its release into the
lumen (step IV). This intermediate recruits more
core lipids to form larger particles (step V).
Prior to ER exit, a fraction of the bound chaperones dissociates
(approximately 50-80% of CRT, GRP94, ERp72, and calreticulin, and
over 90% of BiP and CyPB), and the lipoprotein particles are
transported to the Golgi (step VI). ER-resident
chaperones remain associated with apoB-containing lipoproteins in the
Golgi while additional modifications take place (step VII).
Ultimately, all chaperones dissociate presumably before packaging into
secretory vesicles (step VIII) and nascent VLDL
is secreted.
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