(Received for publication, July 24, 1995)
From the
The site of apolipoprotein B (apoB) degradation was investigated in cultured rat hepatocytes. Brefeldin A plus nocodazole completely blocked apoB degradation suggesting the involvement of a post-endoplasmic reticulum (ER) compartment. Monensin inhibited apoB degradation by 40% implying that a post-Golgi compartment could be involved in degradation of apoB. Ammonium chloride or chloroquine inhibited partially the degradation of apoB100 and apoB48, indicating some degradation in lysosomes, or in an acidic compartment such as trans-Golgi or endosomes. The degradations of apoB100 and apoB48 were blocked completely by (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (EST) during a chase of 90 min demonstrating that a cysteine protease was responsible for apoB degradation. Chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, and aprotinin had no significant effect on the degradation of apoB48. However, leupeptin and pepstatin decreased the degradation of apoB100 by 20-30%. Degradation of apoB100 and apoB48 occurred in isolated Golgi fractions with little degradation in heavy or light ER. Degradation of apoB in Golgi fractions was inhibited by EST and by preincubating hepatocytes with 10 nM dexamethasone. Immunofluorescent microscopy revealed that apoB accumulated in the Golgi region after EST treatment. It is concluded that a major part of apoB degradation in rat hepatocytes occurs in a post-ER compartment via the action of a cysteine protease that is regulated by glucocorticoids.
Apolipoprotein B (apoB) plays a central role in the assembly,
secretion, and metabolism of triacylglycerol-rich lipoproteins
(chylomicrons and VLDL) ()and LDL (1) . There are
two forms of apoB in mammals: the larger molecular weight form,
apoB100, consists of 4536 amino acids, whereas the smaller form,
apoB48, is the amino-terminal 48% of apoB100. Both apoB100 and apoB48
are products of the same gene. ApoB48 mRNA is produced from apoB100
mRNA mainly in the intestine by RNA editing which involves a cytidine
deaminase(2, 3, 4, 5) . Although
most mammalian livers produce only apoB100, rat liver synthesizes both
apoB100 and apoB48. ApoB is synthesized on polyribosomes bound to the
cytoplasmic surface of the ER and then translocates into ER lumen. ApoB
translocation has been suggested to involve specific multiple
pause-transfer sequences that temporarily arrest the translocation
process(6, 7, 8) . Changes in the lipid
composition in microsomal membranes also diminish apoB translocation
across ER membranes(9) . Several studies suggested that
association of apoB with the full complement of lipids occurs in ER (10, 11, 12, 13) . Some experiments,
however, indicated that the majority of triacylglycerols and
phospholipids are assembled into VLDL particles in the
Golgi(14, 15, 16) .
Pulse-chase studies suggest that a significant proportion of the apoB synthesized de novo in rat hepatocytes is degraded intracellularly(17, 18, 19) . Intracellular degradation of apoB has also been observed in HepG2 cells(20, 21, 22) , and the degradation may be important in the regulation of apoB secretion. Treatment of HepG2 cells with oleate increases apoB secretion by decreasing apoB degradation (22, 23) , whereas n-3 fatty acids have the opposite effects (24) . In primary rat hepatocytes, insulin decreases the secretion of apoB and stimulates its degradation(19) . Conversely, glucocorticoids (dexamethasone) stimulate the secretion (25) and decrease apoB degradation(26) . Some studies demonstrated that intracellular degradation of apoB in HepG2 cells occurred in a pre-Golgi compartment (20, 22, 27) . However, considerable differences exist in the control of apoB metabolism and secretion in HepG2 cells and primary hepatocytes. Despite evidence implicating the existence of intracellular apoB degradation in rat hepatocytes(17, 18, 19, 24, 26) , little is known about the pathway responsible for apoB degradation.
In the present study, we investigated the site(s) of degradation of nascent apoB in cultured rat hepatocytes and have characterized the nature of protease for apoB degradation. We used brefeldin A and monensin, inhibitors of protein transport in the secretory pathway, and nocodazole, which inhibits retrograde transport from Golgi to ER in order to identify the location of nascent apoB degradation in whole cells. A cell-free assay system was developed to assess the degradation of labeled apoB in isolated subcellular fractions. The results demonstrate that a major portion of the intracellular degradation of newly synthesized apoB occurs in a post-ER compartment, possibly the Golgi apparatus. A cysteine protease is responsible for this apoB degradation, and this protease activity is decreased by pretreating hepatocytes with dexamethasone.
Figure 1:
Effect of brefeldin A plus
nocodazole and monensin on apoB degradation. Rat hepatocytes were
incubated overnight with Leibovitz L-15 medium containing 0.2% BSA.
Cells were incubated for 40 min in methionine- and cysteine-free
Dulbecco's modified Eagle's medium and then labeled with
[S]methionine (300 µCi/35-mm dish) for 15
min. The medium was removed and hepatocytes were washed quickly with
medium containing 10 mML-methionine and 3
mML-cysteine (chase medium) twice. Hepatocytes were
incubated with control chase medium (
), chase medium containing 5
µg of brefeldin A/ml plus 20 µg of nocodazole/ml (
), or 10
µM monensin (
) for the time indicated. The cell
and medium apoB were immunoprecipitated and separated by
SDS-polyacrylamide gel electrophoresis. Radioactivity associated with
apoB100 and apoB48 was quantified by liquid scintillation counting. The
relative recoveries of labeled apoB100 (A) and apoB48 (B) from cell and medium are expressed as percentages of the
maximum incorporation of [
S]methionine (3467
± 400 dpm/mg of cell protein for apoB100 and 6652 ± 734
dpm/mg of cell protein for apoB48), which was attained after 10 min in
different experiments. Results are means ± S.D. (where large
enough to be shown) of five independent
experiments.
Brefeldin A and monensin were used to elucidate whether protein transport from ER to Golgi is necessary for apoB degradation in rat hepatocytes. Brefeldin A is a fungal metabolite that causes disassembly of the Golgi apparatus by inhibiting vesicle transport from ER to Golgi, thereby blocking protein secretion(44, 45, 46) . Nocodazole, a microtubule assembling inhibitor(47) , blocks the retrograde transport of vesicles from the Golgi to ER. Therefore, treatment with brefeldin A plus nocodazole prevents mixing of ER and Golgi and maintains secretory proteins in the ER compartment. Monensin is a sodium/potassium/proton ionophore that disrupts the ion gradient in trans-Golgi and retains secretory proteins in this network thereby blocking protein secretion(48, 49) .
Treatment with brefeldin A plus nocodazole or monensin blocked the secretion of apoB and albumin by more than 95%. Treatment with brefeldin A plus nocodazole also inhibited completely the degradation of apoB100 and apoB48 (Fig. 1). This result is compatible with the hypothesis that the transport of apoB from ER to Golgi is necessary for apoB degradation in rat hepatocytes. Treatment with monensin only decreased degradation of apoB100 and apoB48 by about 40% after the 120-min chase. The effect of monensin on apoB100 degradation was more prominent than for apoB48 at earlier chase periods. The result from monensin treatment indicated that some newly synthesized apoB may be degraded in a post-Golgi compartment.
Seven protease
inhibitors were employed to determine the nature of the enzyme
responsible for apoB degradation. Cells were pretreated with protease
inhibitor (except EST) for 1 h before labeling to allow time for
interaction with the cells. The percentage recovery of labeled apoB
from cell and medium was measured at the 2-h chase point compared to
the peak of S incorporation at 10 min into the chase (Table 1). Leupeptin (a serine and cysteine protease inhibitor)
and pepstatin (an aspartic protease inhibitor) inhibited intracellular
degradation of apoB100 by about 20-30%, but has no effect on
apoB48 degradation. No significant inhibition of apoB degradation was
observed with chymostatin (inhibitor of chymotrypsin and papain),
phenylmethylsulfonyl fluoride, and aprotinin (serine protease
inhibitors). The penetrations of leupeptin, chymostatin, and pepstatin
into hepatocytes were confirmed by the inhibition of total cell protein
degradation which was determined by the increase of acid-soluble
radioactivity from cell and medium during the 2-h chase. The percentage
inhibition of total protein degradation was 34 ± 11%, 47
± 11%, and 47 ± 8% (n = 3) with
leupeptin, chymostatin, and pepstatin, respectively.
Previous studies showed that apoB degradation was blocked by ALLN (cysteine protease inhibitor) in HepG2 cells(50) . In our hepatocyte system, we were not able to observe a reproducible inhibition of apoB degradation by ALLN. We used, therefore, an alternative cysteine protease inhibitor, EST, to investigate apoB degradation since this agent is more readily permeable to membranes and therefore better able to enter cell compartments. A preincubation with EST seemed not to be required. EST inhibited the intracellular degradation of both apoB100 and apoB48 by 50% after 2 h (Table 1) and blocked apoB degradation completely during the first 90 min of the chase (Fig. 2). Therefore, a cysteine protease was responsible for most of the degradation of apoB100 and apoB48 in this hepatocytes system. It is possible that the action of EST on the cysteine proteases, cathepsin L and cathepsin B, within lysosomes may account for some of the inhibition of apoB degradation. To investigate this possibility, the labeled apoB that remained in the hepatocytes or was secreted into medium was measured from cells which were treated with ammonium chloride plus EST. The recovery of apoB from cells treated with EST plus 20 mM ammonium chloride was higher than cells treated with either EST, or ammonium chloride alone. The additive effect of EST and ammonium chloride on apoB degradation suggests that EST exerts its effect on apoB degradation mainly in a nonlysosomal compartment.
Figure 2:
Time course effect of EST on apoB
degradation. Hepatocytes were cultured overnight and then pulse-labeled
with [S] methionine (300 µCi/35-mm dish) for
15 min. Hepatocytes were washed with chase medium and incubated in
control chase medium (
) or medium containing 40 µg/ml EST
(
) for the time indicated. The radioactivity associated with cell
and medium apoB100 (A) and apoB48 (B) was quantified
as described in the legend of Fig. 1. The results are means
± S.D. (where large enough to be shown) from five independent
experiments.
Two pools of apoB have been reported in microsomal fractions: membrane-bound apoB and luminal apoB. The membrane-bound pool of apoB was suggested to be subjected to intracellular degradation(18) . Two approaches were employed to measure the distribution of apoB between membrane and lumen in purified fractions. Subcellular fractions prepared from cultured hepatocytes were incubated with trypsin and apoB and albumin were detected by immunoblotting (Fig. 3A). Trypsin treatment decreased apoB100 in ERI and ERII by about 90% in these experiments, but about 40% of the apoB100 remained in the Golgi fraction after proteolysis. By contrast, only a small portion of apoB48 was accessible to trypsin, and, in the case of ERI and ERII, a proteolytic fragment was observed. The amount of albumin was not affected significantly in the fractions by trypsin treatment, nor was the total or the latent activity of mannose 6-phosphate phosphohydrolase. The latter result indicates that the membranes remained intact after trypsin treatment. These results indicated that a large proportion of apoB100 was at the cytosolic side of the endoplasmic reticulum, and a significant amount of apoB100 was exposed on Golgi membranes. ApoB48 was located mainly inside the ER and Golgi, and, therefore, it was not degraded by external trypsin.
Figure 3:
Accessibility of apoB to trypsin
proteolysis and distribution of apoB between membrane and luminal
content in subcellular fractions. ERI, ERII (400 µg of
protein/assay), and Golgi fraction (100 µg of protein/assay) were
prepared from hepatocytes cultured for 4 h. A, the fractions
were incubated with, or without, trypsin on ice for 30 min, after which
trypsin inhibitor was added to a final concentration of 0.4 mg/ml.
Samples were centrifuged to remove trypsin and then subjected to
SDS-polyacrylamide gel electrophoresis and Western blot analysis by
using rabbit antiserum against rat apoB and albumin. B, the
fractions were diluted 50-fold with 100 mM sodium carbonate
(pH 11.5) and incubated on ice for 30 min. Samples were centrifuged at
200,000 g for 1 h. Pellets (lanes 1, 3, and 5) and concentrated luminal contents (lanes 2, 4, and 6) were separated by
SDS-polyacrylamide gel electrophoresis and then subjected to Western
blot analysis. A representative photogragh is shown, and the results
from three independent experiments are described in the
text.
The second approach was to separate the membrane and luminal contents of fractions by treatment with sodium carbonate(35) , and this was reflected by albumin existing only in the luminal contents (Fig. 3B). The percentage of apoB100 in the membrane fraction was 85 ± 8.7%, 78 ± 10%, and 43 ± 6.3% (means ± S.D., n = 3) for ERI, ERII, and Golgi, respectively. In contrast, most of apoB48 was present in luminal content (ERI, 91 ± 4%; ERII, 91 ± 3.5%; Golgi, 91 ± 2.3%, n = 3). The results from both experimental protocols showed that apoB48 existed almost exclusively in the lumen of subcellular fractions. However, a large proportion of apoB100 in the ER was associated with the membranes; also, a significant amount of apoB100 was found with the Golgi membranes.
Degradation of labeled apoB in subcellular fractions, which were
isolated from [S]methionine-labeled hepatocytes,
was studied to determine the intracellular site of this process. The
degradation was measured by a method that was developed for the assay
of cysteine protease activities. Fractions were incubated at 40 °C
for 3 h with 0.13 M sodium potassium phosphate buffer (pH 6.5)
containing 2.67 mM dithiothreitol. Labeled apoB was
immunoprecipitated and separated by SDS-polyacrylamide gel
electrophoresis, and the amount of [
S]apoB was
determined by scanning densitometry (Fig. 4). The percentage
distribution of apoB100 in Golgi, ERII, and ERI fraction was 75
± 5%, 14 ± 3%, and 12 ± 3.2% (n =
4), respectively. The equivalent values for apoB48 were 30 ±
6.3%, 42 ± 7.5%, and 28 ± 7.5%. The percentage
degradation of apoB was calculated as the difference in radioactivity
recovered from incubations at 40 °C and 0 °C. The degradation
of apoB100 in Golgi, ERII, and ERI was 33 ± 5.9%, 6.4 ±
4.8%, and 5.2 ± 1.7%, respectively. For apoB48, the equivalent
values were 26 ± 2.8%, 6.9 ± 5.5%, and 3.5 ± 4.9%.
When the percentage degradation in ER was corrected for the percentage
of cross-contamination with the Golgi marker enzyme (Table 2),
the degradation values for apoB100 and apoB48 was decreased to
approximately 3.8% and 4.9% in the ERII fraction, respectively. The
equivalent values for the ERI fraction were 3.3% and 2.0%,
respectively. These results showed that a large portion of labeled
apoB100 was associated with Golgi fraction; however, the sequence of
distribution for labeled apoB48 was ERII fraction > Golgi fraction
> ERI fraction. Degradation of labeled apoB occurred to a
significant extent only in the Golgi-enriched fraction with little
proteolysis in the ERII or ERI fractions. This result and those with
protein traffic inhibitors demonstrate that the major part of apoB
degradation occurs in a post-ER compartment, which is associated with a
Golgi membrane fraction.
Figure 4:
Degradation of
[S]methionine-labeled apoB in purified
subcellular fractions. Rat hepatocytes were cultured in Leibovitz L-15
medium containing 10% fetal bovine serum for 4 h. Cells were pulsed
with [
S]methionine for 40 min, and subcellular
fractions were prepared as described under ``Experimental
Procedures.'' ERI, ERII, and Golgi fractions were suspended in
0.13 M sodium potassium buffer (pH 6.8) containing 2.7 mM dithiothreitol and 0.25 M sucrose and then incubated on
ice or at 40 °C for 3 h. Radioactivity associated with apoB or
albumin was immunoprecipitated and separated by SDS-polyacrylamide gel
electrophoresis. A, a representative autoradiograph of
degradation of apoB in subcellular fractions. Samples in lanes
1, 3, and 5 were incubated at 0 °C, and
those in lanes 2, 4, and 6 at 40 °C. B, the degradation of apoB100 (black bars) and apoB48 (white bars) at 40 °C relative to that at 0 °C was
determined by scanning densitometry. The results are means ±
S.D. from four independent experiments.
Figure 5: Effects of EST and dexamethasone on degradation of apoB in Golgi fraction. Golgi fractions were prepared from hepatocytes cultured for 4 h or from hepatocytes preincubated with or without 10 nM dexamethasone for 16 h. Golgi fractions from control cells were treated with 40 µg of EST/ml on ice for 30 min and then incubated at 40 °C for 3 h. For the Golgi fraction prepared from dexamethasone-treated hepatocytes, the incubation was performed at 40 °C for 3 h in the absence of EST. The relative degradation of apoB100 (black bars) and apoB48 (white bars) was determined as described in the legend of Fig. 4. Results from control and EST treatments are means ± S.D. from four independent experiments. Results from dexamethasone (Dex) treatment are means ± range from two independent experiments.
Our previous studies demonstrated that incubating the hepatocytes for 16 h with dexamethasone decreased the intracellular degradation of apoB100 and apoB48(26) . Therefore, this treatment ought also to decrease apoB degradation in the Golgi fraction. ApoB degradation in Golgi fractions prepared from hepatocytes cultured for 4 h was similar to that from hepatocytes cultured overnight. ApoB degradation in Golgi fractions prepared from hepatocytes which were pretreated for 16 h with 10 nM dexamethasone was decreased by approximately 44% and 72% for apoB100 and apoB48, respectively. Therefore, the combined results from Fig. 5demonstrated that the degradation of apoB as measured in isolated Golgi fractions has the same characters as the intracellular degradation of apoB in cultured hepatocytes.
Figure 6:
Effect of EST treatment on apoB
distribution by immunofluorescence labeling. Hepatocytes were cultured
overnight and incubated for 2 h with control medium (A and D-F), medium containing 100 µM puromycin (B) or 100 µM puromycin plus 40 µg/ml EST (C). Cells were fixed and incubated with rabbit anti-apoB (A-C), rabbit anti--mannosidase II (D),
mouse anti-Bip (E), or rabbit anti-lgp120 antibodies (F). The secondary antibodies were fluorescein-conjugated
sheep anti-rabbit IgG antibodies or Texas Red-conjugated goat
anti-mouse IgG antibodies.
Figure 7:
ApoB accumulated in a perinuclear region
associated with the Golgi apparatus. Hepatocytes were cultured
overnight and incubated with 100 µM puromycin plus 40
µg/ml EST for 2 h. Cells were fixed and incubated with rabbit
anti-apoB antibodies overnight followed by an incubation of mouse
anti--mannosidase II antibodies (A and D), mouse
anti-Bip antibodies (B and E), or 4E4.A6 antibodies (C and F) for 1 h. The secondary antibodies were
fluorescein-conjugated sheep anti-rabbit IgG antibodies and Texas
Red-conjugated goat anti-mouse IgG antibodies. A-C were
stained for apoB; D-F were stained for Golgi, ER, and
lysosomes, respectively.
The degradation of apoB is an important step in the regulation of its metabolism. Treatment of cells with oleate(22, 23, 27) , n-3 fatty acids(24, 51) , insulin(19) , or dexamethasone (26) changes the secretion of apoB and modulates its intracellular degradation. The intracellular site of apoB degradation in HepG2 cells has been studied widely. The use of protein trafficking inhibitors(20, 27) , subcellular fractionation(27) , or permeabilized cells (52) all suggested that the intracellular degradation of nascent apoB in HepG2 cells occurs mainly in the ER. In rat hepatocytes, however, the intracellular site of apoB degradation remains to be elucidated.
Brefeldin A plus nocodazole completely blocked apoB degradation in the rat hepatocyte system. This suggests that apoB degradation could occur in the post-ER compartment. Treatment with monensin partially inhibits apoB degradation implying that a post-Golgi compartment, or lysosomes, could also be involved in apoB degradation. Lysosomotropic agents (ammonium chloride or chloroquine) also decrease apoB degradation, and this also suggests that lysosomes may be involved in this process. However, the vesicles and cisternae of the trans-Golgi are acidic compartments(53) . Weakly basic amines may therefore disrupt the acidic environment of trans-Golgi and may block the normal functions of this compartment(48, 53, 54) . Thus, it is possible that monensin, or lysosomotropic agents elevate the pH value of the trans-Golgi compartment and thereby exert their inhibitory effects on the degradation of newly synthesized apoB.
The assay of
apoB degradation in isolated subcellular fractions from cultured
hepatocytes demonstrates that the major degradation of labeled apoB
occurs in the Golgi fraction rather than in ERI or ERII fractions. This
degradation can be inhibited by the cysteine protease inhibitor, EST,
and by treating hepatocytes with dexamethasone. The extent of
inhibition by EST and dexamethasone on apoB degradation is similar to
the results obtained from intact hepatocytes ((26) , Table 1, Fig. 2). This suggests strongly that the
degradation of apoB in a post-ER compartment accounts for a large part
of intracellular degradation in cultured rat hepatocytes. Marker enzyme
determinations exclude the possibility that the contamination with
lysosomes accounts for the degradation of apoB in isolated Golgi
fractions. Immunohistochemical studies demonstrate that the
accumulation of apoB in hepatocytes incubated with puromycin and EST
occurs in the Golgi region. However, there is not an exact
colocalization with -mannosidase II which in hepatocytes is found
in Golgi stacks(55) . Therefore, apoB may be accumulating in
specific regions of the Golgi apparatus. ApoB accumulation did not
coincide with that of either of the two lysosomal markers employed ( Fig. 6and Fig. 7). We cannot exclude the possibility that
some apoB is shuttled to lysosome for degradation. However, the
majority of the apoB did not accumulate in lysosomes, indicating that
the latter organelles are not the major sites of apoB degradation. We
also cannot exclude completely that there is also an extremely rapid
(less than 15 min) pathway of degradation for apoB in the ER in
cultured rat hepatocytes.
Two approaches were used to investigate the distribution of apoB between membrane and luminal content in isolated fractions: the accessibility of apoB to exogenous trypsin and the separation of membrane-bound and luminal apoB by sodium carbonate treatment. Both results indicate that a large proportion of apoB100 exists in the membrane-bound pool in ER. This result is consistent with the observations from previous studies(13, 16, 18, 56, 57) . Also, a significant amount of apoB100 is membrane-bound in the Golgi fraction which agrees with the results obtained by radioimmunoassay and enzyme-linked immunoassay(16, 56, 57) . In contrast, most of the apoB48 is in the luminal contents of subcellular fractions.
The difference of the distribution of apoB100 and apoB48 may indicate the different fate of apoB100 and apoB48 during the secretory process: the proportion of apoB100 in the membrane is higher as is the extent of degradation of apoB100 compared to apoB48(17, 19, 24, 26) . Also, the mean diameter of secreted apoB100-containing lipoprotein is distinct from that of apoB48-containing lipoprotein. ApoB100 is associated with VLDL, whereas apoB48 distributes in both high density lipoprotein and VLDL(58, 59) . The membrane-bound pool of apoB has been suggested to be degraded intracellularly(18, 21, 60) . Our results agree with this proposition. The existence of apoB in the membrane-bound pool in the Golgi fraction also implies the possibility that Golgi may play a role in the assembly of apoB-containing lipoproteins (14, 15) or in the remodeling process of VLDL particles(61, 62) .
The conclusions that a post-ER compartment is involved in apoB degradation differ from those obtained with HepG2 cells. Brefeldin A was used by Sato et al.(20) who concluded that the degradation pathway for apoB100 in HepG2 cells was in a pre-Golgi compartment. Results obtained by Furukawa et al.(27) by using protein trafficking inhibitors and subcellular fractionation also indicated that the degradation of nascent apoB occurred in the ER in HepG2 cells. Degradation of apoB100 in the ER fraction could be observed by adding Triton X-100(27) . We were also able to observe degradation of apoB100 and apoB48 in the ERI and ERII fractions of rat hepatocytes when Triton X-100 was added, but this degradation was not inhibited by EST. We therefore concluded that this degradation did not have the characteristics that were seen in intact rat hepatocytes. The protease responsible for apoB degradation in HepG2 cells and in CHO cells expressing apoB was ALLN-sensitive, pH-dependent, and ATP-stimulated(50, 52, 63) . This degradation of apoB resembled that of 3-hydroxy-3-methylglutaryl coenzyme A reductase(64) . However, we found no ATP-stimulated hydrolysis of apoB in isolated Golgi fractions or in permeabilized hepatocytes.
Recently, Davis et al.(10) showed that the proteolytic fragments of apoB were detectable in rough and smooth ER fractions and suggested that the site of apoB degradation was ER. We also observe some small fragments of apoB in the ERI and ERII fractions (Fig. 4), but the intensity of the small fragment of apoB showed no change during the degradation assay. Moreover, the results from Verkade et al.(65) showed that a decrease in the number of VLDL particles in choline-deficient rat was observed in Golgi fraction, not in ER. This result also implies that the Golgi apparatus could be a site of apoB degradation and this is consistent with our findings. This conclusion is inconsistent with the results from HepG2 cells(20, 27, 52) . It is well known that there are several differences in apoB metabolism and secretion in rat hepatocytes versus HepG2 cells. The availability of triacylglycerol for VLDL secretion in HepG2 cells is much lower than that in rat hepatocytes. The low mobilization of intracelluar triacylglycerol may account for the inability of HepG2 cells to recruit sufficient triacylglycerol to assemble apoB-containing lipoproteins that are triacylglycerol-rich(66) , and this could lead to degradation of apoB in the ER. This effect can be blocked by adding unesterified fatty acids to the culture medium which stimulates apoB secretion(22, 23) . This level of regulation is not observed with primary cultures of hepatocytes(24) . Reviews of Dixon and Ginsberg (67) and Sparks and Sparks (68) also suggest that the degradation in rat hepatocytes is different from that in HepG2 cells.
There are precedents for the degradation of proteins in a post-ER and non-lysosomal compartments. This degradation is involved in controlling the amount of the protein that is secreted or destined for the cell surface. Such regulation is observed for acetylcholinesterase in cultured muscle cells(69) . In HeLa cells there is a two-phase degradation of ribophorin I: the first phase is in the ER and the second phase is in a post-ER compartment(70) . Furthermore, the degradation of immunoglobulin M in B lymphocytes occurs via a cysteine protease in a post-ER compartment that is distinct from lysosomes (71, 72
It is concluded that a major part of the regulated degradation of apoB in rat hepatocytes occurs in a post-ER compartment via the activity of a cysteine protease. This activity is decreased by long term treatment with glucocorticoids and is accompanied by increased VLDL secretion (26) .