From the Cardiovascular Institute and the Departments
of Medicine and of Cell Biology and Anatomy, Mount Sinai School of
Medicine, New York, New York 10029, the § Department of Cell
Biology and Anatomy, University of Alberta, Edmonton, Alberta T6G
2H7, Canada, the ¶ Department of Medicine, Columbia University
College of Physicians and Surgeons, New York, New York 10032, the
Departments of Surgery, Medicine, and Physiology, University of
California at San Francisco, San Francisco, California 94143, and the ** Department of Medical Biochemistry, Texas A&M University,
College Station, Texas 77843
Received for publication, August 30, 2000, and in revised form, October 4, 2000
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ABSTRACT |
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Hepatic lipoprotein assembly and
secretion can be regulated by proteasomal degradation of newly
synthesized apoB, especially if lipid synthesis or lipid transfer is
low. Our previous studies in HepG2 cells showed that, under these
conditions, newly synthesized apoB remains stably associated with the
endoplasmic reticulum (ER) membrane (Mitchell, D. M., Zhou, M.,
Pariyarath, R., Wang, H., Aitchison, J. D., Ginsberg, H. N.,
and Fisher, E. A. (1998) Proc. Natl. Acad. Sci.
U. S. A. 95, 14733-14738). We now show that independent of
lipid synthesis, apoB chains that appear full-length are, in fact,
incompletely translated polypeptides still engaged by the ribosome and
associated with the ER translocon. In the presence of active lipid
synthesis and transfer, translation and lipoprotein assembly are
completed, and the complexes exit the ER. Upon omitting fatty acids
from, or adding a microsomal triglyceride transfer protein
inhibitor to, culture media to reduce lipid synthesis or transfer,
respectively, apoB was degraded while it remained associated with the
ER and complexed with cytosolic hsp70 and proteasomes. Thus, unlike
other ER substrates of the proteasome, such as major histocompatibility
complex class I molecules, apoB does not fully retrotranslocate to the
cytosol before entering the ubiquitin-proteasome pathway. Although,
upon immunofluorescence, apoB in proteasome-inhibited cells accumulated
in punctate structures similar in appearance to aggresomes (cytosolic
structures containing molecules irreversibly lost from the secretory
pathway), these apoB molecules could be secreted when lipid synthesis
was stimulated. The results suggest a model in which 1) apoB
translation does not complete until lipoprotein assembly terminates,
and 2) assembly with lipids or entry into the ubiquitin-proteasome
pathway occurs while apoB polypeptides remain associated with the
translocon and attached to the ribosome.
Apoprotein B100, the major structural protein of atherogenic very
low density and low density lipoprotein particles, is an unusually
large secretory hepatic protein with a molecular mass ~550 kDa. The
regulation of the assembly and secretion of the apoB100-containing
lipoproteins has been studied in various hepatic cell models, including
the human hepatocarcinoma cell line HepG2. In human intestine,
enterocytes edit the transcript of the apoprotein B gene so that a
shorter protein, apoB48, is formed. Because HepG2 cells do not have
this editing activity (2), throughout this report, the abbreviation
apoB will refer exclusively to apoB100 and its incompletely translated polypeptides.
One of the early steps of apoB-lipoprotein biogenesis in the
endoplasmic reticulum (ER)1
is the association of translocated domains of apoB with its "lipid ligands" in a process mediated by the lipid transfer activity of
microsomal triglyceride transfer protein (MTP) (1). After the initial
co-translational lipidation of apoB, the remainder of the assembly
process is thought to occur post-translationally (3). If either the
synthesis or transfer of lipid ligands is limited, apoB is rapidly
degraded, as illustrated not only by numerous studies in
vitro (e.g. Refs. 4 and 5), but also by the human
genetic disease abetalipoproteinemia, which results from mutations of
MTP (6). We and others have shown in HepG2 cells that most of this
metabolically regulated intracellular degradation of apoB occurs
through the ubiquitin-proteasome pathway (7-9) in a process that
appears to involve the cytosolic chaperone hsp70 (8, 10).
Studies in yeast and mammalian systems have identified the proteasome
as the principal means of disposal of a growing list of ER-associated
proteins that presumably become misfolded because of either a
structural mutation or a failure to assemble properly into a multimeric
complex (11, 12). This disposal process has been called ER-associated
degradation (ERAD). Because the components of the ubiquitin-proteasome
machinery are located in the cytosol, degradation of secretory and
integral membrane proteins by this pathway requires that domains are,
or become, accessible to the cytosol. Studies on the degradation of
major histocompatibility complex class I heavy chains (13) and the
cystic fibrosis transmembrane conductance regulator (14), among other
examples, have shown that multi-ubiquitinylated substrates accumulated
in the cytosol when proteasome activity was inhibited, supporting a
model in which complete retrotranslocation of these proteins from the
ER to the cytosol occurs (14). At least for the cystic fibrosis transmembrane conductance regulator, as undegraded protein accumulated in the cytosol, large aggregates ("aggresomes"), irreversibly lost
from the secretory pathway, formed and were visible by fluorescence microscopy (14, 15).
In contrast to these other ERAD substrates, we and others have recently
shown that, in HepG2 cells, apoB destined for degradation does not
accumulate in the cytosol when the proteasome is inhibited (16), but
remains associated with the ER in close proximity to the translocon
protein Sec61p (1, 17). Thus, complete retrotranslocation may not be a
universal feature of ERAD. Rather, only a subset of domains of the
substrate may become exposed to and engaged by cytosolic components.
This would be consistent with the localization in yeast of the
ubiquitin-conjugating enzymes Ubc6p and Ubc7p to the cytosolic face of
the ER membrane (11, 18) and the demonstration of the 26 S proteasome
itself on the cytosolic surfaces of the nuclear envelope and the ER
membrane in fission yeast (19) and mammalian cells (20).
The focus of this study is the relationship between apoB and the
translational and translocational machinery under metabolic conditions
favoring either lipoprotein assembly or proteasomal degradation. From a
variety of approaches, we have obtained data to support a model in
which apoB translation in HepG2 cells is not completed until
lipoprotein assembly has concluded. If conditions are not favorable to
assemble a lipoprotein, apoB polypeptides destined for degradation form
a complex with the proteasome and the cytosolic chaperone hsp70,
leading to degradation. Overall, then, for apoB in HepG2 cells, both
lipoprotein assembly and ERAD appear to be co-translational rather than
post-translational processes.
General Cell Culture Methods and Immunological
Reagents--
HepG2 cells were maintained in minimal essential
medium (Life Technologies, Inc.) containing 10% fetal bovine serum,
200 µM L-glutamine, and 200 units/ml
penicillin/streptomycin and were studied after achieving ~80%
confluency. In all but the immunofluorescence experiments, cells were
pretreated for 60 min with 10 µM proteasomal inhibitor
(lactacystin), which was then included in all subsequent labeling and
chase media. In addition, unless otherwise noted under "Results,"
all pulse-chase experiments were performed either with oleic acid (OA;
to stimulate lipid synthesis and lipoprotein assembly) complexed to BSA
(0.8 mM final concentration of OA in a 5:1 molar ratio with
BSA) or with 0.16 mM BSA added to the chase medium.
Rabbit antiserum against the 26 S proteasome and the fluorescent
substrate for in vitro proteasome activity assay
(benzyloxycarbonyl-Gly-Gly-Phe-p-aminobenzamide) were
generous gifts from Drs. M. Orlowski and C. Cardozo (Department of
Pharmacology, Mount Sinai School of Medicine, New York). Rabbit antiserum to Sec61
In some apoB immunoprecipitation experiments, we used epitope-specific
monoclonal antibodies, Bsol 14 (specific for the N terminus) and Bsol
22 (specific for the C terminus), which were purchased from the
Lipoprotein Research Facility of the Ottawa Heart Institute (Ottawa,
Canada). Anti-puromycin antiserum was provided by one of us
(W. J. W.) and is described in Ref. 22.
Interaction between Sec61 Immunoprecipitation of ApoB by Epitope-specific
Antibodies--
Proteasome-inhibited HepG2 cells were incubated for 15 min in the presence of 35S-protein labeling mixture and
chased in isotope-free chase medium. At 0, 20, 40, and 60 min,
microsomes were prepared (see below) and lysed in denaturing lysis
buffer, and equal volume aliquots were separately immunoprecipitated
with monoclonal antibodies Bsol 14 and Bsol 22 as described (8). The
immunoprecipitates were analyzed by SDS-PAGE and fluorography as
described above. In pilot experiments, labeled apoB from conditioned
medium was similarly studied, and comparable recoveries were obtained
with both antibodies. In some experiments, the topology of apoB was probed by treating microsomes with trypsin and recovering the remaining
apoB with Bsol 14 or Bsol 22. Briefly, microsomes were resuspended in
trypsin digestion buffer (25 mM KCl, 0.25 M
sucrose, 5 mM HEPES, and 5 mM EDTA), and
trypsin was added to a final concentration of 75 µg/ml. The samples
were incubated on ice for 45 min, and the reaction was arrested by
adding 75 µg/ml soybean trypsin inhibitor and 1× protease inhibitor
mixture. The samples were then divided and processed for denaturing
immunoprecipitation with epitope-specific Bsol 14 and Bsol 22 antibodies. Under these conditions, there was quantitative cleavage of
the cytosolic domain of the transmembrane ER protein calnexin and full
protection of the ER luminal protein Puromycin Incorporation into ApoB--
Proteasome-inhibited
HepG2 cells were incubated for 5 min in the presence of
35S-protein labeling mixture and then incubated in chase
medium. Puromycin (10 µM) was added to the medium either
at the start of the chase period (0-min time point) or after 70 min. In
either case, incubation was continued for an additional 5 min. ApoB and Removal of Ribosomes from the Endoplasmic
Reticulum--
Proteasome-inhibited HepG2 cells were incubated for 15 min in the presence of 35S-protein labeling mixture and
chased for 0, 20, or 60 min. Cells were harvested in 1× PBS containing
0.4% (w/v) digitonin to semi-permeabilize the cells. Equal volume
aliquots were treated with or without 1 mM puromycin and
0.5 M potassium acetate and then incubated on ice for 15 min and at 37 °C for 15 min to remove ribosomes from the ER membrane
(23). Cell pellets were collected by centrifugation at 1500 × g for 10 min and resuspended in DSP-containing buffer, and
apoB cross-linked to Sec61 Interaction between Microsomal ApoB and the
Proteasome--
Proteasome-inhibited HepG2 cells were incubated for 15 min in the presence of 35S-protein labeling mixture and
chased for 20 or 60 min. At each time point, plates of cells were
placed on ice and sequentially washed with ice-cold PBS, buffer
containing 0.25 M sucrose and 5 mM EDTA, and SH
buffer (0.25 M sucrose and 5 mM HEPES (pH
8.0)). Cells were scraped in SH buffer containing 1× protease
inhibitor mixture and sonicated twice on ice at 50% duty cycle for 30 pulses using a Vibra CellTM sonicator (Sonics & Materials,
Danbury, CT). The cell suspension was centrifuged at 10,000 × g at 4 °C for 10 min to remove the nuclei and unbroken
cells. Microsomes were separated by ultracentrifugation at 100,000 × g for 1 h at 4 °C in a Beckman TLA 100.4 rotor.
The pellets were resuspended in 1 ml of 0.5 M sucrose and 5 mM HEPES (pH 8.0) and centrifuged again for 1 h at
100,000 × g at 4 °C.
Microsomal pellets were resuspended in nondenaturing lysis buffer
(0.5% (w/v) deoxycholate, 150 mM NaCl, and 50 mM Tris-HCl (pH 8)) overnight with rocking at 4 °C. The
suspension was clarified by centrifugation for 5 min at 1500 × g. The supernatant containing the microsomal membranes was
diluted in nondenaturing immunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl (pH 8), and 5 mM EDTA) and incubated with anti-proteasome antiserum for
3 h with rocking at 4 °C. Immunocomplexes captured by protein
A-Sepharose were eluted by boiling in buffer containing 2% (v/v)
Interactions among Microsomal ApoB, hsp70, and the
Proteasome--
Proteasome-inhibited HepG2 cells were labeled to
steady state by incubation for 2 h in the presence of
35S-protein labeling mixture. The cells were then
permeabilized (4 °C for 15 min) with buffer containing 1× PBS,
0.4% (w/v) digitonin, and 1× protease inhibitor mixture with or
without 50 units/ml apyrase (Sigma). Microsomal membranes were prepared
as described above and subjected to immunoprecipitation with the
anti-hsp70 monoclonal antibody. One-half of the immunoprecipitate was
used for Western blotting (Renaissance kit, PerkinElmer Life Sciences) with anti-proteasome antiserum (to detect hsp70-proteasome
interactions), and the other half was used for a second
immunoprecipitation with anti-apoB antiserum (to detect hsp70-apoB
interactions). The immunoprecipitate was then analyzed by SDS-PAGE and
fluorography as described above.
Immunofluorescence Studies--
HepG2 cells grown on
collagen-coated coverslips were preincubated for 2 h with 10 µM lactacystin in minimal essential medium containing 1%
fetal bovine serum. Cells were then washed, and the medium was replaced
with one containing either 0.16 mM BSA or 0.8 mM OA (as a complex with BSA) to stimulate lipid synthesis. The cells were fixed 0, 30, or 90 min after OA addition using 3%
paraformaldehyde in 1× PBS. Fixed cells were permeabilized with 0.1%
Triton X-100 and incubated with anti-apoB antibody (primary antibody)
and then with Texas Red-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) as the secondary antibody as described (1). In control experiments, the same protocol was performed
using anti-human apoA-I antiserum as the source of primary antibody.
Kinetics of the Close Association of ApoB with the Translocon
Protein Sec61
The interaction of translocating chains with Sec61
HepG2 cells pretreated with lactacystin were incubated in the presence
of [35S]methionine/cysteine for 15 min and chased for up
to 60 min in isotope-free medium containing excess methionine/cysteine
(chase medium). Cells were harvested at 0-, 20-, or 60-min time points, and apoB adjacent to Sec61
To exclude the possibility that OA was affecting apoB-translocon
interactions independent of its promotion of lipoprotein assembly, such
as by disrupting the ER membrane, the above experiment was repeated in
the presence of 0.1 µM BMS-200150, an inhibitor of MTP
lipid transfer activity (kindly provided by Drs. David Gordon and John
Wetterau, Bristol-Myers Squibb Co.) (27). If the effect of OA were
nonspecific, blocking MTP activity and lipoprotein assembly would not
alter the interaction of apoB with the translocon. As shown in Fig. 1
(lane 6), however, the decrease in apoB-Sec61 Differential Recovery of ApoB by Epitope-specific Monoclonal
Antibodies--
The above and previous (1) results demonstrate that
apoB remains adjacent to the translocon during lipoprotein assembly. We
wondered whether the apoB bands in Fig. 1 included some incompletely translated apoB that was indistinguishable upon SDS-PAGE from bona fide full-length apoB100. If translation were
incomplete, we would expect that in a pulse-chase study, the appearance
of an epitope at the extreme C terminus should be abnormally delayed relative to one at the N terminus. Therefore, we compared the relative
recoveries of the N- and C-terminal epitopes from microsomes isolated
at different time points during the 60-min chase. As in the experiments
above, proteasome-inhibited HepG2 cells were pulse-labeled; and after
0, 20, 40, and 60 min of chase, cells were harvested, and microsomes
were collected by ultracentrifugation (see "Experimental
Procedures"). Equivalent aliquots of the resuspended microsomal
pellets were immunoprecipitated with Bsol 14 or Bsol 22 monoclonal
antibodies, which recognize the N-terminal (amino acids 405-539) and
C-terminal (amino acids 4521-4536) regions of apoB, respectively. The
signals on the resulting fluorograms were quantified by densitometry,
and the abundance of labeled apoB recovered by the anti-C terminus
antibody was divided by the abundance recovered by the anti-N terminus
antibody. This fraction was multiplied by 100 to derive the parameter
we have termed the "C/N ratio."
The results are summarized in Fig.
2A. The C/N ratio was 100 for
apoB in the conditioned medium (i.e. medium collected after 2 h of continuous labeling in the presence of OA), demonstrating equal recovery efficiencies of the antibodies when both epitopes were
present on fully translated apoB. At the end of the 15-min labeling
period (i.e. 0 min of chase), the C/N ratio was only 50, indicating that only 50% of the microsome-associated apoB molecules
contained the C-terminal epitope. At 20 min of chase, there was still a
relative deficiency of the C-terminal epitope. Based on the average
elongation rate of hepatic proteins, apoB translation would be expected
to be finished by ~11 min. Therefore, the data indicate a greater
than expected lag in the appearance of the C-terminal epitope,
reflecting a delay in completing apoB translation. In the presence of
OA, the C/N ratios at the 20- and 40-min time points were similar to
those in Fig. 2A, but were lower at 60 min, most likely
representing the exit from the ER of full-length apoB molecules
assembled into lipoproteins (data not shown).
As shown in Fig. 2A, some apoB polypeptides were gradually
elongated to include the C-terminal epitope, as evidenced by the C/N
ratio approaching 100 at the 60-min time point. We were interested in
the topology of this extreme C-terminal region, so protease protection
studies using these epitope-specific antibodies were performed (see
"Experimental Procedures"). Representative results are shown in
Fig. 2B. Note that at the 60-min time point, relative to the
N-terminal epitopes, the C-terminal epitopes were more sensitive to
trypsin digestion under conditions of limited lipid synthesis
(i.e. without OA). When OA was added to stimulate
lipid synthesis and lipoprotein assembly, the relative protection of the C-terminal epitope was significantly increased.
In control experiments, if microsomes were pretreated with Triton X-100
to disrupt membranes, the concentration of trypsin used resulted in
100% disappearance of the signal for apoB. In addition, this
concentration was not damaging to microsomal integrity, as it degraded
only the cytosolic tail of calnexin, an integral ER membrane protein
(data not shown). Thus, the results shown in Fig. 2B most
likely reflect the topological relationship between apoB and the ER
membrane and imply that when lipid synthesis is inadequate, there is
incomplete translocation and cytosolic exposure of the extreme
C-terminal domain of apoB. In contrast, when lipid synthesis is
stimulated, this domain is shielded from the cytosol, most likely
because it was fully translocated.
ApoB Remains Functionally Bound to the Ribosome throughout the Time
Required for Lipoprotein Assembly--
The prolonged proximity of apoB
polypeptides to the translocon and the kinetics of the differential
recoveries of the C- and N-terminal epitopes suggested that the nascent
apoB polypeptides were still functionally bound to the ribosome as
peptidyl-tRNAs. This possibility was tested by assessing whether
puromycin could be incorporated into labeled apoB chains during the
chase period, particularly at the later time points. For comparison
purposes, puromycin incorporation into another secretory protein,
HepG2 cells pretreated with lactacystin were incubated for 5 min in the
presence of [35S]methionine/cysteine and then incubated
for up to 70 min in chase medium containing either OA/BSA or BSA
alone. Puromycin (10 µM) was added to the medium after 70 min of chase, and incubation was continued for another 5 min. Labeled
apoB or
As shown in Fig. 3 (lanes 6 and 7), in the presence or absence of OA, there were no
If OA was present in the chase medium, puromycin incorporation into
apoB was significantly lower (Fig. 3, lane 4 versus
lane 3), consistent with the loss of association of apoB
with the translocon at the time when lipoprotein assembly should be
completed, resulting in exit from the ER (Fig. 1) (1, 24). In contrast
to these results, if puromycin was added at the end of the labeling
period, both labeled nascent apoB and
These results imply that apoB translation either slows or pauses very
close to its carboxyl terminus. Overall, the data in Figs. 1-3 suggest
that when lipid synthesis is stimulated, apoB translation is completed,
and assembled lipoproteins are transported out of the ER.
Association of ApoB with the Translocon Is Independent of the
Presence of the Ribosome--
Since nascent apoB is bound to the
ribosome via the tRNA, and the ribosome is bound to the translocon, the
prolonged proximity of apoB to the translocon (Fig. 1) may not be due
to direct apoB-translocon interactions. To determine whether the
release of the ribosome from the ER membrane would result in the loss
of apoB-Sec61 Interactions of Microsome-associated ApoB with the Proteasome and
the Cytosolic Chaperone hsp70--
The results above and in our
previous reports (1, 8, 16) imply that apoB can be directed to either
lipoprotein assembly or degradation while still associated with the
translocon and the ribosome. Thus, rather than interacting with
cytosolic factors, such as hsp70 and proteasomes, only after being
completely retrotranslocated, apoB may be engaged by these factors
while still bound to the ER membrane in a "bitopic" state
(i.e. with part of apoB accessible to the cytosol and part
in the ER lumen). To test this possibility, HepG2 cells were labeled to
steady state for 2 h in the presence of 10 µM
lactacystin and then semi-permeabilized with digitonin. To test for the
ATP dependence of interactions among apoB, hsp70, and the proteasome,
some samples were treated with 50 units/ml apyrase to deplete ATP.
Microsomes were collected by ultracentrifugation from both
apyrase-treated and buffer-treated lysates, and the pellets were
resuspended in nondenaturing lysis buffer. hsp70-associated proteins
were isolated by immunoprecipitation with anti-hsp70 monoclonal
antibody, and one-half of this immunoprecipitate was resolved by
SDS-PAGE and immunoblotted with anti-proteasome antiserum (see
"Experimental Procedures").
As shown in Fig. 5, 26 S proteasome
subunits ranging from 25 to 35 kDa were detected. This interaction with
hsp70 was specific as shown by its ATP dependence (Fig. 5, lane 3 versus lane 4). The other half of the hsp70
immunoprecipitate was diluted, and a second immunoprecipitation was
performed with anti-apoB antiserum. Labeled apoB was recovered
(lanes 1 and 2), and the amount recovered increased after apyrase treatment (lane 1 versus lane
2), indicating an ATP dependence consistent with previous results
for hsp70-apoB interactions (10).
To investigate the kinetics of the association of microsomal apoB with
the proteasome, HepG2 cells were pretreated with lactacystin; pulse-labeled for 15 min; and then chased for 0, 20, or 60 min as
before. Microsomes were isolated at these time points and subjected to
sequential immunoprecipitation under nondenaturing conditions (see
"Experimental Procedures") using anti-proteasome antiserum in the
first step and anti-apoB antiserum in the second step. As shown in Fig.
6, proteasomes and labeled microsomal
apoB co-immunoprecipitated at all time points, and the extent of this
interaction was lower in the presence of OA. These results are
consistent with the decreased degradation of apoB (29) and the
decreased cytosolic exposure of the C-terminal domain when HepG2 cells
are incubated with OA (Fig. 2B).
The interaction between apoB and the proteasome was specific as judged
by the absence of the apoB band if nonimmune serum was used in the
first immunoprecipitation step (Fig. 6, lane 1). We also
tested whether proteasomes co-immunoprecipitating with microsomal apoB
were functional by performing assays for the chymotrypsin-like activity
of the proteasome. By incubating the co-immunoprecipitated proteasomes
with a synthetic substrate
(benzyloxycarbonyl-Gly-Gly-Phe-p-aminobenzamide) (30),
lactacystin-inhibitable activity was readily detected, suggesting that
the microsomal apoB-associated proteasomes were indeed functional (data
not shown).
Overall, these results imply that the proteasome and hsp70, both of
which we have previously shown to be involved in ERAD of apoB (8),
interact with apoB while it is still associated with the ER membrane.
In other words, complete retrotranslocation of apoB is not required for
its interaction with cytosolic factors and its entry into the
ubiquitin-proteasome pathway.
ApoB Accumulates in Non-aggresomal Structures when Proteasome
Activity Is Inhibited--
We have previously shown by indirect
immunofluorescence in HepG2 cells that when proteasomes are inhibited,
apoB accumulates and assumes a punctate appearance that
co-localizes with ER markers (1, 8). Biochemical studies suggested that
apoB accumulating under these conditions can still be recruited to
lipoprotein assembly and the secretory pathway when OA is added to
stimulate lipid synthesis (1). This implies that the intracellular
localization of proteasome-protected apoB, as detected by
immunofluorescence, will be changed by OA.
To test this, HepG2 cells were treated with lactacystin for 2 h,
and then the cells were chased in either BSA- or
OA/BSA-containing medium for 30 or 90 min. The cells were
immunostained using either anti-apoB or anti-apoA-I antibody (control
protein) as the primary antibody. As shown in Fig.
7 (left panel), a punctate
appearance of apoB, similar to that shown previously (1, 8), was
apparent at the start of the chase period. Note that in the presence of BSA, there were no obvious changes in the level or distribution of the
immunofluorescent signals over time (upper center and
right panels). In marked contrast, with OA, the signal
became diffuse by 30 min and was significantly lower in intensity by 90 min, consistent with the mobilization and secretion of the accumulated apoB after lipid synthesis was stimulated. Supporting this
interpretation is that the apoB signal at 30 min overlapped with a
marker (ERGIC53) (31) for the intermediate compartment, which is
enriched in secretory vesicles (data not shown). These results were not
due to nonspecific effects of OA, based on the lack of changes in the
intensity or distribution of the signal for apoA-I (data not shown).
Taken together with the published biochemical results (1, 8), these
data demonstrate that apoB accumulating in punctate structures after
proteasomal inhibition can be recruited to lipoprotein assembly and
secretion. As will be described further under "Discussion," the
apoB-associated punctate structures are unlikely to represent aggresomes, which are also punctate in immunofluorescent appearance. Although they also consist of an ERAD substrate (the cystic fibrosis transmembrane conductance regulator) that accumulates when the proteasome is inhibited, they contain protein molecules irreversibly lost from the secretory pathway (14, 15).
As polypeptides emerge from the translocon, they encounter ER
chaperones, such as BiP and calnexin, and undergo a "quality control" process in which conformation-dependent sorting
results in either a native folded state that exits the ER or a
malfolded state that is retained and subject to ERAD (see Ref. 32 for a
recent review). We have previously shown that when HepG2 cells are
deprived of fatty acids, the association of apoB with its lipid ligands
is reduced, and ERAD mediated by the ubiquitin-proteasome pathway
ensues (1, 8). Complete assembly of apoB with lipids to form a hepatic
lipoprotein particle culminating in ER exit has been estimated to take
~40 min based on the kinetic analysis of subcellular fractionation
data (24). It has also been shown that the initiation of lipid
association with nascent apoB polypeptides in HepG2 cells is
co-translational (33), consistent with the interaction of metabolically
labeled apoB with either Sec61 The data in this report argue strongly that not only the initial, but
the bulk, if not all, of the assembly process in HepG2 cells occurs
co-translationally. A key line of evidence to support this view derives
from the finding that apoB is adjacent to translocon proteins
throughout the time period required for lipoprotein assembly. Furthermore, the disappearance of apoB-translocon interactions appears
to coincide with the expected exit of the completed lipoprotein particle from the ER. A simple explanation for the persistent interaction between apoB and the translocon is that apoB does not leave
the translocon until translation has terminated. Yet, in many studies
of HepG2 cells, including our own, labeled apoB appears to be
full-length (i.e. apoB100, ~550 kDa) at early time points
in the chase period. SDS-PAGE, however, does not have the resolution to
reveal minor differences in size among apoB polypeptides that would
occur if translation slowed or paused near the carboxyl terminus of the
protein. Thus, we determined directly whether apoB polypeptides were
still bound to functional ribosomes as peptidyl-tRNAs late in the chase
period by their ability to react with puromycin, an aminoacyl-tRNA
analog, and by the kinetics of C-terminal synthesis.
As shown in Figs. 2 and 3, the results independently support the notion
that the assembly of a lipoprotein particle and the termination of apoB
translation are coupled events. In proteasome-inhibited cells,
puromycin could be incorporated into 35S-labeled apoB
polypeptides as long as 70 min following pulse labeling, indicating the
functional engagement of nascent apoB chains with the ribosome well
into the chase period. In the presence of OA, the decrease in
incorporation of puromycin added after 70 min of chase (Fig. 3), a time
sufficient for the complete assembly of a lipoprotein, most likely
reflects the completion of translation and exit from the ER. Because OA
is not totally effective in sorting apoB to lipoprotein assembly (29),
the residual apoB at 70 min in +OA samples most likely represents
nascent apoB that is not associated with a full complement of lipid ligands.
Another indication of a delay in completing apoB translation is the
significant lag in the appearance of the C-terminal epitope of apoB
relative to that of the N-terminal epitope (Fig. 2). This lag can only
be explained by a slower rate of, or pause in, the translation of the
region of apoB mRNA encoding the extreme carboxyl-terminal portion
of the full-length protein. Interestingly, the location of the
C-terminal epitope is just distal to one of the strongest pause-transfer sequences detected by Lingappa and co-workers (36) in
their survey of the entire length of apoB. In a cell-free assay system,
they have shown that sequences dispersed throughout the length of apoB
can mediate pauses in translocation (37, 38). In addition,
we (39) and others (40) have observed pauses in translation
of apoB. Both types of pauses have been hypothesized to allow time for
the assembly of nascent apoB chains with lipids (3). Making an analogy
to the coordinated efforts of the ribosome and translocon during the
insertion of a transmembrane domain into the ER membrane bilayer (41),
the results (puromycin incorporation, delayed C-terminal epitope
appearance, and prolonged interaction with Sec61p) imply that the
translational and translocational apparatuses cooperate to stabilize a
topographical state of apoB necessary to achieve the final stage of
lipoprotein assembly in HepG2 cells. Relevant to this point is a
proposed "stop-translation" sequence (in the bacterial
The prolonged binding of the nascent apoB chain to the ribosome via the
tRNA and the affinity of the ribosome for the translocon lead, not
surprisingly, to the positioning of the apoB within or near the
translocon late in the assembly process. Yet, when ribosomes were
stripped off the ER membrane, apoB was still adjacent to Sec61 Our results also speak to the fate of nascent apoB chains not
successfully assembled into a lipoprotein particle. In contrast to the
paradigm for other substrates of ERAD, such as major histocompatibility complex class I molecules in cytomegalovirus-infected cells and mutant
carboxypeptidase Y in yeast (see Ref. 49 for a recent review), we have
previously shown in proteasome-inhibited HepG2 cells maintained in
OA-deficient medium that apoB does not appear to be completely
retrotranslocated or "dislocated" to the cytosol prior to
degradation (1). The proteasome and cytosolic factors involved
in apoB degradation therefore appear to interact with ER-associated
apoB polypeptides, as supported by the finding of ATP-dependent interactions among microsome-bound apoB,
proteasomes, and cytosolic hsp70 (Figs. 5 and 6). It should be noted
that the pretreatment of fatty acid-deficient HepG2 cells with
lactacystin allowed the study of apoB intermediates of greater length
than would have been ordinarily present in this metabolic state, based on data showing that targeting of apoB polypeptides to the
ubiquitin-proteasome pathway can occur as early as midway through
translation (16, 50). Nonetheless, many features of the proteasomal
targeting of apoB appear to be independent of polypeptide length,
namely, stimulation by a deficiency in lipid synthesis or transfer, the increased exposure of the C-terminal region relative to the N terminus
when lipoprotein assembly is not successful, and an increased interaction with hsp70 (16, 51). We have previously shown that
increased hsp70 expression promotes apoB ubiquitinylation (16) and
proteasomal degradation (8), and we presume that the hsp70 bound to
microsomal apoB (Fig. 5) is fulfilling functional roles, consistent
with the decrease in apoB-proteasome interaction when OA is present
(Fig. 6).
The lack of cytosolic accumulation of apoB in proteasome-inhibited
cells (1) indicates that, unlike other ERAD substrates, such as the
cystic fibrosis transmembrane conductance regulator (14, 15) and
influenza virus antigen (52), apoB does not form aggresomes,
cytosolic structures containing proteins that are irreversibly lost to
the secretory pathway. Consistent with this was the finding that the
stimulation of lipid synthesis and lipoprotein assembly by OA resulted
in the disappearance of the apoB-containing punctate immunofluorescent
structures that formed after proteasome inhibition (Fig. 7). That this
disappearance represented the rapid recruitment of the accumulated apoB
to lipoprotein assembly and secretion is supported by our two previous
studies (1, 8) in which there was quantitative recovery of
metabolically labeled apoB from the conditioned medium of HepG2 cells
treated similarly to the cells in Fig. 7. Because these apoB-containing punctate structures co-localized with ER markers in previous
immunofluorescence studies (1, 8), it is plausible that they are part
of the recently described "ER-associated bodies" formed at exit
sites in which another proteasomal ERAD substrate, a mutant form of a
yeast ABC transporter Ste6p, has been shown to accumulate (53). It is
therefore intriguing to speculate that the diffuse apoB immunofluorescent signal observed 30 min after OA addition represents a
plethora of secretory vesicles containing apoB-lipoprotein
complexes that budded off the ER after lipid synthesis and
assembly were stimulated.
In summary, we have used proteasome-inhibited HepG2 cells to study the
molecular events in the two possible paths of apoB metabolism:
lipoprotein assembly or ERAD. Both paths appear to be co-translational.
The assembly process, which occurs when there is sufficient transfer of
lipid ligands, functions while apoB polypeptides are incompletely
translated and are interacting with the ribosome, translocon, ER
chaperones, and, perhaps, the lipid bilayer. Lipoprotein assembly, apoB
translocation, and apoB mRNA translation appear to be coordinated
events that serve to maintain apoB in an appropriate functional and
topographical state. If lipid ligands are insufficient, then
ER-associated apoB polypeptides interact with cytosolic factors that
target it to the ubiquitin-proteasome pathway. Given the potential of
the proteasome (54) or chaperones (55, 56) to act as molecular motors
by virtue of their intrinsic ATPase activities, apoB is most likely
extracted from the ER membrane rather than becoming fully
retrotranslocated into the cytosol prior to its degradation.
A schematic summarizing the essential features of the proposed model is
given in Fig. 8. Basically, lipoprotein
assembly and apoB degradation resemble the quality control mechanisms
governing the molecular sorting of other secretory and transmembrane
proteins in the ER, in that exit and retention (and ultimate
degradation) represent the successful separation of conformational
variants. The differences between apoB and other ERAD substrates most
likely reflect special requirements of lipoprotein assembly, unusual structural features of the apoB protein, and the regulation of the
quality control process by the lipid metabolic state.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was prepared by Research Genetics (Huntsville, AL) and used as described previously (21). Goat anti-human apoB antiserum and monoclonal antibody for immunoprecipitation and immunofluorescence, respectively, and antisera to human apoA-I and
calnexin were purchased from Calbiochem. Monoclonal antibody to hsp70
was purchased from Stressgen Biotech Corp. (Victoria, Canada).
Antiserum to human
2-macroglobulin (
2M)
was purchased from BIODESIGN International (Saco, ME).
and ApoB--
Proteasome-inhibited
HepG2 cells were incubated for 15 min in the presence of 100 µCi/ml
Express 35S-protein labeling mixture (labeled
methionine and cysteine; PerkinElmer Life Sciences). Isotope-containing
medium was then removed, and the cells were incubated in chase medium
(minimal essential medium containing 5 mM methionine and 2 mM cysteine) for 20 or 60 min. At the end of the chase
period, cells were harvested in cross-linking buffer (1× PBS, 5 mM EDTA, and 0.4% (w/v) digitonin). The homobifunctional cross-linking agent DSP (Pierce) was added to a final concentration of
0.25 mM, and the cells were incubated on ice for 40 min.
The cross-linking reaction was quenched by adding ice-cold Tris (pH 8)
to a final concentration of 50 mM and incubating on ice for 15 min. Cells were then lysed in denaturing lysis buffer (150 mM NaCl, 50 mM Tris (pH 8), 5 mM
EDTA, 0.5% (w/v) deoxycholate, 1% (v/v) Triton X-100, and 0.1% (w/v)
SDS containing 1× protease inhibitor mixture (Roche Molecular
Biochemicals)) with shaking overnight at 4 °C. The cell lysates were
clarified by centrifugation at 1500 × g for 3 min, and
the supernatants containing equivalent trichloroacetic
acid-precipitable counts were then used for sequential immunoprecipitations. The sample was first exposed to antiserum against
Sec61
as described (21), and the resulting immunocomplexes were
captured by protein A-Sepharose and released from the beads by boiling
in elution buffer (10% (v/v)
-mercaptoethanol, 4% (w/v) SDS, and
20% (v/v) glycerol). The eluate was diluted in 1× NET, and the
second immunoprecipitation was performed with anti-apoB antiserum as
described previously (8). The eluate from the second
immunoprecipitation was analyzed by 3-17% gradient SDS-PAGE and
fluorography. The signals were quantified using a densitometer (Bio-Rad
GS 700) or a PhosphorImager (Molecular Dynamics, Inc.).
2M (data not shown).
2M (control protein) polypeptides that had incorporated
puromycin were isolated by sequential immunoprecipitation using
anti-apoB or anti-
2M antibodies, respectively, in the
first step, followed by anti-puromycin antiserum (22) in the second
step. The proteins in the final immunoprecipitate were resolved by
SDS-PAGE and detected by fluorography as described above. To verify the
specificity of anti-puromycin antiserum, 100 µM puromycin
was added to an aliquot of a control sample before incubation with
anti-puromycin antiserum (see Fig. 3).
was isolated by sequential immunoprecipitation.
-mercaptoethanol. To isolate proteasome-associated apoB, the eluates
were diluted, and a second immunoprecipitation was performed using
antiserum to apoB. The final immunoprecipitate was analyzed by SDS-PAGE and fluorography as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
We have previously reported that pulse-labeled
apoB that is protected from degradation by proteasomal inhibitors stays
associated with the ER translocon protein Sec61
for at least 1 h after the start of the chase period (1). This prolonged association
with Sec61
was disrupted after 40 min when triglyceride synthesis, and therefore lipoprotein assembly, was stimulated by including OA in
the chase medium. Based on the time estimated for hepatic lipoprotein
assembly and ER exit (~37 min) (24), this result suggested that
throughout the biogenesis of an apoB-lipoprotein complex in the
HepG2 cell, apoB remained in close proximity to the translocon.
and Sec61
can
vary depending on the length of the polypeptide or its transmembrane
topology (e.g. Ref. 25). Thus, to test whether the previous
results for Sec61
represented a special relationship of that protein
with apoB or a more general association of apoB with the translocon,
the pattern of interaction of pulse-labeled apoB with Sec61
was determined.
was detected by DSP-based cross-linking and sequential immunoprecipitation (see "Experimental Procedures"). As shown in Fig. 1, similar to the
published results for Sec61
(1), a substantial amount of
Sec61
-associated apoB (~23% of the total labeled apoB pool) (Fig.
1B) could be detected as late as the 60-min time point when
OA was omitted from the chase medium. In the presence of OA, the
fraction of apoB adjacent to Sec61
was significantly reduced at 60 min (Fig. 1A, lane 5 versus lane 4). Note that the efficiency of chemical cross-linking of proteins with disuccinimide-based reagents typically averages no more than 10%
(26), strongly suggesting that the relatively robust results for the
apoB-Sec61
interactions summarized in Fig. 1 are
representative of the behavior of the bulk of apoB polypeptides studied
under these conditions.
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Fig. 1.
Association of newly synthesized apoB with
Sec61 depends on lipid synthesis and
transfer. A, proteasome-inhibited HepG2 cells were
labeled with [35S]methionine/cysteine for 15 min and
chased with excess unlabeled methionine/cysteine for 20 or 60 min in
the presence or absence of 0.8 mM OA (OA stimulates lipid
synthesis) and the MTP inhibitor BMS-200150 (which decreases lipid
transfer). At each time point, cells were harvested and subjected to
cross-linking and sequential immunoprecipitation (first step,
anti-Sec61
antiserum; second step, anti-apoB antiserum) to detect
labeled apoB associated with Sec61
(see "Experimental
Procedures"). After SDS-PAGE resolution of the second
immunoprecipitate, fluorography was performed to visualize the apoB
that was covalently bound to Sec61
. Note the significant decrease in
the apoB signal at 60 min when OA was present along with
BMS-200150; however, the decrease in the signal for apoB bound
to Sec61
was not observed (lane 6 versus lane
5). The results shown are representative of three separate
experiments. The increase in apoB signal at 20 min in this and other
figures is typical (24) and represents continued incorporation of
[35S]methionine/cysteine into apoB polypeptides in the
early chase period. B, the fluorograms from the three
separate experiments were quantified by densitometry, and the signals
for apoB bound to Sec61
were normalized to the recovery of total
labeled apoB at the corresponding time points. The results are shown as
the mean ± S.E.
cross-linking at 60 min could be reversed by adding BMS-200150 to the
medium in the presence of OA. Thus, the decreased proximity of apoB to
Sec61
in the presence of OA most likely reflects successful lipoprotein assembly and exit from the ER, and not a nonspecific effect
of the fatty acid on apoB-Sec61
association.
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Fig. 2.
Differential recovery and protease
sensitivity of N- and C-terminal epitopes of apoB after pulse
labeling. A, proteasome-inhibited HepG2 cells were
labeled for 15 min with [35S]methionine/cysteine and
chased for 20, 40, or 60 min. Microsomes were isolated at each time
point, and equal volume aliquots were immunoprecipitated with either
Bsol 14 (N-terminal antibody) or Bsol 22 (C-terminal antibody).
Immunoprecipitates were resolved by SDS-PAGE; apoB signals were
quantified by densitometry; and the ratio of the recovery attained with
Bsol 22 to that attained with Bsol 14 (referred to as the C/N ratio)
was calculated for each time point. The y axis is
the (mean C/N ratio × 100%) ± S.E. (n = 4). Note the relative deficiency of C-terminal epitopes at the 20- and
40-min time points and the equal recoveries of N- and C-terminal
epitopes from the conditioned medium. B, the topology of the
N- and C-terminal epitopes of microsomal apoB was analyzed by protease
sensitivity studies using epitope-specific N- and C-terminal
antibodies. Equivalent volume aliquots of microsomes isolated from
proteasome-inhibited HepG2 cells that had been pulse-labeled and chased
for 60 min were incubated on ice for 45 min with or without 75 µg/ml
trypsin. Microsomes were then lysed, immunoprecipitated (IP)
with Bsol 14 (N-terminal specific antibody (Ab)) or Bsol 22 (C-terminal specific antibody), and analyzed on 3-17%
SDS-polyacrylamide gel (left panel). ApoB signals were
quantified, and the percentage protection was calculated for each
epitope in the absence of OA (lipid deficiency) and in the presence of
OA (lipid sufficiency). The protection of the C-terminal epitope is
expressed as the mean fraction ± S.E. (three separate
experiments) relative to the average percentage protection of the
N-terminal epitope in the absence and presence of OA (right
panel). Note that the C-terminal epitope was highly susceptible to
trypsin digestion in the absence of OA, whereas the majority of the
C-terminal epitopes were resistant to trypsin digestion in the presence
of OA.
2M, was also assessed.
2M chains that had incorporated puromycin were
recovered by sequential immunoprecipitation with either anti-apoB or
anti-
2M antibody in the first step and anti-puromycin
antibody in the second step.
2M-puromycin conjugates detected. Surprisingly, there
were apoB-puromycin conjugates (lanes 3 and 4),
with the labeled apoB species appearing full-length (based on the
migration of apoB recovered from the conditioned medium). The percent
of apoB conjugated to puromycin was ~20% of total labeled apoB,
consistent with the 23% recovery of apoB cross-linked to Sec61
late
in the chase period when OA was absent (Fig. 1). The comparable
quantitative recoveries under essentially the same experimental
conditions strongly imply that the apoB polypeptides conjugated to
puromycin or cross-linked to Sec61
belong to the same pool of
molecules.
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Fig. 3.
ApoB remains functionally bound to the
ribosome throughout the time required for lipoprotein assembly.
Proteasome-inhibited HepG2 cells were labeled with
[35S]methionine/cysteine for 5 min and chased for either
0 or 70 min in the presence or absence of OA. Puromycin was then
added, and incubation was continued for 5 min. Cells were lysed, and
labeled apoB and 2M polypeptides that incorporated
puromycin were isolated by immunoprecipitation with anti-apoB or
anti-
2M antiserum, respectively, in the first step and
anti-puromycin antiserum in the second step. Immunoprecipitates were
resolved by SDS-PAGE, and labeled polypeptides were visualized by
fluorography. A representative result is shown. Note that when
puromycin was added at 0 min, labeled apoB and
2M chains
of various lengths that had incorporated puromycin were visible
(lanes 2 and 5, respectively). In contrast, when
puromycin was added after 70 min of chase, only apoB
polypeptides were visible (lanes 3 and 4 versus lanes 6 and 7). Note that the
2M lanes were deliberately overexposed to ensure that
there was no residual signal detectable at the 70-min time point. In
the presence of OA, apoB-puromycin conjugates were decreased
(lane 3 versus lane 4). To control for the
specificity of the anti-puromycin antiserum, excess puromycin was added
to some samples before the second immunoprecipitation to compete
against labeled apoB-puromycin conjugates; note the absence of signal
(lane 1).
2M chains
incorporated puromycin, as expected, and a range of incomplete
translation products from 45 to 550 kDa for apoB and from 70 to 180 kDa
for
2M was observed (Fig. 3, lanes 2 and
5). That the sequential immunoprecipitation was specific for
apoB-puromycin conjugates was indicated by a large reduction in the
recovery of apoB when excess puromycin was added to the reaction
mixture just before adding the anti-puromycin antibody (lane
1).
cross-linking, HepG2 cells were pretreated, labeled,
and chased for the Sec61
cross-linking experiment as described
above. At the indicated time points (Fig.
4), cells were scraped into buffer, and
the suspension was divided into two aliquots. One was treated with 1 mM puromycin in the presence of 0.4 M potassium
acetate prior to cross-linking (to disrupt the ribosome-translocon
interaction) (23), and the other processed was similarly, but without
adding puromycin (see "Experimental Procedures"). Both samples were
then treated with the DSP cross-linker, lysed, and subjected to
sequential immunoprecipitation with anti-Sec61
and anti-apoB
antisera (see "Experimental Procedures"). As shown in Fig. 4, the
puromycin/high salt treatment, which releases ribosomes from
translocons (28), did not affect the extent of cross-linking of apoB to
Sec61
in samples taken at the different chase points. This suggests
that along with apoB engagement by the ribosome (Fig. 3), there are also interactions with ER proteins or lipids that serve to retain apoB
in the translocon during lipoprotein assembly.
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Fig. 4.
Effect of the removal of ribosomes on the
proximity of apoB to Sec61 .
Proteasome-inhibited HepG2 cells were labeled with
[35S]methionine/cysteine for 15 min and chased for 0, 20, or 60 min. Cells were harvested in 1× cross-linking buffer. One-half
of the cells were treated with 1 mM puromycin
(Puro) and 0.5 M potassium acetate and then
incubated (15 min on ice, followed by 15 min at 37 °C) to release
the ribosomes from the nascent polypeptide chains. The other half were
treated similarly, but without puromycin. After cross-linking with DSP
(see "Experimental Procedures"), cell lysates were sequentially
immunoprecipitated with anti-Sec61
antiserum in the first step and
anti-apoB antiserum in the second step. Immunoprecipitates were
resolved by SDS-PAGE, and a representative fluorogram is shown.
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Fig. 5.
Interactions among microsomal apoB,
proteasomes, and hsp70 in HepG2 cells labeled to steady state.
Proteasome-inhibited HepG2 cells were labeled to steady state by
incubation with [35S]methionine/cysteine for 2 h.
The cells were treated with permeabilization buffer with or without 50 units/ml apyrase before microsomes were prepared as described under
"Experimental Procedures" and subjected to nondenaturing
immunoprecipitation (IP) with anti-hsp70 antibody. The
immunoprecipitate was divided in two aliquots; one was processed for a
second immunoprecipitation with anti-apoB antiserum, and the other was
used for immunoblotting (Western blotting (WB)) with
anti-proteasome antiserum. Both apoB (lanes 1 and
2) and 26 S proteasome subunits ranging from 25 to 35 kDa
(lanes 3 and 4) were recovered from the hsp70
immunoprecipitate. Note that these signals increased in apyrase-treated
samples, indicating that the interactions with hsp70 were
ATP-dependent.
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Fig. 6.
Kinetics of the interaction between
microsomal apoB and the proteasome. Microsomes were isolated from
proteasome-inhibited HepG2 cells that had been labeled with
[35S]methionine/cysteine for 15 min and chased for 0, 20, or 60 min in the presence or absence of OA. Proteasome-associated apoB
was recovered by sequential immunoprecipitation using either
anti-proteasome antiserum (lanes 2-6) or preimmune serum
(lane 1; negative control) in the first step and anti-apoB
antiserum in the second step. Immunoprecipitates were resolved by
SDS-PAGE, and the signals corresponding to labeled apoB were displayed
by fluorography. Note that proteasome-associated apoB was detected at
all time points, but that the level was significantly reduced at 60 min
in the presence of OA.
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Fig. 7.
After the proteasome is inhibited, apoB
accumulates in punctate structures that are reversed when lipid
synthesis is stimulated. HepG2 cells were treated with 10 µM lactacystin for 2 h to promote apoB accumulation.
The medium was replaced with medium containing lactacystin and either
0.8 mM OA or 0.2% (w/v) BSA. Cells were fixed 0, 30, or 90 min later and immunostained with anti-apoB monoclonal antibody,
followed by Texas Red-conjugated anti-mouse IgG. Note that the apoB
immunofluorescent signal in the BSA-treated cells remained similar in
intensity and distribution throughout the time course. In marked
contrast, in the presence of OA, the punctate appearance of the apoB
fluorescent signal changed by 30 min to a diffuse pattern; and by 90 min, only a residual Golgi region signal was present, consistent with
the mobilization of the majority of accumulated apoB to lipoprotein
assembly and secretion from the cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(1) or Sec61
(Fig. 1A)
(17). In the common models proposed for hepatic lipoprotein formation
(e.g. Refs. 3, 34, and 35), however, it has been proposed
that the initial lipidation is co-translational, but subsequent
lipidation (referred to as a "second step") occurs post-translationally to complete the assembly process.
-receptor), which is thought to halt translation to allow sufficient
time for important interactions between the nascent polypeptide and
membrane lipids and proteins that are required for secretion (42). The
fact that a considerable length of apoB polypeptide is translated prior
to a final assembly stage is consistent with recent studies showing
that glycosylation sites in apoB as distal as apoB68 are modified in
HepG2 cells (43) in the absence of OA.
(Fig.
4). Thus, the cross-linking of apoB to Sec61
results not only from
their spatial proximity in the ribosome-translocon complex, but also
from protein-protein interactions. These interactions with the nascent
chain may be mediated by translocon components or by
translocon-associated proteins, as suggested by studies on
-sheet
and pause-transfer sequences contained in apoB (44, 45). Also
consistent with the data, apoB may remain in association with the ER
membrane by interacting with ER proteins in the vicinity of the
translocon (as suggested by studies of apoB and ER chaperones) (46, 47)
or with lipid components (because of the many hydrophobic domains of
apoB). This last possibility is compatible with the inability of urea
to effectively extract apoB from
microsomes.2 Delayed
translation (and hence termination of translation) then may not reflect
the need of the ribosome to be a structural anchor of apoB
polypeptides, but may provide the length of polypeptide and time
required to position nascent apoB properly so that it interacts with a
variety of protein and lipid factors that help achieve or maintain the
conformation needed to complete lipoprotein assembly. This would not be
surprising, given that many transmembrane and secretory proteins,
albeit more typical than apoB, undergo chaperone-mediated
conformational maturation to the native folded state before ER exit is
allowed (32). That the maturation process is prolonged for apoB is
consistent with its previously reported tardiness (relative to other
secretory proteins) in exiting the ER of HepG2 and rat hepatic cells
(24, 48).
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Fig. 8.
Model of the targeting of apoB polypeptides
to lipoprotein assembly or proteasomal degradation in HepG2 cells.
Right panel, when lipid synthesis and transfer are
adequate, apoB co-translationally associates with its lipid ligands.
Translation appears to be paused at the extreme C terminus, perhaps
mediated by a pause-transfer sequence (indicated in red).
When lipoprotein assembly is finished, translation completes, and the
apoB-lipoprotein complex exits the ER. Left panel, when
lipid synthesis and transfer are inadequate, apoB polypeptides become
exposed to the cytosol and interact with hsp70 and the proteasome while
still associated with the translocon. PDI, protein
disulfide isomerase.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Xinye Wu for technical assistance. We thank Dr. Tom A. Rapoport (Harvard Medical School) for the protocol and advice on the experiment shown in Fig. 4. We are also grateful to Dr. C. Cardozo for advice on the proteasome activity assays.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL 58541 (to E. A. F.), HL 55638 (to H. N. G.), and GM 26494 (to A. E. J.); American Heart Association Grant 0050415N (to A. E. J.); and Texas Advanced Research Program Grant 010366-131 (to A. E. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Cardiovascular
Inst., Mount Sinai School of Medicine, 1 Gustave Levy Place, P. O. Box
1269, New York, NY 10029. Tel.: 212-241-7152; Fax: 212-828-4178; E-mail: edmd-phd.fisher@mssm.edu.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M007944200
2 H. Wang, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
ER, endoplasmic reticulum;
MTP, microsomal triglyceride transfer protein;
ERAD, endoplasmic reticulum-associated degradation;
OA, oleic acid;
BSA, bovine serum albumin;
2M,
2-macroglobulin;
PBS, phosphate-buffered saline;
DSP, dithiobis(succinimidyl propionate);
PAGE, polyacrylamide gel
electrophoresis.
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