Cytosolic Components Are Required for Proteasomal Degradation of
Newly Synthesized Apolipoprotein B in Permeabilized HepG2 Cells*
Nobuhiro
Sakata,
J. Daniel
Stoops, and
Joseph L.
Dixon
From the Department of Food Science and Human Nutrition, University
of Missouri, Columbia, Missouri 65211
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ABSTRACT |
Recent studies have proposed that
post-translational degradation of apolipoprotein B100 (apoB) involves
the cytosolic ubiquitin-proteasome pathway. In this study,
immunocytochemistry indicated that endoplasmic reticulum
(ER)-associated proteasome molecules were concentrated in perinuclear
regions of digitonin-permeabilized HepG2 cells. Signals produced by
antibodies that recognize both
- and
-subunits of the proteasome
co-localized in the ER with specific domains of apoB. The mechanism of
apoB degradation in the ER by the ubiquitin-proteasome pathway was
studied using pulse-chase labeling and digitonin-permeabilized cells.
ApoB in permeabilized cells incubated at 37 °C in buffer alone was
relatively stable. When permeabilized cells were incubated with both
exogenous ATP and rabbit reticulocyte lysate (RRL) as a source of
ubiquitin-proteasome factors, >50% of [3H]apoB
was degraded in 30 min. The degradation of apoB in the intact ER of
permeabilized cells was much more rapid than that of extracted
[3H]apoB incubated with RRL and ATP in vitro.
The degradation of apoB was reduced by clasto-lactacystin
-lactone, a potent proteasome inhibitor, and by ubiquitin K48R
mutant protein, an inhibitor of polyubiquitination. ApoB in HepG2 cells
was ubiquitinated, and polyubiquitination of apoB was stimulated by
incubation of permeabilized cells with RRL. These results suggest that
newly synthesized apoB in the ER is accessible to the cytoplasmic
ubiquitin-proteasome pathway and that factors in RRL stimulate
polyubiquitination of apoB, leading to rapid degradation of apoB in
permeabilized cells.
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INTRODUCTION |
Apolipoprotein B100
(apoB)1 is a unique secretory
protein because of its large size (540 kDa), requirement for lipids for
secretion, and inefficient secretion resulting from rapid
post-translational degradation (1, 2). Previous studies showed that
>50% of newly synthesized apoB in HepG2, a hepatoma cell line, was
degraded within 20 min when cells were cultured under lipid-poor
conditions (3). Recent studies have indicated that newly synthesized
apoB is degraded post-translationally by a cytosolic proteolytic
system, the ubiquitin-proteasome pathway (4-8). This pathway has
recently been identified to be involved in the degradation of many
different proteins, including proteins within the endoplasmic reticulum (ER) compartment (9-15).
Because apoB is largely located in the ER lumen, the precise mechanism
of apoB degradation by the ubiquitin-proteasome pathway has not been
worked out. Recently, ER proteins have been shown to undergo retrograde
transport from the ER to the cytosol for proteasomal degradation (9,
13, 15-17). Because apoB is located primarily in the ER lumen, it also
would require retrograde transport before proteasomal degradation.
Recent reports have shown that apoB associates with an ER membrane
translocon component, Sec61 (7, 18). ApoB has also been shown to
associate with the cytosolic chaperone HSP70 and the ER chaperones
calnexin, ERp72, GRP94, calreticulin, and BiP (5, 7, 8, 19, 20). These
chaperones may be involved in both normal and retrograde transport of
apoB in the ER.
Studies on the ubiquitination of target proteins and their subsequent
recognition and degradation by the proteasome are difficult in intact
cells and would be even more pronounced with the large apoB molecule
(540 kDa). Therefore, to investigate the degradation of apoB in the ER
by the ubiquitin-proteasome pathway, we have studied apoB degradation
in digitonin-permeabilized pulse-labeled HepG2 cells utilizing rabbit
reticulocyte lysate (RRL) as source of ubiquitin-conjugating enzymes
and other factors involved in proteasomal degradation (21, 22).
The questions that we sought to answer are as follows. 1) How can newly
synthesized apoB that is located largely in the ER lumen be degraded by
the ubiquitin-proteasome pathway in the cytosol? 2) Does newly
synthesized apoB need to be transported to another location in the cell
or are apoB and the proteasome in close proximity in the liver cell?
This study demonstrates that cytosolic components in RRL are required
for proteasomal apoB degradation in permeabilized cells with an intact
ER membrane. Newly synthesized apoB in the ER is "primed" for rapid
degradation by the proteasome, suggesting that efficient retrograde
transport of apoB occurs in permeabilized cells.
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EXPERIMENTAL PROCEDURES |
Materials--
RRL was obtained from Promega (Madison, WI). ATP,
creatine kinase, creatine phosphate, and bovine serum albumin were
purchased from Sigma. L-[4,5-3H]Leucine and
luminol reagent (ECL kit) were purchased from Nycomed Amersham
(Princeton, NJ). Ubiquitin aldehyde, clasto-lactacystin
-lactone, and anti-proteasome
-subunit antibody were obtained from Calbiochem-Novabiochem. Ubiquitin K48R mutant protein was obtained
from Boston Biochem (Cambridge, MA). Anti-proteasome
-type subunit
(zeta) and
-type subunit (HC10) antibodies were purchased from
Affiniti (Mamhead, United Kingdom). Anti-ubiquitin antibody was
purchased from Roche Molecular Biochemicals. Monospecific anti-human
apoB B4 region antibody was raised in a rabbit (23). Sheep anti-human
apoB antibody was purchased from Serotec Ltd. (Raleigh, NC). The CC3.4
monoclonal antibody to apoB was a gift of Dr. Gustav Schonfeld
(Washington University, St. Louis, MO). Monoclonal antibody to p63
(G1/296) was a gift of Dr. Hans-Peter Hauri (Department of
Pharmacology, Biocenter of the University of Basel, Basel,
Switzerland). Protein A-Sepharose CL-4B was obtained from Amersham
Pharmacia Biotech. Minimal essential medium and leucine-free medium
(minimal essential medium Selectamine kit) were purchased from
Life Technologies, Inc. Anti-mouse and anti-rabbit secondary antibodies
conjugated with the fluorochrome Cy3 (indocarbocyanine) or Cy5
(indodicarbocyanine) were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). All other chemicals were of the
highest purity available.
Cell Culture--
HepG2 cells were grown on collagen-coated
tissue culture dishes (for labeling experiments) or coverslips (for
immunocytochemistry) as described previously (23). Briefly, HepG2 cells
were cultured in 24- or 6-well tissue culture plates with complete
medium containing minimal essential medium with 0.1 mM
nonessential amino acids, 1.0 mM sodium pyruvate, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine
serum. After 4 days of culture in a CO2 incubator, cells
were used for pulse-chase experiments. For immunocytochemistry, HepG2
cells were used at lower confluency (20-30%).
Permeabilization of HepG2 Cells--
The cell permeabilization
procedures were described previously (23). In this study, we used
digitonin to perforate the plasma membrane and saponin to perforate
both plasma and intracellular membranes. For permeabilization of cells
with digitonin for immunocytochemistry, subconfluent HepG2 cells were
washed twice with phosphate-buffered saline and treated for 5 min at
4 °C with digitonin (60 µg/ml) in intracellular buffer (75 mM potassium acetate, 2.5 mM magnesium acetate,
1.8 mM calcium chloride, and 25 mM HEPES, pH
7.2). After removing digitonin, the cells were washed three times with
intracellular buffer and then fixed with 2% paraformaldehyde for 30 min at room temperature and processed for immunocytochemistry. For
permeabilization of cells with saponin for immunocytochemistry,
subconfluent cells were washed twice with phosphate-buffered saline,
fixed in 2% paraformaldehyde, washed three times with intracellular
buffer, and processed for immunocytochemistry. All subsequent steps for saponin-permeabilized cells were performed in intracellular buffer containing 0.1% saponin and 0.1% bovine serum albumin. For
permeabilization of cells with digitonin for apoB degradation studies,
confluent HepG2 cells were washed twice with phosphate-buffered saline, treated for 5 min at 4 °C with digitonin (60 µg/ml) in
intracellular buffer, washed three times with intracellular buffer, and
used immediately.
Immunocytochemistry--
Digitonin- or saponin-permeabilized
cells were incubated for 4 h or overnight with primary antibodies
and then incubated for 4 h with the appropriate anti-mouse or
anti-rabbit IgG secondary antibodies conjugated with fluorochromes (Cy3
or Cy5) and examined with an MRC-600 confocal microscope (Bio-Rad) as
described in detail by Du et al. (23).
Degradation of ApoB in Permeabilized Cells--
HepG2 cells were
preincubated with serum-free medium for 1 h and then pulsed with
[3H]leucine (100 µCi/ml) in leucine-free medium for 10 min and chased for 10 min with serum-free medium at 37 °C. After
labeling, cells were permeabilized with digitonin at 4 °C as
described above and incubated at 37 °C with intracellular buffer
(total of 250 µl/well) containing RRL (10-25 µl/well), 2 mM ATP, 100 µg/ml creatine kinase, and 10 mM
creatine phosphate. When effects of inhibitors were studied, cells were
preincubated with intracellular buffer (250 µl/well) in the absence
or presence of inhibitors for 20 min before incubation with RRL and
ATP. The incubation buffer (intracellular buffer containing RRL) had
also been treated with inhibitors for 20 min and then was added to
inhibitor-pretreated permeabilized cells. After incubation,
[3H]apoB in the cells was extracted with lysis buffer,
immunoprecipitated, separated by SDS-PAGE, and detected by
fluorography. Film images were scanned by an image scanner (Epson
Expression 636) and analyzed by the NIH Image program (Version 1.61) on
a Power Macintosh 9600 system (Apple Computer).
Degradation of ApoB in Vitro--
The degradation of extracted
[3H]apoB was monitored in vitro as described
previously (24). Briefly, HepG2 cells were pulsed with 75 µCi/ml
[3H]leucine for 10 min and chased for 10 min. Newly
synthesized [3H]apoB was isolated by immunoprecipitation
using anti-human apoB antiserum and protein A-Sepharose CL-4B (3, 24,
25). Extracted [3H]apoB (~60,000 dpm/40 µl) in 0.1 M glycine, 0.02 M Tris, and 1% Triton X-100,
pH 7.4, was incubated with 45 µl of RRL, 2 mM ATP, 5 mM MgCl2, 100 µg/ml creatine kinase, and 10 mM creatine phosphate in a final volume of 90 µl. After
incubation at 37 °C, 200 µl of electrophoresis sample buffer was
added, and it was boiled for 4 min. Samples were separated by SDS-PAGE
and visualized by fluorography.
Immunoblotting--
Unlabeled HepG2 cells were permeabilized
with digitonin and incubated for 0 or 30 min at 37 °C in
intracellular buffer plus rabbit reticulocyte lysate and ATP as
described for the radiolabel studies. ApoB in cells was extracted with
lysis buffer, immunoprecipitated with anti-human apoB antibody, and
separated by SDS-PAGE. Proteins in the gel were transferred to
polyvinylidene fluoride Immobilon-P membranes as described previously
(23). ApoB and apoB-bound ubiquitin in the membrane were detected
by anti-apoB and anti-ubiquitin antibodies and visualized with
horseradish peroxidase-conjugated second antibodies and ECL luminol reagent.
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RESULTS |
Recent studies have indicated that the proteasome is largely
located in the cytosol and that a small percentage of proteasome molecules are associated with the ER membrane (26, 27). It is not clear
whether ER membrane-associated proteasomes are involved in the
degradation of ER proteins. To investigate this question, we first
studied the locations of apoB and the proteasome in
digitonin-permeabilized HepG2 cells using immunocytochemistry.
In a previous study, two domains in apoB were shown to be exposed on
the cytosolic side of the ER membrane in subconfluent streptolysin
O-permeabilized HepG2 cells (23). In the current experiments, digitonin
was used to permeabilize cells because only a maximum of ~50% of the
cells in confluent monolayers of HepG2 cells were permeabilized using
streptolysin O, whereas almost 100% of the cells can be permeabilized
with digitonin (data not shown).
Two anti-apoB antibodies were used for immunocytochemistry. The CC3.4
antibody to apoB recognizes amino acids 690-797 in the N-terminal
region of the apoB molecule (28), whereas the B4 anti-peptide antibody
recognizes a more limited region of apoB (amino acids 3221-3240) (23).
Although these regions of apoB were shown to be cytosol-facing, a
majority of each apoB molecule is located in the lumen of the ER (23).
Both apoB antibodies (CC3.4 (Fig.
1A) and B4 (Fig. 1,
D and G)) produced bright concentrated staining
in the perinuclear regions of cells, with less intense reticular
staining in peripheral regions, as previously observed in streptolysin
O-permeabilized cells (23).

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Fig. 1.
Proteasome and cytosol-facing domains of apoB
co-localize in the ER. HepG2 cells were either permeabilized with
digitonin (60 µg/ml) for 5 min, washed, and fixed with 2%
paraformaldehyde (A-C) or fixed first, permeabilized, and
washed (D-I) before processing for immunocytochemistry.
Permeabilized cells were incubated with anti-apoB antibodies
(A, CC3.4 (1:200 final dilution); D and
G, B4 (1:50 dilution)) and anti-proteasome antibodies
(B, -subunit; E, zeta ( -type subunit);
H, HC10 ( -type subunit); all at 1:200) as described under
"Experimental Procedures." Cells were then incubated with the
appropriate anti-mouse or anti-rabbit IgG secondary antibodies (1:100)
conjugated with fluorochromes (Cy3 or Cy5) and examined with an MRC-600
confocal microscope. C, F, and I are
superimposed pictures of A and B, D
and E, and G and H, respectively. The
areas of co-localization of apoB and proteasome are
yellow.
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The 26 S proteasome is primarily a cytosolic protease (11, 12) that
consists of a large complex of proteins that compose a central 20 S
core and two terminal PA700 complexes (29, 30). We asked the question
whether the proteasome would still be present in
digitonin-permeabilized cells. For some experiments, cells were
permeabilized first and then fixed for immunocytochemistry to allow
removal of soluble cytosolic constituents (Fig. 1, A-C). When digitonin-permeabilized cells were probed with a polyclonal antibody to the 20 S proteasome
-subunit, bright signals were observed in the perinuclear regions of cells (Fig. 1B).
Monoclonal antibodies to the zeta (
-type) and HC10 (
-type)
subunits of the proteasome (Fig. 1, E and H) gave
similar signals in the perinuclear regions and more diffuse staining in
the peripheral secretory membranes. These results indicate that a
significant population of proteasome molecules remains associated with
the ER membranes in the perinuclear regions of digitonin-permeabilized cells.
When the signals due to the
-subunit of the proteasome were
superimposed with the signals produced by the CC3.4 antibody to apoB
(Fig. 1C), there was significant overlap in the areas closest to the nucleus of the cell. There was also substantial overlap
of signals from the zeta and HC10 subunits of the proteasome with the
signals produced by the B4 region of apoB (Fig. 1, F and
I). These immunocytochemical observations suggest that
proteasome molecules that are associated with the ER may be in close
proximity to cytosolic domains of apoB in the perinuclear regions of
HepG2 cells.
A series of biochemical experiments were performed to investigate the
degradation of apoB in digitonin-permeabilized cells. Cells were
cultured under standard media conditions without added fatty acid to
mimic conditions that allow maximal early apoB degradation (3). First,
immunocytochemistry was used to determine whether the ER membrane was
intact in digitonin-permeabilized cells (23). Whereas a bright signal
for the luminal portion of the ER membrane protein p63 was observed
when cells were permeabilized with saponin (Fig.
2, right), only a background
signal was observed in digitonin-treated cells (Fig. 2,
left). These data indicated that ER membranes were not
permeabilized by digitonin treatment.

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Fig. 2.
Digitonin permeabilization does not perforate
the ER membrane. HepG2 cells were permeabilized with digitonin (60 µg/ml) for 5 min (left) or with saponin (0.1%) treatment
(right). Cells were washed with intracellular buffer three
times and fixed with 2% paraformaldehyde. Fixed cells were incubated
with anti-p63 antibody (1:1000), followed by incubation with anti-mouse
IgG (1:100) conjugated with Cy3. Cells were examined with an MRC-600
confocal microscope.
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The degradation of apoB in permeabilized cells was measured as
described under "Experimental Procedures" and shown in Fig. 3. Digitonin treatment itself did not
decrease the content of [3H]apoB in HepG2 cells (Fig.
4A, lanes 1 and
2). When digitonin-permeabilized cells were incubated in
buffer at 37 °C with no additions, [3H]apoB was
observed to be relatively stable (Fig. 4A, lane
3). This observation is similar to our early observation that apoB was stable in preparations of intact microsomes (25). This result suggests that there is a requirement for cytosolic components for the
degradation of apoB in permeabilized cells.

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Fig. 3.
ApoB degradation in permeabilized HepG2
cells. HepG2 cells were pulse-labeled with
[3H]leucine, chased, permeabilized with digitonin (60 µg/ml), washed, and incubated with RRL ± factors as described
under "Experimental Procedures." After incubation, the buffer was
removed, and apoB was extracted from cells, immunoprecipitated, and run
on a 3-15% gradient SDS-polyacrylamide gel.
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Fig. 4.
[3H]ApoB is degraded in
permeabilized cells in the presence of RRL and ATP. A,
HepG2 cells were pulsed with [3H]leucine for 10 min,
chased for 10 min, permeabilized with 60 µg/ml digitonin for 5 min,
washed with intracellular buffer, and incubated for 0 or 1 h with
RRL and ATP at 37 °C. [3H]ApoB in the cells was
extracted, immunoprecipitated, and analyzed by SDS-PAGE (3-15%).
Lane 1, no digitonin treatment (control); lane 2,
digitonin treatment only; lane 3, digitonin treatment plus
incubation at 37 °C with buffer alone; lane 4, digitonin
treatment plus incubation at 37 °C with RRL and ATP. The
asterisk denotes a 150-kDa apoB degradation product in
lane 4. B, shown is the in vitro
degradation of extracted apoB. ApoB was isolated from HepG2 cells that
had been pulse-labeled for 10 min with [3H]leucine and
chased for 10 min with serum-free medium. [3H]ApoB was
incubated with RRL and ATP at 37 °C for 0 or 3 h.
[3H]ApoB was analyzed as described under "Experimental
Procedures." Lane 1, no incubation (control); lane
2, incubation at 37 °C with buffer alone; lane 3,
RRL added, but no incubation; lane 4, incubation at 37 °C
with RRL and ATP. The mobilities of molecular mass markers and plasma
low density lipoprotein are indicated to the right of each gel.
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Previously, we utilized RRL to study apoB degradation in
vitro (24) and observed that the degradation of isolated apoB was primarily dependent upon the ubiquitin-proteasome system present in
RRL. Therefore, we added RRL to permeabilized cells to provide a source
of the ubiquitin-proteasome system and other factors. Other additions
(such as ATP) were made as noted in the figure legends. In contrast to
permeabilized cells incubated in buffer alone, newly synthesized apoB
was rapidly degraded in digitonin-permeabilized cells incubated at
37 °C in buffer containing RRL and the ATP-generating system (ATP)
(Fig. 4A, lane 4). ApoB degradation was not
observed when permeabilized cells were treated with a preparation of
boiled RRL, suggesting that the required factors were heat-sensitive (data not shown). As previously observed (24) when apoB was extracted
from HepG2 cells and incubated with RRL and ATP, apoB degradation was
dependent on RRL, but occurred at a much slower rate (Fig.
4B, lane 4). Approximately 75% of apoB was
degraded after 3 h using the in vitro assay. A moderate
size 150-kDa apoB degradation product (Fig. 4A) and, in some
experiments, a less prominent 380-kDa band (see Fig. 7B)
were observed in permeabilized cells treated with RRL, but were not
produced in the in vitro assay (Fig. 4B).
A time course study indicated that newly synthesized apoB in
permeabilized cells was degraded more rapidly than isolated apoB in the
in vitro assay (Fig. 5). After
30 min of incubation, ~70% of apoB was degraded in permeabilized
cells versus only 25% in the in vitro assay.
This difference in rate occurred despite the fact that less RRL was
added to permeabilized cells compared with the amount of RRL added to
the in vitro assay. To determine whether the loss of apoB
signal was due to degradation of apoB or to release of apoB after ER
membrane rupture, the effects of incubation with RRL on newly
synthesized albumin were monitored in permeabilized cells. We
previously showed that if secretory membranes are ruptured, albumin
will be lost from permeabilized cells (23). Incubation of permeabilized
cells for 30 min with RRL did not lead to loss of albumin signal (Fig.
6). We also measured release of apoB into the incubation buffer. Only 2-3% of the total cellular apoB counts were immunoprecipitated from the incubation buffer regardless of the
presence of RRL (data not shown). These results show that ER membranes
remained impermeable during and after incubation of cells with RRL.

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Fig. 5.
Time course of the degradation of
[3H]apoB in permeabilized cells and the in
vitro study. ApoB degradation was assessed as described
in the legend to Fig. 4. Newly synthesized apoB in the ER membrane of
permeabilized cells in the presence of RRL and ATP was degraded much
more rapidly than isolated free apoB in the presence of RRL and ATP in
the in vitro study. See assay details under "Experimental
Procedures."
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Fig. 6.
[3H]Albumin in permeabilized
HepG2 cells is stable in the presence of RRL and ATP at 37 °C.
HepG2 cells were labeled and permeabilized as described in the legend
to Fig. 3. The permeabilized cells were incubated for 0 or 30 min with
RRL and ATP at 37 °C. [3H]Albumin in the cells was
extracted with lysis buffer, immunoprecipitated, and analyzed by
SDS-PAGE (3-15%). Lane 1, digitonin treatment only;
lane 2, digitonin treatment plus incubation at 37 °C with
buffer alone; lane 3, digitonin treatment plus incubation at
37 °C with RRL and ATP.
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The next series of experiments utilized inhibitors of the
ubiquitin-proteasome pathway to determine whether the degradation of
apoB in RRL-treated permeabilized cells was proteasomal in nature. ApoB
degradation in permeabilized cells (Fig.
7) was reduced by incubation with 50 µM clasto-lactacystin
-lactone, a potent inhibitor of the proteasome (31-33). ApoB immunoprecipitated from cells treated with clasto-lactacystin
-lactone (Fig.
7A, lane 3) showed increased signals both for the
main apoB band and for a higher molecular mass apoB species that may
represent polyubiquitinated apoB (see Fig. 8). ApoB degradation was
also inhibited by ubiquitin K48R mutant protein, which perturbs
polyubiquitination of proteins (34, 35). Incubation with 50 µM ubiquitin K48R mutant protein (Fig. 7B,
lane 3) produced an increased apoB signal without increasing the signal of the higher molecular mass apoB species that had been
observed in Fig. 7A (lane 3). This observation is
consistent with inhibition of polyubiquitination of apoB by ubiquitin
K48R mutant protein. In contrast to apoB degradation in the in
vitro system (24), degradation of apoB in permeabilized cells was not significantly affected by ubiquitin aldehyde, an inhibitor of
ubiquitin hydrolases (data not shown).

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Fig. 7.
Degradation of [3H]apoB in
permeabilized cells is reduced by a proteasome inhibitor,
clasto-lactacystin -lactone,
and by ubiquitin K48R mutant protein. After preincubation
with/without inhibitors as described under "Experimental
Procedures," permeabilized cells were incubated for 0 or 30 min with
RRL and ATP in the absence or presence of inhibitors of the
ubiquitin-proteasome pathway. Lane 1, digitonin treatment
only; lane 2, digitonin treatment plus incubation at
37 °C with RRL and ATP; lane 3, digitonin treatment plus
incubation at 37 °C with RRL and ATP plus inhibitor. A,
clasto-lactacystin -lactone ( -LC; 50 µM); B, ubiquitin (Ub) K48R (50 µM). After incubation, apoB was extracted,
immunoprecipitated, and analyzed by SDS-PAGE. The asterisk
and double asterisks denote 150- and 380-kDa apoB
degradation products, respectively.
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Collectively, these results indicate that apoB degradation in
RRL-treated permeabilized cells involves polyubiquitination, followed
by efficient proteasomal proteolysis. Interestingly, there were only
limited effects of the two inhibitors on the formation of the 150-kDa
apoB degradation intermediate (Fig. 7), indicating that another process
may be involved in the generation of this fragment.
In the next experiment, we wished to examine the ubiquitination state
and degradation of total cellular apoB in RRL-treated permeabilized
cells (Fig. 8, A and
B). As observed with
[3H]apoB, apoB degradation could be observed by
immunoblotting of digitonin-permeabilized cells after addition of RRL
and ATP (Fig. 8A, lane 3), but was not observed
during incubation without RRL and ATP (lane 2). Immunoblots
of immunoprecipitated apoB probed with anti-ubiquitin antibody showed
that apoB in permeabilized cells was ubiquitinated prior to incubation
with RRL (Fig. 8B, lane 1). Despite loss of
intact apoB (Fig. 8A, lane 3), ubiquitinated apoB
increased during incubation with RRL (Fig. 8B, lane
3). The slower gel mobility of the signal produced by
anti-ubiquitin antibody suggested that the ubiquitinated apoB formed
during incubation with RRL was primarily polyubiquitinated apoB. When
incubated without RRL, apoB appeared to become de-ubiquitinated
(lane 2). These results indicate that a certain percentage
of apoB is ubiquitinated in intact HepG2 cells and that, during
incubation of RRL-treated permeabilized cells, ubiquitination of apoB
is maintained or possibly increased, leading to the accumulation of
polyubiquitinated apoB.

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Fig. 8.
Polyubiquitination of apoB is increased in
permeabilized cells following incubation with RRL and ATP.
Unlabeled HepG2 cells were permeabilized with digitonin and incubated
for 0 or 30 min with RRL and ATP at 37 °C. ApoB in the cells was
immunoprecipitated and then separated by SDS-PAGE. Proteins in the gel
were transferred to polyvinylidene fluoride Immobilon-P membranes. ApoB
(A) and ubiquitin (B) were detected by anti-apoB
and anti-ubiquitin antibodies and visualized with horseradish
peroxidase-conjugated second antibodies and ECL luminol reagent.
Lane 1, digitonin treatment only; lane 2,
digitonin treatment plus incubation at 37 °C with buffer alone;
lane 3, digitonin treatment plus incubation at 37 °C with
RRL and ATP.
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DISCUSSION |
Co-localization of Proteasome with ApoB--
In mammalian cells, a
majority of the cellular proteasome was shown to be present in the
cytosol (26, 27, 36). Immunogold electron microscopy studies indicated
that 69-83% of proteasomes in rat hepatocytes were located in the
cytoplasm, whereas the ER contained ~11-14% of the gold particles
(26, 27). A study in yeast showed that proteasomal subunits labeled
with green fluorescent protein accumulated mainly in the nuclear
envelope-ER network (37).
We did not measure what percent of total cellular proteasomes is
represented by the signals produced by anti-proteasome antibodies in
our immunocytochemistry photographs. Whether the cells were permeabilized or fixed first, a large amount of soluble proteasome complexes may have been released into the washes during the
permeabilization process. The results of this study provide evidence
that populations of proteasomes are associated with the ER membrane and
that they are located in regions of the cell that are important in the
early metabolism of newly synthesized apoB.
Degradation Studies--
ApoB was relatively resistant to
degradation in permeabilized cells incubated without RRL and ATP.
Therefore, although the ER membrane still retained associated
proteasomes, some other factor(s) appeared to be required for efficient
degradation of apoB in permeabilized cells. We had previously used RRL
as a source of ubiquitin-proteasome components in an in
vitro apoB degradation assay (24). In the current study, after
addition of RRL and ATP, [3H]apoB in intact ER membranes
of permeabilized cells was degraded much more rapidly than extracted
apoB that was not associated with secretory membranes (Fig. 5). The
decline in apoB in permeabilized cells represents the degradation of a
large fraction of newly synthesized apoB. Similar experiments in intact
cells under lipid-poor conditions (3, 25) showed remarkably similar
early apoB kinetics (0-30 min), but different later decay (> 30 min)
due to the presence of a small, more degradation-resistant apoB pool in
intact cells. Because only short pulse-chase periods were used in this
study, the apoB degraded was primarily newly synthesized apoB. This
pool of apoB in the membrane of the ER was primed for rapid
degradation, but required cytosolic components.
The degradation of apoB in permeabilized cells incubated with RRL and
ATP was primarily by the ubiquitin-proteasome system, as it was reduced
by both clasto-lactacystin
-lactone (Fig. 7A) and ubiquitin K48R mutant protein (Fig. 7B). ApoB in
permeabilized cells became polyubiquitinated during incubation with RRL
(Fig. 8). The results with ubiquitin aldehyde, which had minimal
effects on apoB degradation in permeabilized cells, but had significant effects on the degradation of extracted apoB in the in vitro
study (24), suggest either that de-ubiquitination of apoB is different in permeabilized cells or that the inhibitor does not have access to
the ubiquitin hydrolases. These studies suggest that RRL provides one
or more factors (additional proteasome complexes, ubiquitin-conjugating enzymes, or possibly a cytosolic chaperone) to permeabilized cells that
are required for rapid proteasomal degradation of apoB.
ApoB is one of an expanding group of proteins (misfolded, mutant, or
overproduced membrane or secretory proteins) that may be transported
from the ER lumen to the cytosolic side of the ER membrane for
proteolysis by the ubiquitin-proteasome pathway. Recent reports showed
ubiquitination of apoB (4, 5, 7, 8, 19) and inhibition of apoB
degradation by proteasome inhibitors (4-7, 38) using intact cells.
These observations all suggest the involvement of the
ubiquitin-proteasome pathway in apoB degradation (5). Questions that
remain are how does the primarily luminal apoB molecule gain access to
the proteasome and what cell conditions or signals target apoB for
degradation? A recent study reported that expression of
cholesterol-7
-hydroxylase NADPH:oxygen oxidoreductase reduces
proteasomal degradation of heterologous apolipoprotein B53 in Chinese
hamster ovary cells by inhibiting ubiquitination (39). This may be a
possible mechanism by which lipid metabolism influences early apoB
degradation by the proteasome. The previous reports concerning apoB
degradation in cells (4-8, 19) and the current results obtained with
permeabilized cells support the paradigm that apoB, either entirely or
partially, must undergo retrograde transport before degradation by the
proteasome in the cytosol.
The mechanism of retro-transport of apoB is still unknown. Chen
et al. (7) and Mitchell et al. (18) recently
showed that apoB co-immunoprecipitated with Sec61 and that treatments
that slowed apoB-lipoprotein assembly increased this association.
Because apoB is transported cotranslationally through the translocon, it was not determined whether the apoB found associated with Sec61 was
being transported inward or outward. But the observation that the
ubiquitination state of apoB associated with the Sec61 complex increased as proteasomal degradation of glycosylated apoB increased is
evidence that retro-transport may occur through the translocon (7).
Recently, we reported that two limited regions in apoB are exposed on
the cytosolic side of the ER membrane (23). It is possible that the
CC3.4 and B4 domains of apoB play an important role in targeting poorly
lipidated apoB molecules for proteasomal degradation. These domains may
be used as attachment sites for proteins involved in apoB
retro-transport. Zhou et al. (8) demonstrated that apoB is
associated with HSP70, a cytosolic chaperone. It is intriguing to
speculate that these cytosol-facing domains of apoB interact with HSP70
or ubiquitin. We also observed that a small portion of apoB degradation
(the 150-kDa apoB fragment) was insensitive to ubiquitin-proteasome
pathway inhibitors (Fig. 7). This apoB degradation peptide is not seen
in intact cells and may be the result of another protease in RRL or a
native protease in the ER of permeabilized cells that is activated by
factors in RRL. A recent study showed that apoB was associated with an ER resident protease, ER60 (40). We reported that a
dithiothreitol-sensitive protease in the ER might be partially involved
in the degradation of apoB fragment (41).
This study shows that ER membrane-bound proteasomes co-localize with
cytosol-facing domains of apoB that may play a role in co-translational
or post-translational apoB degradation (Fig. 9). When lipid availability is
inadequate, non-lipidated or poorly lipidated apoB is targeted for
degradation by the proteasome. Proteasomal degradation of apoB in
permeabilized cells may involve either ER-bound HepG2-derived
proteasomes or RRL-derived proteasomes (Fig. 9). Studies investigating
which population of proteasomes is responsible for apoB degradation and
the factors in RRL that are required for degradation of apoB in
permeabilized cells are now being conducted.

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|
Fig. 9.
Model for proteasomal degradation of apoB in
permeabilized HepG2 cells. ApoB is synthesized in the ER on
membrane-bound ribosomes and co-translationally transported into the ER
lumen. Depending upon lipid availability, apoB can either be assembled
into a nascent lipoprotein or be degraded in the ER membrane.
Ub, HSP, and MTP denote ubiquitin,
heat shock protein, and microsomal triglyceride transfer protein,
respectively. Ovals without labels are putative ER luminal
chaperones.
|
|
 |
ACKNOWLEDGEMENTS |
We thank JoAnn Lewis for typing the
manuscript, Dr. Thomas E. Phillips for advice concerning confocal
microscopy, Dr. Gustav Schonfeld for the gift of the CC3.4 anti-apoB
antibody, and Dr. Hans-Peter Hauri for the gift of the anti-p63 antibody.
 |
FOOTNOTES |
*
This work was supported by NHLBI Grant HL-47586 (to
J. L. D.) from the National Institutes of Health.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: Dept. of Food Science
and Human Nutrition, University of Missouri, 122 Eckles Hall, Columbia,
MO 65211. Tel.: 573-882-4113; Fax: 573-882-0596; E-mail:
DixonJ{at}missouri.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
apoB, apolipoprotein
B100;
ER, endoplasmic reticulum;
RRL, rabbit reticulocyte lysate;
PAGE, polyacrylamide gel electrophoresis.
 |
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