(Received for publication, May 22, 1995; and in revised form, September 8, 1995)
From the
Previous studies show that translocation and degradation of
apolipoprotein B (apoB), two processes occurring on or within the
endoplasmic reticulum, determine how much de novo synthesized
apoB is secreted. We determined which of these processes regulates the
intracellular fate of apoB by examining whether degradation determines
how much apoB is translocated or if translocation determines how much
apoB is degraded. HepG2 cells, treated with the cysteine active site
protease inhibitor ALLN, previously shown to block the degradation of
translocation-arrested apoB in Chinese hamster ovary cells (Du, E.,
Kurth, J., Wang, S.-L., Humiston, P., and Davis, R. A.(1994) J.
Biol. Chem. 269, 24169-24176), showed a 10-fold increase in
the accumulation of de novo synthesized
[S]methionine-labeled apoB. The majority (80%)
of the apoB accumulated in response to ALLN was in the microsomal
fraction. In contrast, ALLN did not effect apoB secretion. Since ALLN
did not effect the intracellular accumulation of
[
S]methionine-labeled albumin and other proteins
(trichloroacetic acid-precipitable
[
S]methionine-labeled proteins), its effect on
apoB was specific. Pulse-chase studies showed that ALLN dramatically
reduced the first-order rate of removal of
[
S]methionine-labeled apoB from the cell but did
not effect its rate of secretion. The finding that ALLN caused the
intracellular accumulation of incompletely translated chains of apoB
suggests that at least some of the degradation occurs at the ribosomal
level. Moreover, 85% of the apoB that accumulated in isolated
microsomes in response to ALLN was accessible to exogenous trypsin,
indicating this pool of apoB was incompletely translocated. The
combined data suggest that translocation, not degradation, determines
the intracellular fate of de novo synthesized apoB.
apoB ()is the major structural protein responsible
for the assembly of triglyceride-rich lipoproteins by the intestine and
liver(1, 2, 3) . After secretion into blood
plasma by the liver, VLDL triglycerides are rapidly hydrolyzed into
free fatty acids, which are taken up by peripheral tissues where they
are utilized for energy and anabolic purposes. The remaining VLDL
remnants, containing apoB100, are then either removed by the liver or
converted into LDL, a major risk factor for atherosclerosis (reviewed
in (4) ). Because of the importance of hepatic secretion of
VLDL apoB in determining blood levels of LDL, a major goal of our
research has been directed toward identifying the regulatory factors
and processes.
We have made two observations, both of which have shown unusual characteristics governing the hepatic secretion of apoB: 1) a large amount of de novo synthesized apoB is not secreted but is degraded intracellularly (5) and 2) the translocation of apoB across the endoplasmic reticulum is unusually inefficient(6) . These results led us to hypothesize that as a result of incomplete translocation, apoB is diverted from the secretory pathway into a degradative pathway that occurs in the endoplasmic reticulum(3, 6) . Regulated translocation of apoB across the endoplasmic reticulum could explain the post-translational control of apoB secretion. In several studies, the amount of apoB that is secreted is less than the amount that is synthesized(5, 7, 8, 9, 10, 11, 12, 13, 14) , leading to the conclusion that the unaccounted for apoB is degraded intracellularly. The consistent observation that in different types of cultured cells and perfused organs obtained from several species, a diverse group of hormones, nutritional states, stimulatory and inhibitory lipids, and mutations in the coding region of apoB alter the rate of apoB secretion by reciprocal changes in its rate of intracellular degradation suggests that this unusual regulatory mechanism of secretion is of general importance (reviewed in Refs. 3, 15-17).
An important question toward which this research was directed was to determine whether translocation governs degradation or if degradation governs translocation. To this end we employed the proteolytic inhibitor ALLN, which we have shown blocks the degradation of apoB in CHO cells, which normally degrade all of the apoB expressed by a plasmid construct(18, 19) . Based on the results showing that ALLN also blocks the degradation of apoB100 in HepG2 cells(20) , we examined if blocking the intracellular degradation of apoB would either increase the amount secreted or would cause more to accumulate as an incompletely translocated form. If blocking degradation leads to increased secretion, the data would indicate that degradation determines the fate of apoB. Conversely, if blocking degradation leads to an accumulation of incompletely translocated apoB, the data would indicate that translocation determines the fate of apoB.
Additional aliquots of cells and medium were precipitated with 10% trichloroacetic acid(5) .
First-order rate constants and half-lives were calculated using Sigma Plot program having least-squares analysis.
Figure 1:
Immunoprecipitation of apoB100 and
albumin from HepG2 cells. HepG2 cells were cultured in methionine-free
DMEM containing 8% serum with (+) or without(-) 50 µg/ml
ALLN for 0.5 h. [S]Methionine was added, and the
cells were incubated for an additional 4 h. Equal portions of cell
lysates (C) and medium (M) were immunoprecipitated
using a polyclonal anti-human apoB or an anti-human albumin antibody,
separated by SDS-PAGE.
We examined if ALLN caused S-labeled apoB100 to accumulate in the endoplasmic
reticulum, where the majority of apoB in liver cells
resides(5, 22) . Cells were treated with ALLN for 20
min and then labeled with [
S]methionine for 40
min. Cells were isolated and disrupted by nitrogen decavitation, and
microsomes were isolated by ultracentrifugation. Total cell homogenates
and microsomes were subjected to immunoprecipitation. In three separate
experiments, during the 40-min labeling period, ALLN increased the
accumulation of
S-labeled apoB100 in cells by 3.0 ±
0.4-fold in whole cells and 2.3 ± 0.9-fold in microsomes (Table 1). These data show that the majority (77%) of the apoB
that accumulated in cells was isolated in the microsomal fraction.
Analysis of the S-labeled apoB100 during the pulse-chase experiment shows
that immediately after the pulse period, there are several small
molecular weight forms of apoB that disappeared during the chase period (Fig. 2A). These smaller molecular weight forms of apoB
are likely to be incompletely translated nascent chains of apoB
residing on the ribosome, as demonstrated by the increased amount of
S-labeled apoB100 that appears in cells after 20 min of
chase (Fig. 3). After this time, in both groups of cells (with
and without ALLN)
S-labeled apoB100 was rapidly lost from
the cell (Fig. 2A). After a 30-min period,
S-labeled apoB100 appeared in the medium obtained from
cells treated both with and without ALLN (Fig. 2). Since even in
the presence of ALLN, the amount of
S-labeled apoB100 that
appeared in the medium was less than the amount that was lost from the
cells, under the conditions of this experiment ALLN does not appear to
block all of the intracellular degradation of apoB100. It should be
pointed out that the time course and amount of ALLN used for our
experiments was determined empirically to not cause cytotoxicity as
determined by no effect on the amount of
S-labeled albumin
that accumulated in either cells or medium (Fig. 2B) or
the amount of trichloroacetic acid-precipitable
S-labeled
proteins (data not shown). We have found that higher concentrations of
ALLN will inhibit protein synthesis. Because of the cytotoxicity of
ALLN, it is not possible to conclude that the dose of ALLN used in our
studies was sufficient to block all ALLN-inhibitable proteolysis.
Figure 2:
Pulse-chase labeling and
immunoprecipitation of apoB100 and albumin from HepG2 cells. HepG2
cells were cultured in methionine-free DMEM containing 8% serum with
(+) or without(-) 50 µg/ml ALLN for 0.5 h.
[S]Methionine was added to the culture medium,
and the cells were incubated 10 min. After the labeling period, cells
were chased with medium containing a 1000-fold excess of cold
methionine and the same amount of ALLN as was present in the initial
incubation. A, cells and medium were harvested after 0, 15,
30, 60, and 120 min of chase. Equal portions of cell lysates (C) and medium (M) were immunoprecipitated using a
polyclonal anti-human apoB antibody and separated by SDS-PAGE. B, cells and medium were harvested after 0, 15, 30, and 120
min of chase. Equal portions of cell lysates (C) and medium (M) were immunoprecipitated using an anti-human albumin
antibody and separated by SDS-PAGE.
Figure 3:
Cell and medium content of S-labeled apoB100. Autoradiographs from triplicate
pulse-chase experiments, including that shown in Fig. 2, were
analyzed on a Molecular Dynamics densitometer. Bands corresponding to
immunoprecipitated apoB100 were quantitated; the quantitation is given
in arbitrary units (solid lines). Results are expressed as the
mean S.D. of three individual experiments. A, the best-fit
line for first-order exponential decay is shown (dashed
lines). Open symbols represent cells treated with ALLN,
while filled symbols designate cells not treated with the
inhibitor. B, secretion of
S-labeled apoB100 into
the culture medium during the pulse-chase experiment. Open symbols represent cells treated with ALLN, while filled symbols designate cells not treated with the
inhibitor.
Estimation of the first-order rate of loss of S-labeled
apoB peptides (all molecular weight forms) showed that it was 6-fold
greater in cells not treated with ALLN compared to cells treated with
ALLN (0.05 min
, t
=
13.4 min without ALLN; 0.008 min
, t
= 90 min with ALLN). In three separate plates of cells, we
examined the quantitative effects of ALLN on apoB accumulation in cells
and secretion into the medium during the pulse-chase experimental
protocol. ALLN did not significantly affect the rate nor the relative
amount of
S-labeled apoB100 that appeared in the cultured
medium (Fig. 3). These pulse-chase study results confirm the
previous data (Fig. 1) showing that while ALLN increases the
cellular accumulation of
S-labeled apoB100, there is no
effect on secretion. Moreover, the data extend these observations by
showing that the accumulation of cellular
S-labeled
apoB100 is caused by a decreased rate of removal (i.e. degradation).
It is interesting to note that during the chase
period, ALLN blocked the rate of disappearance of the incompletely
translated forms of S-labeled apoB that appear as several
bands of lower molecular weight than apoB100 (Fig. 2A).
With the proviso that ALLN did not affect the rate of apoB translation,
these data suggest that a portion of the intracellular degradation of
apoB occurs before it is completely translated.
Figure 4:
Susceptibility of apoB100 and albumin in
microsomes to trypsin digestion. HepG2 cells were cultured in
methionine-free DMEM containing 8% serum with (+) or
without(-) 50 µg/ml of the inhibitor ALLN for 20 min.
[S]Methionine was added to the culture medium,
and the cells were incubated for an additional 40 min. Microsomes were
isolated and incubated with the indicated presence of trypsin and/or
soybean trypsin inhibitor. The reaction was stopped by the addition of
soybean trypsin inhibitor, and microsomes were reisolated. Equal
portions were immunoprecipitated using a polyclonal anti-human apoB or
an anti-human albumin antibody and separated by
SDS-PAGE.
The results presented in this report indicate that the translocation step is responsible for directing apoB into either the secretory or degradation pathways. Furthermore, the data show that under the conditions used to culture HepG2 cells, the amount of apoB that is synthesized is in excess of the amount that is fully translocated, and the ``excess'' incompletely translocated pool of apoB is shunted into a degradative pathway that can be blocked by ALLN. Taken together, these results indicate that the capacity of HepG2 cells to translocate apoB determines its two major intracellular fates: secretion and degradation. These conclusions are based on the following observations. First, blocking intracellular degradation of apoB by treating HepG2 cells with ALLN caused apoB100 to accumulate intracellularly in microsomes ( Fig. 1and Table 1). Most (77%) of the apoB100 that accumulated in the microsomes following ALLN treatment was susceptible to degradation by exogenous trypsin (Fig. 4), indicating that portions of the protein were exposed on the cytoplasmic surface (i.e. it remained incompletely translocated). A previous study indicates that the apoB100 that accumulates in microsomes from both ALLN-treated and untreated HepG2 cells assumes an orientation in which 69 kDa of the N terminus is intralumenal, with the remaining C-terminal portions residing on the cytoplasmic surface(19) .
The finding that secretion of apoB and albumin is not affected by ALLN supports the view that under the conditions of these experiments this proteolytic inhibitor did not cause a general impairment of protein synthesis, translocation, or secretion. With the proviso that ALLN does not specifically inhibit apoB translocation, we are compelled to conclude that the inability of ALLN treatment to augment apoB100 secretion is because one or more factors that determine the ability of HepG2 cells to translocate apoB is operating at its capacity (both with and without ALLN treatment).
Our results are consistent with those of a previous study showing that ALLN did not affect the secretion of apoB100 but did increase its intracellular accumulation(20) . This study did not examine the translocation status of the accumulated apoB100. Our results showing that essentially all of the apoB100 that accumulates in ALLN-treated HepG2 cells is incompletely translocated extends this earlier work by demonstrating for the first time that translocation, not degradation, determines how much apoB is secreted. The additional finding that ALLN caused several small apoB peptides to accumulate in pulse-labeled cells suggests that at least some of the degradation of apoB occurs before it is completely translated. These data raise the interesting possibility that translocation arrest and degradation may be concerted processes.
For most constitutive secretory proteins, rates of secretion are linked to rates of synthesis(23, 24, 25, 26) . In contrast, under most circumstances the rate of apoB secretion is not linked to its rate of synthesis (reviewed in (3) ). As our studies presented here suggest, increasing the availability of apoB by blocking its rapid degradation with ALLN does not lead to increased secretion. Under the conditions used in our experiments, translocation of apoB cannot adapt to accommodate more apoB for entrance into the secretory pathway. However, there are several conditions in which the secretion (hence translocation) of apoB can be augmented (e.g. nutritional status, reviewed in (15) ). In a previous study, adding oleic acid to the medium of ALLN-treated HepG2 cells increased the secretion of apoB100 to a greater extent than in HepG2 cells not treated with ALLN(20) . These results led to the proposal that oleic acid increased the translocation of apoB100(20) . Moreover, the finding that oleic acid but not ALLN increases the translocation of apoB suggests that lipid, not the amount of apoB, signals an increase in this apparently rate-limiting process.
Two potential recipients for this signal are pause-transfer sequences in apoB (27, 28) and microsomal triglyceride transfer protein (MTP) (29, 30, 31) . The effect of lipid on pause-transfer has been inferred(27, 28) , but its demonstration has not been reported. Lipid has been shown to increase the expression and activity of MTP(32) . MTP is a lipid transfer protein (33) found in the lumen of the endoplasmic reticulum associated with protein disulfide isomerase(29, 30, 31) . Several lines of evidence suggest that MTP is essential for apoB translocation. CHO cells, which do not express MTP, have a complete inability to translocate and secrete intact apoB53(18) . In the absence of proteolytic inhibitors, the intralumenal N terminus of translocation-arrested apoB is cleaved and secreted(19) . However, in the presence of ALLN, apoB53 accumulates as a stable, translocation-arrested, transmembrane protein(19) . Additional studies show that COS (34) and Hela (35) cells, like CHO cells, do not express MTP and cannot secrete intact apoB. Moreover, expression of MTP in COS and Hela cells complements their inability to secrete intact apoB, suggesting that this gene product is essential for apoB secretion. If the basis for the inability of non-MTP-expressing COS and Hela cells to secrete apoB-containing lipoproteins is the same as has been described for CHO cells(18, 19) , it would be likely that MTP either directly or indirectly facilitates apoB translocation. Since the activity and expression of MTP are increased by dietary fat(32) , MTP-facilitated translocation can explain why oleic acid in combination with ALLN but not ALLN alone increases the translocation of apoB100 in HepG2 cells.
While our studies clearly show that the capacity of the translocation step limits the secretion of apoB100 in HepG2 cells cultured under the conditions used in our experiments, there may be situations where other steps in the apoB secretory pathway become limiting. The nutritional and phenotypic state of the cell may have profound influences on one or more steps of the apoB secretory pathway. For example, while in HepG2 cells oleic acid stimulation of lipogenesis augments apoB secretion(8, 10, 20, 36, 37) , there is no effect in primary rat hepatocytes(38, 39) . Additional studies show that oleic acid increases apoB secretion when infused into livers from fasted rats but not when infused into livers from fed rats(40) . A rate-limiting or paused translocation step (27, 28) may act in concert with MTP and the availability of specific lipids to provide the opportunity to efficiently package lipid into lipoproteins for export from the liver and intestine.