From the
Translocation of apolipoprotein (apo) B across the endoplasmic
reticulum membrane is a likely site for regulation of secretion of very
low density lipoproteins from the liver. When primary rat hepatocytes
are enriched with the phospholipid phosphatidylmonomethylethanolamine,
the secretion of apoB, but not other proteins such as apoprotein A1 and
albumin, is disrupted (Vance, J. E.(1991) J. Lipid Res. 32,
1971-1982). Moreover, less apoB enters the microsomal lumen and
the intracellular degradation of apoB is increased (Rusiol, A. E.,
Chan, E. Y. W., and Vance, J. E. (1993a) J. Biol. Chem. 268,
25168-25175). In the present study we have used McArdle 7777 rat
hepatoma cells stably transfected with carboxyl-terminal-truncated
variants of human apoB100 and have demonstrated that the reduction in
apoB secretion induced by phosphatidylmonomethylethanolamine is not a
function of assembly of the apoB into a buoyant lipoprotein particle.
In addition, inhibition of the intracellular degradation of the
apoproteins B does not restore apoB secretion, suggesting that the
effect of phosphatidylmonomethylethanolamine enrichment on apoB
degradation is secondary to the effect on translocation of the protein
into the endoplasmic reticulum lumen. Furthermore, supplementation of
the culture medium with oleic acid does not increase apoB secretion,
reduce the intracellular degradation of apoB or reverse the effects of
phosphatidylmonomethylethanolamine enrichment on these processes. Our
data support the hypothesis that translocation of apoB protein across
the endoplasmic reticulum membrane, regardless of the association of
the apoB with neutral lipids, may be a key regulatory step in very low
density lipoprotein secretion.
A major risk factor for the development of atherosclerosis is
the presence of high levels of apolipoprotein (apo)
As a step toward understanding how the secretion
of apoB-containing lipoproteins from the liver is regulated, we have
recently developed a model in which the secretion of apoproteins B100
and B48, but not other secretory proteins such as albumin and apoA1, is
specifically inhibited when the concentration of the phospholipid,
PMME, is increased in rat hepatocyte membranes (Vance, 1991). The
defect in apoB secretion upon enrichment of the cells' membranes
with PMME was shown to be associated with (i) a disruption in
translocation of apoB into the microsomal lumen, (ii) an increased
exposure of apoB on the cytosolic surface of the microsomes, and (iii)
an increased intracellular degradation of apoB (Rusiol et al.,
1993a).
We have now investigated further the mechanism by which PMME
inhibits apoB secretion by dissociation of the processes of
translocation of the protein into the lumen and acquisition of neutral
core lipids by apoB. McArdle rat hepatoma cell lines that had been
stably transfected with carboxyl-truncated variants of human apoB100
(apoB15, apoB18, apoB23, and apoB28, representing the amino-terminal
15, 18, 23, and 28% of human apoB100, respectively) were examined for
the effect of PMME enrichment on secretion of the truncated apoproteins
B. ApoB15, apoB18, and apoB23 are secreted from these hepatoma cells at
a density of >1.23 g/ml, implying that essentially no neutral lipids
(i.e. triacylglycerols and cholesteryl esters) are associated
with the secreted apoB (Yao et al., 1991). ApoB28 is secreted
in the form of a lipoprotein particle of size and density corresponding
to that of high density lipoprotein (density of 1.17 g/ml). ApoB28 is
therefore secreted in association with some neutral ``core''
lipids and presumably also with some surface lipids (phospholipids and
cholesterol) (Yao et al., 1991). In the present study, we have
found that secretion of all four truncated variants of apoB was
inhibited and that the intracellular degradation of the apoproteins B
was increased, when the cells were enriched with PMME. These
observations indicate that the inhibitory effect of PMME enrichment on
apoB secretion is not dependent upon acquisition of the neutral lipid
core, but is related to a defect in translocation of newly synthesized
apoB across the endoplasmic reticulum membrane. In addition, inhibition
of the intracellular degradation of apoB with the cysteine protease
inhibitor, ALLN, did not reverse the inhibition of secretion caused by
PMME, suggesting that increased degradation of the truncated
apoproteins B induced by PMME is secondary to the effect of PMME on
translocation of the proteins across the endoplasmic reticulum
membrane.
We have attempted to distinguish
between these two possibilities by examining the effect of PMME
enrichment of the endoplasmic reticulum membrane on the translocation
and secretion of four carboxyl-truncated variants of human apoB100:
apoB15, apoB18, apoB23, and apoB28. McArdle 7777 hepatoma cells
expressing these apoproteins were cultured for 16 h in the absence or
presence of MME (400 µM). In the MME-treated cells the
amount of PMME was 3.18 ± 0.21 nmol/mg of protein and in cells
not incubated with MME the PMME content was 0.21 ± 0.08 nmol/mg
of protein. The amounts of apoB variants secreted into the culture
media were measured by an enzyme-linked immunosorbent assay
(Fig. 1A) using a polyclonal antibody directed against
human apoB100. As shown in Fig. 1A, the amounts of B15,
B18, B23, and B28 secreted were reduced by 55.8, 42.9, 44.8, and 57.3%,
respectively, as a result of incubation with MME. Since the mass of
apoproteins B in the medium, as shown in Fig. 1A,
represents the truncated apoB species, as well as small amounts of
endogenous, native rat apoproteins B100 and apoB48, samples of the
culture medium isolated after 16-h incubation were examined by
immunoblotting specifically for the amount of truncated apoprotein B
secreted. The data presented in Fig. 1B, in agreement
with the data shown in Fig. 1A, confirm that PMME
enrichment significantly reduced the amount of the truncated
apoproteins B secreted into the culture medium. These data demonstrate
that the inhibitory effect of PMME enrichment previously observed for
the secretion of apoB100 and apoB48 from rat hepatocytes is also
evident for these truncated apoB species in transfected McArdle 7777
cells. The secretion of native apoB100 and apoB48 from the transfected
McArdle cell lines was also inhibited by approximately 50% upon PMME
enrichment of the cells (data not shown). Since secreted apoB15 and
apoB18 are not appreciably associated with a neutral lipid core (Yao
et al., 1991), our observation implies that the effect of PMME
on apoB secretion is not related to binding of apoB to neutral lipid.
Rather, PMME enrichment of the membranes most likely affects the
process of translocation of apoB across the membrane.
We have previously demonstrated that incubation of primary rat
hepatocytes with 400 µM MME overnight did not inhibit the
synthesis of apoproteins B100 or B48 (Rusiol et al., 1993a).
Since the amount of intracellular apoB reflects both synthesis and
removal (by intracellular degradation and secretion), we examined
whether or not the decreased rate of secretion of the truncated apoB
variants in the presence of MME could be explained by a decreased rate
of synthesis. In a continuous labeling experiment McArdle cells
expressing apoB28 were incubated for 16 h in the presence of 0 or 400
µM MME, then 100 µCi/dish of
[
In an attempt to examine a cause and
effect relationship between translocation and degradation of the
truncated apoproteins B, we investigated whether or not inhibition of
apoB degradation would promote the secretion of truncated apoproteins B
and thereby reverse the impact of PMME enrichment on this process. The
cysteine protease inhibitor ALLN (calpain inhibitor I), a tripeptide
which blocks the intracellular degradation of apoB in Chinese hamster
ovary cells (Thrift et al., 1992) and Hep G2 cells (Sakata
et al., 1993), was used as an inhibitor of apoB degradation.
McArdle 7777 cell lines expressing apoB28 were incubated for 1 h in the
presence or absence of ALLN. The cells were subsequently pulse-labeled
for 1 h with [
We conclude from the data presented
in Fig. 4that inhibition of the intracellular degradation of
apoB28 and apoB18 by ALLN did not reverse the defect in secretion
caused by PMME enrichment. The increased degradation of apoproteins B
induced by PMME enrichment was, therefore, secondary to the effect of
PMME on translocation of the protein into the endoplasmic reticulum
lumen.
A prime candidate for regulation of very low density
lipoprotein secretion from the liver is the movement of apoB across the
endoplasmic reticulum membrane into the lumen (Thrift et al.,
1992). We have investigated whether or not the translocation process is
linked to the ability of apoB to associate with neutral lipids during
formation of a floating lipoprotein particle. We have used the rat
hepatoma cell line, McArdle 7777, stably transfected with truncated
apoB variants, some of which do not require assembly with core lipids
(triacylglycerols and cholesteryl esters) for secretion. A direct
linear correlation has been reported previously between the size of the
apoB molecule and the extent of its association with lipids. In cells
expressing various truncated variants of human apoB100, as the length
of the apoB variant increased the density of the secreted
apoB-containing particles decreased and the size of the particles
increased. For example, apoproteins B15, B18, and B23 were isolated
from the bottom of a salt gradient, at a density of 1.23 g/ml, whereas
apoproteins B28, B31, B37, B48, and B53 (Yao et al., 1991), as
well as apoproteins B60, B72, B80, B88, B94, and B100 (McLeod et
al., 1994) exhibited a progressive decrease in density from 1.17
g/ml for apoB28 to 1.02 g/ml for apoB100. These studies implied that
apoB15, apoB18 and apoB23 are secreted essentially devoid of
significant amounts of associated lipid, whereas apoB variants larger
than apoB23 are associated with progressively more lipid as the size of
the apoprotein B increases.
In our studies, when rat hepatoma cells
expressing apoB15, apoB18, apoB23, and apoB28 were enriched in PMME,
the secretion of all four truncated apoproteins B was inhibited by
42-57%, compared with that in the same cells not enriched in
PMME. Secretion of native apoB100 and apoB48 by the transfected McArdle
7777 cell lines was also inhibited by PMME enrichment, as has been
observed previously in primary rat hepatocytes (Vance, 1991).
Therefore, the PMME effect was clearly independent of the assembly of
apoB with significant amounts of neutral lipids or formation of a
floating lipoprotein particle. PMME enrichment of endoplasmic reticulum
membranes specifically disrupted the translocation process per se and appeared to be specific for apoB.
A second significant
observation from the present study was that inhibition of intracellular
apoB degradation did not reverse the inhibitory effect of PMME on
secretion. PMME enrichment of cells expressing apoB18 and apoB28 caused
an increased intracellular degradation of the apoproteins B. In
addition, in PMME-enriched microsomes, apoB28 and apoB18 were more
susceptible to degradation by exogenously added trypsin, than in
control microsomes, suggesting that PMME enrichment impaired the
translocation of these apoproteins. When the protease inhibitor ALLN,
which inhibits apoB degradation, was added, the PMME-induced increase
in degradation was prevented and the apoproteins B accumulated
intracellularly. ALLN did not, however, restore the rate of secretion
of apoB28 or apoB18 to the level of secretion from cells not treated
with MME. These experiments suggest that the increased degradation of
apoB18 and apoB28 was the consequence, not the cause, of the decreased
rate of movement of these proteins through the secretory pathway. Our
observation complements the report of Sakata et al.(1993) that
in Hep G2 cells apoB100 secretion is regulated independently by
proteolysis and by lipid supply. Parallel experiments in primary rat
hepatocytes were not possible, because incubation of these cells with
ALLN, even at concentrations as low as 10 µg/ml, inhibited the
synthesis and secretion of proteins in general and induced cell death.
Oleic acid has been reported to increase the secretion of apoB100
and to decrease its intracellular degradation in the human hepatoma
cell line Hep G2 (Dixon et al., 1991; Sakata et al.,
1993). Oleate presumably increases the secretion of apoB-containing
lipoproteins from these cells because the increased supply of fatty
acids stimulates lipid synthesis. In contrast, in cultured rat
hepatocytes, oleate does not increase apoB secretion,
A novel mechanism for
regulation of apoB translocation across the endoplasmic reticulum
membrane by the process of translocational pausing has been described
by Chuck et al.(1990). The existence of translocational
pausing has, however, been challenged by others (Pease et al.,
1991; Shelness et al., 1994). Although the process is unusual,
translocational pausing has also been observed for the prion protein
and for GRP94, a luminal endoplasmic reticulum protein widely
distributed in eukaryotic cells (Nakahara et al., 1994).
ApoB100 has been proposed to contain throughout its sequence multiple
(perhaps 40) topological domains designated as pause-transfer sequences
which are degenerate sequences that act independently of one another
(Chuck and Lingappa, 1993). Six putative pause-transfer sequences have
been identified in apoB15 (Chuck and Lingappa, 1993). The presence of a
pause-transfer sequence in a protein has been reported to cause the
temporary arrest of the protein within the aqueous, proteinaceous
translocation channel in the membrane during co-translational
translocation; after pausing has occurred, translocation resumes. The
pausing process was inferred, because discrete fragments of apoB15 were
detected in the microsomal lumen from in vitro translation
experiments in which the microsomes were treated post-translationally
with an exogenous protease (Chuck et al., 1990; Chuck and
Lingappa, 1992). When a pause-transfer sequence of apoB was engineered
into a chimeric secretory protein, translocational pausing of the
protein was induced, whereas the same chimera lacking the
pause-transfer sequence did not pause during translocation (Chuck and
Lingappa, 1992). A possible function of the multiple pause-transfer
sequences in apoB might be to allow time for the protein either to
become ``painted'' with (phospho)lipid or to become correctly
folded so that the large hydrophobic protein assumes a stable
conformation and can enter the aqueous lumen. An attractive hypothesis
related to our present study is that PMME enrichment of membranes might
specifically affect translocational pausing so that apoB would remain
paused in the translocation channel and that restarting of
translocation after the pausing might be delayed. As a result, the apoB
would be exposed to the protease, which is present either in the
cytosol or in the endoplasmic reticulum membrane, for an increased
length of time and the apoB would be degraded.
We conclude that the
defect in apoB translocation induced by PMME enrichment is dependent on
neither the length of the apoB variant nor on the acquisition of
neutral core lipids by the apoB. Moreover, the increase in the
intracellular degradation of apoB is the result, rather than the cause,
of the decreased translocation of apoB into the microsomal lumen.
We thank Zemin Yao (University of Alberta) for the
gift of the McArdle 7777 cell lines and Penney Bandura for excellent
technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
B in the circulation (Sniderman et al., 1980). The
plasma concentration of apoB is determined by the balance of its rate
of secretion, primarily from the liver, and its rate of removal from
the blood via low density lipoprotein receptor-mediated endocytosis
(Brown and Goldstein, 1976). ApoB is synthesized in the liver and
intestine and is thought to be assembled with lipids into very low
density lipoprotein particles in the endoplasmic reticulum (Alexander
et al., 1976; Borchardt and Davis, 1987; Boren et
al., 1992; Rusiol et al., 1993b). ApoB secretion from the
liver is apparently not normally regulated by alteration in the rate of
apoB synthesis or in the level of apo-mRNA. Instead, apoB is
synthesized constitutively and in excess of its needs for lipoprotein
secretion (Pullinger et al., 1989; Dashti et al.,
1989; Sorci-Thomas et al., 1989). Superfluous apoB that is not
assembled into lipoprotein particles is degraded intracellularly, most
likely in the endoplasmic reticulum (Davis et al., 1990;
Furukawa et al., 1992; Sato et al., 1990). Two
possible scenarios might link the degradation and secretion of apoB. In
the first, the intracellular degradation of apoB might limit the
secretion of apoB. In the second, another factor, such as translocation
of apoB across the endoplasmic reticulum membrane, or the association
of apoB with lipids, might control how much apoB is secreted.
Consequently, any apoB that is not secreted would be degraded. In the
latter case, translocation of newly translated apoB across the
endoplasmic reticulum membrane and into the lumen would be a key
regulatory process.
Materials
Stably transfected McArdle 7777 rat
hepatoma cells expressing apoB15, apoB18,(
)
apoB23, and apoB28 were generous gifts from Dr. Zemin Yao,
University of Alberta. [
S]Methionine and Amplify
were purchased from Amersham, Canada, and Tran
S-label was
obtained from ICN, Irvine, CA. Polyvinylidene difluoride membranes were
from Millipore. Antibodies directed against rat apoB100 and rat apoA1
were prepared and characterized in our laboratory by standard
procedures (Rusiol et al., 1993a). The antibody directed
against human apoB was purchased from Boehringer Mannheim, as was the
calpain inhibitor I, ALLN. Monomethylethanolamine and water-soluble
oleic acid (encapsulated in
-cyclodextrin) were from Sigma. The
reagents used for electrophoresis were supplied by Bio-Rad. All other
chemicals and solvents were purchased from either Sigma or Fisher.
Culture of Hepatoma Cells
Transfected McArdle 7777
cells were plated on 60-mm culture dishes (Falcon) at a concentration
of approximately 3 10
cells/dish. The cells were
cultured in Dulbecco's modified Eagle's medium, DMEM,
containing 10% fetal bovine serum (Life Technologies, Inc.) and 10%
heat-inactivated horse serum (Life Technologies, Inc.). The cells were
grown at 37 °C in an atmosphere of 5% CO
. When growth
reached 70% confluence, the cells were incubated overnight (16 h) in
the presence of 400 or 0 mM MME in DMEM and used the next
morning. The culture media were harvested and centrifuged at 10,000
g for 15 min to remove cell debris. Cells were scraped
from the dishes and pelleted by centrifugation for 5 min at 1,000
g.
Pulse-Chase Experiments
The monolayer of cells was
preincubated for 1 h in methionine-free DMEM, then incubated with 1 ml
of the same medium containing 200 µCi/dish of
TranS-label for 1 h. Radioactivity was chased for defined
periods of time, up to 4 h, in DMEM containing 400 µM
unlabeled methionine and various additives, as defined for each
experiment. ApoB products were immunoprecipitated from cells and
culture medium with polyclonal antibody directed against rat apoB using
a published method (Borchardt and Davis, 1987; Rusiol et al.,
1993b). After immunoprecipitation the proteins were separated by
electrophoresis on a 10% polyacrylamide gel containing 0.1% sodium
dodecyl sulfate. The gel was stained and subsequently impregnated with
Amplify for 15 min and dried. Autoradiography of the dried gel was
performed at -80 °C for 16-32 h. The apoB-containing
bands were excised from the gel, the gel was digested with 20% hydrogen
peroxide (Rusiol et al., 1993b), and radioactive incorporation
into apoB was measured.
Studies on the Rate of ApoB Synthesis
McArdle 7777
cells expressing apoproteins B15, B18, and B28 were incubated in the
presence of 0 or 400 µM MME for 16 h. Newly synthesized
proteins were subsequently radiolabeled by addition of 100 µCi/dish
of [S]methionine. After time intervals of from
0.5 to 6 h, apoproteins B were immunoprecipitated from the cells with a
polyclonal antibody directed against human apoB100; the proteins were
electrophoresed on a 10% polyacrylamide gel containing 0.1% sodium
dodecyl sulfate, and the apoB-containing bands were excised from the
gels for measurement of radioactivity. In an alternative experimental
protocol, apoB18-expressing cells that had been incubated in the
presence or absence of MME were labeled with
[
S]methionine for 5-20 min, and
radioactivity in apoB18 was measured after immunoprecipitation and
polyacrylamide gel electrophoresis.
Measurement of the Mass of ApoB Variants and ApoA1 in
Culture Medium
The amounts of apoproteins B and A1 secreted into
the medium were determined by enzyme-linked immunosorbent assays using
polyclonal antibodies directed against human apoB and rat apoA1,
respectively, as described previously (Rusiol et al., 1993b).
The mass of apoproteins B secreted into the medium was also examined by
immunoblotting of aliquots of culture medium as described previously
using a polyclonal antibody directed against human apoB100 (Rusiol
et al., 1993b).
Proteolysis of Microsomal ApoB28, ApoB18, and ApoA1 with
Exogenously Added Trypsin
McArdle 7777 cells expressing apoB28
or apoB18 were incubated for 16 h with or without MME (400
µM). Microsomes were isolated as described previously
(Rusiol et al., 1993a) and 80 µg of protein was treated
with 0.1 volume of trypsin (0.5 mg/ml) for 30 min at 0 °C.
Proteolysis was terminated by addition of 0.1 volume of soybean trypsin
inhibitor (4 mg/ml), and microsomes were re-isolated by centrifugation
for 15 min at 400,000 g in a Beckman TL 100.3 rotor.
The membrane pellet was boiled for 5 min in a solution containing 8
M urea and 2% sodium dodecyl sulfate. Aliquots of the extract
were subjected to electrophoresis on an 8.5% polyacrylamide gel
containing 0.1% sodium dodecyl sulfate. Proteins were analyzed by
immunoblotting using a polyclonal antibody raised against human apoB100
(Boehringer Mannheim) and a polyclonal antibody specific for rat apoA1,
raised in our laboratory. The amounts of apoB and apoA1 were estimated
by densitometric scanning of the immunoblots.
Other Methods
The protein content of the cells was
measured by the method of Lowry et al.(1951).
MME Inhibits the Secretion of Truncated ApoB
Variants
We have previously shown that enrichment of microsomal
membranes of primary rat hepatocytes with PMME inhibited the secretion
of apoproteins B100 and B48 as a result of a defective movement of the
apoproteins B into the endoplasmic reticulum lumen (Vance, 1991; Rusiol
et al., 1993a). These studies did not, however, distinguish
between whether the defect was due to a problem in translocation of the
apoB protein across the membrane or whether the defect was the result
of the inability of the protein to associate with lipid and assemble
into a lipoprotein particle.
Figure 1:
Secretion of truncated apoB variants,
but not apoA1, from McArdle 7777 cells is inhibited by enrichment of
membranes in PMME. Stably transfected McArdle 7777 cells expressing the
amino-terminal 15, 18, 23, and 28% of human apoB100 (i.e. apoB15, apoB18, apoB23, and apoB28, respectively) were cultured
for 16 h in medium containing no or 400 µM MME. The amount
of apoB variants and native apoA1 secreted into the culture medium was
determined by enzyme-linked immunosorbent assays (A) and
immunoblotting (B). The data in A represent averages
± S.D. of six individual experiments. Open bars represent cells incubated without MME, solid bars represent cells incubated with 400 µM MME. For the
immunoblotting experiment (B) the cells were incubated for 16
h, and medium equivalent to the following amounts of cell protein was
applied to the 3-15% polyacrylamide gel: apoB15, 75 µg;
apoB18, 30 µg; apoB23, 47 µg; apoB28, 20 µg. The locations
of endogenous apoproteins B100 and B48 on the gel are indicated by the
notations at the left-hand side.
The defect in
secretion induced by PMME appeared to be specific for apoB. In primary
rat hepatocytes PMME enrichment did not impair the
translocation/secretion of other secretory proteins, such as albumin
and apoAI (Vance, 1991). Furthermore, in the McArdle cells expressing
the truncated apoproteins B we measured the secretion of apoA1 into the
culture medium by an enzyme-linked immunosorbent assay. PMME enrichment
of these cells did not inhibit the secretion of apoA1
(Fig. 1A).
Intracellular Degradation of Truncated ApoB Variants Is
Increased upon MME Treatment
ApoB is constitutively synthesized
by hepatocytes (Pullinger et al., 1989) in excess of the
requirement for lipoprotein secretion. ApoB that is not assembled into
lipoprotein particles for secretion is apparently degraded
intracellularly, most likely in the endoplasmic reticulum (Davis et
al., 1990; Sato et al., 1990; Dixon et al.,
1991; Furukawa et al., 1992). The intracellular degradation of
apoB15, apoB18, and apoB28 was examined in transfected McArdle 7777
hepatoma cells. Cells were pulse-labeled with
[S]methionine for 1 h, after which the
radioactive medium was removed, and the incubation was continued for a
chase period of up to 4 h. ApoB proteins were immunoprecipitated from
cells and culture medium, separated by polyacrylamide gel
electrophoresis and the apoB-containing bands were excised from the gel
for measurement of radioactivity (Fig. 2). In cells incubated
without MME, radioactivity in all three cellular apoproteins B decayed
as a function of time with a concomitant increase in radioactivity in
secreted apoB. Almost all of the decrease in radioactivity in the
intracellular apoproteins B (94.5% for apoB15, 80.9% for apoB18, and
84.5% for apoB28) was recovered in the secreted apoproteins B.
Therefore, only 5-19% of the newly synthesized, truncated
apoproteins B was degraded intracellularly, in agreement with the
report of Yao et al. (1991). The experiment was repeated with
cells that had been preincubated with MME for 16 h. When these data
were compared with those from cells incubated in the absence of MME, a
smaller percentage of radioactive apoB present in the cells at the end
of the 1-h pulse was secreted from cells treated with MME
(Fig. 2). Of the radioactivity present in apoB15, apoB18, and
apoB28 at the end of the 1-h pulse, 40.3, 50.1, and 55.0%,
respectively, had been degraded intracellularly by the end of the 4-h
chase period. These data demonstrate that the intracellular degradation
of the apoB variants was increased upon enrichment of cells with PMME
and that the efficiency of secretion was decreased.
Figure 2:
Intracellular degradation of truncated
apoB variants is increased upon MME treatment. McArdle 7777 cells
harboring truncated apoB constructs (apoB15, apoB18, and apoB28) were
cultured for 16 h in the presence of 400 µM (closed
symbols) or 0 µM (open symbols) MME.
Methionine-free medium without or with 400 µM MME, and
containing 200 µCi of [S]methionine/60-mm
dish, was added for 1 h. The medium was removed, and cells were
incubated for the indicated times with medium containing unlabeled
methionine. Apoproteins B were immunoprecipitated from cells
(circles) and media (triangles) and separated by
polyacrylamide gel electrophoresis on 10% gradient gels containing 0.1%
sodium dodecyl sulfate. Data are plotted as radioactivity in apoB as a
percentage of total radioactivity in cellular apoB at the end of the
1-h pulse period (T
). Radioactivity in cellular
apoB15, apoB18, apoB23, and apoB28 at the end of the pulse of cells
incubated without MME was: 16,328, 17,532, and 20,023 dpm/dish,
respectively, and in cells incubated with MME was 17,415, 18,160, and
21,394 dpm/dish, respectively. Similar results were obtained when the
experiment was repeated.
As confirmation
that the truncated apoproteins B were not being degraded in the culture
medium, apoB28-expressing cells that had been enriched in PMME were
incubated for 16 h with [S]methionine. One-half
of the medium was used directly for immunoprecipitation of apoB28. The
other half of the medium was added to unlabeled cells, then re-isolated
after 4 h incubation. ApoB was immunoprecipitated and separated by
polyacrylamide gel electrophoresis. The recovery of radioactivity in
apoB28 from medium incubated with cells for 4 h was 89.3%, indicating
that little degradation of apoB had occurred in the culture medium.
S]methionine was added. After time intervals of
30 min to 6 h apoB28 was immunoprecipitated from the cells, the
proteins were separated by polyacrylamide gel electrophoresis, and the
apoB28-containing band was excised for measurement of radioactivity.
The labeling curves for apoB28 were essentially identical for cells
incubated in the presence and absence of MME (data not shown). We also
examined the synthesis of apoB18 by labeling the cells with
[
S]methionine for 5-20 min. At all times,
the radioactivity in apoB18 in MME-treated cells was approximately 20%
higher than in control cells (data not shown). We conclude, therefore,
that the synthesis of the truncated apoBs is not inhibited by MME
treatment. Rather, for apoB18, the synthetic rate appears to be
slightly enhanced in MME-treated cells compared with untreated cells.
Sensitivity of ApoB18 and ApoB28 to Exogenously Added
Trypsin Is Increased in PMME-enriched Microsomes
In primary rat
hepatocytes (Thrift et al., 1992) and in Hep G2 human hepatoma
cells (Dixon et al., 1991) a pool of microsomal apoB is
apparently exposed to the cytosolic surface and is consequently
susceptible to degradation upon treatment of intact microsomes with
exogenously added proteases. In primary rat hepatocytes treated with
MME, microsomal apoB100 and apoB48 are more susceptible to degradation
by exogenous trypsin than is apoB in microsomes from hepatocytes not
treated with MME (Rusiol et al., 1993a). These data indicated
that PMME enrichment of microsomal membranes impaired the translocation
of apoB into the microsomal lumen so that a smaller fraction of the
apoB was protected from degradation by the exogenously added protease.
Microsomes were isolated from apoB28- and apoB18-expressing McArdle
7777 cells that had been incubated for 16 h with or without MME. The
sensitivity of apoB28 and apoB18 in these microsomes to digestion by
exogenously added trypsin was examined. The results presented in
Fig. 3
revealed that in cells not treated with MME essentially all
of the microsomal apoB28 (83.6 ± 2.9%) and apoB18 (99.0 ±
3.6%) was protected from proteolysis. In contrast, in PMME-enriched
microsomes the majority of microsomal apoB28 (89.4 ± 1.6%), and
39.4 ± 9.1% of apoB18 (according to densitometric scanning of
the immunoblots), was degraded by trypsin, apparently as a result of
being exposed on the cytosolic surface of the microsomes. The
resistance of the microsomal apoproteins B to trypsin digestion was not
due to an inherent inability of trypsin to degrade the proteins,
because when the microsomes were disrupted with the detergent Triton
X-100, all the microsomal apoB28 and apoB18 was proteolyzed
(Fig. 3). As a note of caution when interpreting these results,
some investigators have questioned whether or not the susceptibility of
apoB to exogenous protease is indeed due to exposure of the protein on
the cytoplasmic side of the membrane (Pease et al., 1991;
Shelness et al., 1994). However, the accessibility of apoB,
but not apoA1, to exogenous protease is clearly different in
PMME-enriched cells compared with that in control cells. We did not
examine the protease sensitivity of apoB15 in transfected McArdle
cells. However, in other studies we have shown that protection of
in vitro translated apoB15 from protease is decreased upon
PMME enrichment of microsomes.(
)
Figure 3:
Protease digestion of apoB28 and apoB18,
but not ApoA1, is increased in PMME-enriched microsomes. Microsomes
were prepared from apoB28- and apoB18-expressing McArdle 7777 cells
that had been incubated for 16 h in the presence or absence of MME (400
µM). Aliquots of microsomal protein were incubated with
trypsin, and proteolysis was terminated by addition of trypsin
inhibitor. Microsomes were re-isolated by centrifugation, and
microsomal proteins (20 µg/lane) were subjected to polyacrylamide
gel electrophoresis, then transferred to a polyvinylidene difluoride
membrane. ApoB28 (A) and apoB18 (B) on the membranes
were probed with anti-human apoB polyclonal antibody. ApoA1 was
detected with a polyclonal antibody directed against rat apoA1. In some
samples, as indicated, trypsin digestion was performed in the presence
of 0.5% Triton X-100. The amounts of apoproteins B and A1 were
estimated by densitometric scanning of the immunoblots from three
independent experiments for apoB18 and apoB28 and from six independent
experiments for apoA1. Data given in the text are averages ±
S.D.
The secretion
of endogenous, native apoA1 from the McArdle cells was not affected by
PMME enrichment (Fig. 1A). Moreover, microsomal apoA1 in
the transfected McArdle cells was almost completely protected from
exogenous trypsin in both control and PMME-enriched microsomes (by 94.3
± 7.5% and 87.3 ± 2.3%, respectively) (Fig. 3,
A and B). These observations indicate that the
increased degradation of apoB18 and apoB28 observed in PMME-enriched
microsomes was not the result of ``leaky'' microsomes, but
was due to an increased exposure of the apoproteins B on the cytosolic
surface of the microsomes.
Inhibition of Intracellular Degradation of ApoB18 and
ApoB28 Does Not Reverse the Inhibitory Effect of MME on
Secretion
Two alternative explanations might account for the
increased degradation and decreased secretion of apoproteins B in
PMME-enriched cells. First, the increased content of PMME in the
endoplasmic reticulum membrane might have stimulated apoB degradation
making less apoB available for secretion. In this case, the degradation
of apoB would have limited its secretion. Alternatively, enrichment of
PMME in the membranes might have impaired the translocation process
per se, resulting in less apoB being secreted. As a
consequence of the blocked translocation process, apoB would reside in
the translocation channel, or become associated with the membrane, for
an increased length of time, and the susceptibility of apoB to
degradation would be increased.
S]methionine in the presence or
absence of ALLN, then chase medium containing 40 µg/ml of ALLN was
added. After 1 h, the medium was replaced with fresh medium containing
ALLN (40 µg/ml), and the incubation was continued for an additional
3 h. ApoB28 was immunoprecipitated from cells and culture medium,
separated by polyacrylamide gel electrophoresis, and excised from the
gel for measurement of radioactivity. In cells not exposed to MME, of
the radioactivity present in intracellular apoB28 at the end of the 1-h
pulse period, approximately 20% had been degraded intracellularly by
the end of the 4-h chase period (i.e. the total radioactivity
in cells plus medium decreased by approximately 20%)
(Fig. 4B), in agreement with the data shown in
Fig. 2
. ALLN did not significantly influence either the rate of
secretion of apoB28 into the medium or the rate of decay of
radiolabeled intracellular apoB28. Similar results were obtained for
experiments with cells expressing apoB18 (data not shown).
Figure 4:
ALLN
reduces the intracellular degradation of apoB28 in MME-treated cells
but does not reverse the effect of MME on secretion. McArdle 7777 cells
expressing apoB28 were cultured for 16 h in medium without MME
(B) or with 400 µM MME (A), after which
the cells were preincubated for 1 h in the same media that contained
(solid symbols) or lacked (open symbols) ALLN (40
µg/ml). The proteins were pulse-labeled for 1 h with
TranS-label (200 µCi/dish) in the same media, and
radioactivity was chased in media that contained unlabeled methionine.
After 1 h, the ALLN-containing medium was replenished with fresh ALLN.
At the indicated times apoB28 was immunoprecipitated from the cells
(circles) and media (triangles), and radioactivity in
apoB28 was determined after separation of the proteins by
polyacrylamide gel electrophoresis. Data are plotted as radioactivity
in apoB as a percentage of total radioactivity in cellular apoB at the
end of the 1-h pulse period (T
). Radioactivity in
apoB28 at the end of the pulse for MME-treated cells was 16,909
dpm/dish for cells incubated without ALLN and 16,234 dpm/dish for cells
incubated with ALLN. For cells not treated with MME, radioactivity at
the end of the pulse was 17,320 dpm/dish and 16,390 dpm/dish for cells
incubated with and without ALLN, respectively. Similar results were
obtained when the experiment was repeated.
In
parallel experiments, hepatoma cells expressing apoB28 were incubated
for 16 h in the presence of MME before the beginning of the pulse-chase
experiment described above with ALLN. In agreement with the data shown
in Fig. 2, PMME enrichment increased the intracellular
degradation of apoB28 (Fig. 4A compared with
Fig. 4B). By the end of the 4-h chase period the total
radioactivity in apoB28 in cells plus medium had declined by
approximately 60%. However, ALLN inhibited the majority of the
intracellular degradation of apoB28 for the first 2 h
(Fig. 4A). The increased rate of degradation of apoB28
that occurred after 2 h of the chase period may have been the result of
ALLN losing some of its potency. Importantly, in spite of apoB28 being
protected from degradation, the secretion of apoB28 increased only
marginally, if at all, in the presence of ALLN
(Fig. 4A), and did not begin to approach the level of
secretion from the cells incubated without MME (Fig. 4A compared with Fig. 4B). For example, in cells
incubated in the absence of MME, after 4 h of the chase period,
approximately 50% of radioactive cellular apoB28 present at the end of
the pulse had been secreted. In contrast, in MME-treated cells, only
20% of the radiolabeled apoB28 present at the end of the pulse had been
secreted, either in the presence or absence of ALLN. Similar results
were obtained for apoB18. The intracellular location of the accumulated
apoproteins B in ALLN-treated cells is not known but is presently being
investigated in our laboratory.
Oleic Acid Treatment Does Not Reverse the Effects of MME
on ApoB28 Secretion or Degradation
Incubation of Hep G2 human
hepatoma cells with oleic acid increases the secretion of apoB100 and
causes a reciprocal reduction in the rate of intracellular apoB100
degradation (Furukawa et al., 1992). Addition of oleate to
McArdle 7777 cells transfected with several truncated apoBs (including
apoB13, apoB17, apoB23, apoB29, apoB32, and apoB36) promoted the
secretion, and reduced the intracellular degradation, of only those
apoBs large enough to produce buoyant lipoproteins (i.e. apoB29, apoB32, and apoB36) (White et al., 1992). The
mechanisms responsible for these effects are not completely understood,
but the stimulation of secretion by oleate in Hep G2 cells can be
clearly dissociated from the inhibitory effect of oleate on degradation
(Sakata et al., 1993). As a means of gaining further
information on the mechanism which apoB secretion is inhibited by PMME
enrichment, we investigated whether or not oleate would reverse the
effect of MME on apoB28 secretion. McArdle 7777 cells expressing apoB28
were incubated overnight in the presence or absence of MME, after which
the cells were pulse-labeled for 1 h with TranS-label, and
radioactivity was chased in the presence or absence of 0.8 mM
oleate. The results shown in Fig. 5B for cells incubated
without MME demonstrate that oleate did not significantly enhance the
secretion of apoB28 or significantly reduce the degree of its
intracellular degradation. In agreement with the data presented in
Fig. 1
, 2, and 4, however, MME inhibited the secretion of apoB28
(Fig. 5). The addition of oleate to both MME-treated, and
untreated, cells neither stimulated the secretion, nor inhibited the
degradation, of apoB28. Therefore, oleate did not reverse the effect of
PMME on either secretion or degradation of apoB28.
Figure 5:
Oleic acid neither increases apoB28
secretion nor reverses the defect in secretion induced by MME
treatment. McArdle 7777 cells expressing apoB28 were incubated for 16 h
in the presence (A) or absence (B) of MME. The cells
were pulse-labeled for 1 h with TranS-label (200
µCi/dish), then radioactivity was chased in the presence
(closed symbols) or absence (open symbols) of oleic
acid (0.8 M) for the indicated times. ApoB28 was
immunoprecipitated from cells (circles) and media
(triangles), and radioactivity was measured after apoB28 had
been separated by polyacrylamide gel electrophoresis. Data are
presented as radioactivity in apoB28 as a percentage of total
radioactivity (T
) in cellular apoB28 at the end of
the 1-h pulse period. Radioactivity in cellular apoB28 at the end of
the pulse of MME-treated cells was 19,300 dpm/dish and 18,343 dpm/dish
for cells incubated with and without oleate, respectively. For cells
not treated with MME, radioactivity in apoB28 at the end of the pulse
was 20,110 dpm/dish and 19,433 dpm/dish, for cells incubated with and
without oleate, respectively. Similar results were obtained when the
experiment was repeated.
The effect of
oleate and MME on the secretion of endogenous rat apoB48 by the McArdle
7777 cells was also examined. In contrast to the effect of oleate on
the secretion of truncated apoproteins B, but in agreement with the
effect of oleate on apoB100 secretion from Hep G2 cells (Furukawa
et al., 1992), the addition of oleate to the McArdle cells
(both untreated and MME-treated) did increase the secretion of native
apoB48 by approximately 2-fold (Fig. 6, A and
B). However, the amount of radiolabeled apoB48 secreted from
the MME-treated cells was only approximately 50% of that from untreated
cells (Fig. 6, A compared with B).
Figure 6:
Oleic acid increases the secretion of
native apoB48 from McArdle 7777 cells. McArdle cells expressing apoB28
were incubated for 16 h with 400 µM MME (A) or
without MME (B). The cells were pulse-labeled with
TranS-label for 1 h, then radioactivity was chased in the
presence (closed symbols) or absence (open symbols)
of 0.8 M oleic acid. ApoB was immunoprecipitated from cells
(circles) and media (triangles), the proteins were
separated by polyacrylamide gel electrophoresis and radioactivity in
apoB48 was measured. Data are presented as radioactivity in apoB48 as a
percentage of total radioactivity (T
) present in
cellular apoB48 at the end of the pulse period. Radioactivity in
cellular apoB48 at the end of the pulse for MME-treated cells was 8,302
dpm/dish and 8,520 dpm/dish in cells incubated with and without oleate,
respectively. For cells not treated with MME, radioactivity in apoB48
at the end of the pulse was 7,532 dpm/dish in cells incubated without
oleate and 8,007 dpm/dish for cells incubated with oleate. Similar
results were obtained when the experiment was
repeated.
(
)
even though the synthesis of phospholipids and
triacylglycerols is increased (Pelech et al., 1983). In the
present study, we examined whether or not oleate was capable of
restoring the level of secretion of the truncated apoproteins B from
McArdle cells enriched in PMME to the levels of secretion found for
cells not enriched in PMME. Oleate did not stimulate the secretion of
apoB28 from either control or PMME-enriched McArdle 7777 cells, nor did
oleate reverse the inhibition of secretion of apoB28 or its accelerated
degradation, caused by the increased cellular content of PMME. The
secretion of native rat apoB100 and apoB48 from cell lines expressing
the truncated apoproteins B was, however, stimulated approximately
2-fold by incubation of the cells with oleate. These data might reflect
the finding that, unlike apoB100 and apoB48, the truncated apoproteins
B used in our study do not require the association with large amounts
of neutral lipid for secretion (Yao et al., 1991).
Interestingly, we have recently shown that in vitro translated
apoB15 isolated from the lumen of rat liver microsomes is associated
with some phosphatidylcholine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.