Degradation of Newly Synthesized Apolipoprotein B-100 in a Pre-Golgi Compartment*

The synthesis and secretion of apolipoprotein (ape) B-100 have been studied in a human hepatoblastoma cell line, the Hep G2 cells. Pulse-chase analysis showed that apoB-100 was not quantitatively recovered in the culture and Secretion of ApoB-100-In order to examine the effect of exogenous LDL on the synthesis and secretion of apoB-100 in the cells which had never been exposed to BFA, confluent cells were preincubated with serum- free medium in the presence or absence of LDL (80 pg of cholesterol/ ml medium) for 18 h, followed by pulse-chase studies in the absence of LDL. Pulse-chase Studies on the Effect of LDL on Degradation of ApoB- IOO-Confluent cells were preincubated with serum-free medium in the presence or absence of LDL (80 pg of cholesterol/ml medium) for 48 h and the medium replaced by fresh medium every 24 h. Pulse-chase studies were carried out in the presence of BFA as described above. LDL was removed from the medium during the pulse and chase period to prevent interference of cold apoB-100 with the im- munoprecipitation analysis. The cells were preincubated with Met- free medium containing 1 kg/ml BFA followed by pulse-chase studies.

The synthesis and secretion of apolipoprotein (ape) B-100 have been studied in a human hepatoblastoma cell line, the Hep G2 cells. Pulse-chase analysis showed that apoB-100 was not quantitatively recovered in the culture medium.
To reveal the intracellular degradation of apoB-100 prior to secretion, cells were incubated with 1 @g/ml Brefeldin A (BFA) which impeded protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus and the fate of apoB-100 retained in the cells was traced at 37 V. A significant amount of intracellular apoB-100 (40-60%/h) was degraded during the chase period, whereas apoA-1 remained intact. ApoB-100 degradation was temperature dependent, no degradation was observed below 20 "C. This degradation process was not inhibited by chloroquine, leupeptin, pepstatin, and chymostatin, suggesting that iysosomal proteases were not involved and that apoB-100 was degraded in a pre-Golgi compartment which is either part of, or closely related to, the ER. Preincubation of cells with low density lipoproteins (LDL) induced a 22-32% increase in the degradation of apoB-100. This result raised the possibility that secretion of apoB-100 might be regulated through the intracellular degradation of apoB-100. These results suggest the existence of the degradation pathway for apoB-100 in a pre-Golgi compartment and an unique regulatory mechanism for apoB-100 secretion.
Apolipoprotein B-100 is a major protein component of LDL' and very low density lipoprotein of human plasma. It consists of 4536 amino acid residues and has been shown to be essential for the formation and secretion of these lipoproteins in the liver (l-3). It has been established that apoB-100 is synthesized on the rough ER and then transported from the ER, through the Golgi apparatus, to secretory vesicles. Immunochemical studies have indicated that the assembly of apoB-loo-containing lipoproteins occurs at the border between the rough and smooth ER (4). Several studies support the hypothesis that apoB-100 is bound to the rough ER and Golgi apparatus membrane, and this association between apoB-100 and the membrane may be necessary for the addition of lipid components to the nascent lipoprotein particles (5-8). Furthermore, the findings reported by Janero and Lane (9) and Higgins (10)  tein particles occurs within the Golgi region. These results support the view that apoB-loo-containing lipoproteins are assembled by the sequential association of their components in both the ER and the Golgi apparatus.
It is, however, as yet unclear how synthesis, intracellular transit and secretion of apoB-100 are regulated in the liver. Borchardt and Davis (11) have observed in pulse-chase experiments that a significant proportion of de nova synthesized apoB is lost from rat hepatocytes, but not recovered in the culture medium. Davis et al. (12) have recently shown that the degradation products of apoB were detectable in rough and smooth ER and suggested that the site of apoB degradation was ER. We also have been studying the regulation of synthesis and secretion of apoB-100 in Hep G2 cells (13) and found that a proportion of apoB-100 was degraded prior to secretion. In this study we have used Brefeldin A (BFA) in order to further investigate the precise site of intracellular degradation of apoB-100 in Hep G2 cells. BFA strongly inhibits protein transport from the ER to the Golgi apparatus (14)(15)(16)(17). In these studies we examined the fate of apoB-100 retained in the ER. In addition we further investigated the possible roles of lysosomes and LDL in the intracellular degradation of apoB-100.  Basu et al. (18) and Sato et al. (13). In order to avoid proteolytic breakdown during incubation with intiserum, protease inhibitors and EDTA were added at the same concentrations as described above. Briefly, after 2 h incubation at room temperature with antiserum, each tube received a suspension of protein A-Sepharose CL-4B and was rotated at room temperature for 40 min. The immunoprecipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (19), followed by fluorography at -80 "C with Kodak XAR-5 film for 1 or 2 days. At this stage, the relevant protein bands were cut from the dried gel and transferred to glass vials. The gel slices were treated with Protosol (Du Pont-New England Nuclear) prior to radioactivity counting. In some experiments the bands corresponding to the relevant protein were quantified by densitometric scanning.
Preparation of LDL-LDL was isolated from human serum by NaBr density gradient ultracentrifugation (20). The serum was adjusted to density = 1.21 g/ml with solid NaBr. A discontinuous NaBr density gradient was formed by layering the NaBr solutions (density = 1.063, 1.019, and 1.006 g/ml) above the serum. Centrifugation was carried out at 23,000 rpm at 4 "C for 24 h (Hitachi RPS 27). The LDL fractions (density 1.019-1.063 g/ml) were pooled and the density of this fraction adjusted to 1.21 g/ml with NaBr. The second centrifugation was carried out at described above. The LDL fractions (density 1.030-1.063 g/ml) were dialyzed at 4 "C against PBS containing 0.04% EDTA-and sterilized by passage through a 0.45-pm filter (Millinore Corn.). The LDL contained onlv anoB-as checked by SDS-PAGE. Thi ratios of total cholesteroiaid phospholipids to triglyceride content on a weight basis were 4.4:1.7:1.0, respectively.
Pulse-chase Studies on the Effect of Certain Agents on the Degradation of ApoB-100-Confluent cells were preincubated with 1 pg/ml BFA and one of the following agents for 1 h: leupeptin (100 pg/ml), chloroquine (100 PM), pepstatin (100 rg/ml), and chymostatin (100 fig/ml) (21). Pulse-chase studies were carried out as described above in the presence of BFA and one of these agents.
Effect of Certain Agent on Lysosomal Cell Protein Degradation-Cell protein degradation by lysosomes was monitored by the increase in release of acid-soluble radioactivity from radiolabeled cells during withdrawal of serum from medium (22). Cells were labeled with 10 &i of [?S]Met in Met-free medium containing 10% serum for 16 h and chased in 10% serum medium for 3 h. The cells were then incubated with one of the above-listed agents in the presence or absence of 10% serum. After 1 h preincubation, the medium was removed and replaced with the same fresh medium. Aliquots of media were collected at 2 h and the acid-soluble material was obtained by precipitation with a mixture of 5% trichloroacetic acid. Serum was added to the serum-free medium before precipitation. The increase in release of acid-soluble radioactivity during serum deprivation was measured and the percent inhibition of this increase by each agent treatment (relative to no treatment) was calculated.
Pulse-chase Studies on the Effect of LDL on Synthesis and Secretion of ApoB-100-In order to examine the effect of exogenous LDL on the synthesis and secretion of apoB-100 in the cells which had never been exposed to BFA, confluent cells were preincubated with serumfree medium in the presence or absence of LDL (80 pg of cholesterol/ ml medium) for 18 h, followed by pulse-chase studies in the absence of LDL.
Pulse-chase Studies on the Effect of LDL on Degradation of ApoB-IOO-Confluent cells were preincubated with serum-free medium in the presence or absence of LDL (80 pg of cholesterol/ml medium) for 48 h and the medium replaced by fresh medium every 24 h. Pulsechase studies were carried out in the presence of BFA as described above. LDL was removed from the medium during the pulse and chase period to prevent interference of cold apoB-100 with the immunoprecipitation analysis. The cells were preincubated with Metfree medium containing 1 kg/ml BFA followed by pulse-chase studies.
Analytical Methods-The total cholesterol content of LDL was determined by V-cholestase kit (Nissui Seiyaku Co., LTD., Tokyo, Japan).

RESULTS
Pulse-chase Studies on the Synthesis and Secretion of ApoB-100 and A-l-We followed the synthesis and secretion of pulse-labeled apoB-100 in Hep G2 cells (Fig. 1). A small amount of radiolabeled apoB-100 appeared in the culture medium at 30 min into the chase and continued to be secreted at a linear rate up to 120 min. On the other hand, the cellular content of radiolabeled apoB-100 rapidly decreased during the second 30 min (30-60 min into the chase) and continued to fall more gradually to 120 min. Consequently the total radioactivity recovered in the cells and medium decreased during the second 30 min (30-60 min into the chase), but did not decrease thereafter. At 120 min, 65% of the total radiolabeled apoB-100 observed at 30 min into the chase was recovered in the cells and medium. The pattern of synthesis and secretion of apoA-1 was quite different from that of apoB-100. The total radioactivity of apoA-1 (sum of cells and medium) remained virtually constant throughout the chase period ( Fig. 1, right panel).
Effect of BFA on the Processing of cu,-Antitrypsin-Firstly, we examined the inhibitory effect of BFA on the processing of oligosaccharide chains of al-antitrypsin in Hep G2 cells.
Pulse-chase studies with [35S]Met were carried out to confirm that 1 pg/ml BFA was sufficient to inhibit protein transport from the ER to the Golgi apparatus. At 30 min into the chase, two forms of cq-antitrypsin, mature and precursor forms, were detected in the cells which had never been exposed to BFA, whereas only the precursor form, whose oligosaccharide chains had not yet been processed in the Golgi apparatus, was observed in the presence of the drug (Fig. 2). At 1.5 h into the chase, mature cyl-antitrypsin was observed in the medium in the absence of BFA, whereas precursor form was still detected in the cells in the presence of the drug. In addition, preincubation of cells with 1 pg/ml BFA for 1 h had little effect on

Intracellular
Degradation of Apolipoprotein B-100 incorporation of [""S]Met into total cell proteins and blocked the secretion of radiolabeled proteins almost completely (97%) (data not shown).

Intracellular
Degradation of ApoB-loo--As shown in Fig. 1, a significant amount of pulse-labeled apoB-100 was lost from the cells during the chase period. This gives rise to the following three possibilities: (i) apoB-100 is intracellularly degraded prior to secretion into the culture medium; (ii) apoB-100 secreted into the medium is rapidly taken up and degraded by the cells; (iii) after secretion, apoB-100 is degraded in the medium. In preliminary experiments we examined the reuptake of radiolabeled apoB-100 secreted into medium. After cells were pulsed with [3"S]Met for 30 min and chased in serum-free medium for 3 h, the culture medium was administered to fresh Hep G2 cells. Ninety-two and three percent of radiolabeled apoB-100 were recovered in the medium and the cells at 2 h, respectively. These findings suggest that very little radiolabeled apoB-100 was taken up by the cells and degraded in the culture medium. In addition, the total radioactivities of apoB-100 did not decrease after 60 min into the chase (Fig. 2). These results suggest that apoB-100 is degraded intracellularly, probably at an early stage in the secretory pathway for apoB-100. To further investigate the intracellular degradation of apoB-100, cells were incubated with 1 pg/ml BFA and the fate of radiolabeled apoB-100, retained in the ER, was traced. The radioactivity of intracellularly retained apoB-100 by BFA decreased during the chase period, whereas that of apoA-1 remained constant (Fig. 3A). The whole gel after immunoprecipitation of apoB-100 is shown in Fig. 3B 100, which were not immunoprecipitated in the presence of LDL, was observed during the chase period. The protein smaller than intact apoB-100 (arrow), which was immunoprecipitated specifically by apoB-100 antiserum, was detected at 40 and 65 min. It also disappeared during the chase period and might be the proteolytic fragment of apoB-100.

Temperature
Dependence of ApoB-100 Degradation-Temperature affects many events, including proteolytic cleavage by enzymes, membrane transport of proteins and protein structure. To investigate the temperature dependence of apoB-100 degradation, we pulsed cells with [3JS]Met at 37 "C and chased for various times at the different temperatures: 37,20, and 4 "C in the presence of BFA. At 37 "C, the amount of radiolabeled intracellular apoB-100 was reduced to 60% of the value at 4 "C after only 15 min and continued to decrease up to 65 min (Fig. 4). Below 20 "C no degradation was observed, suggesting that apoB-100 is degraded in a temperature-dependent manner.

Effects of Several Protease Inhibitors
on ApoB-100 Degradation-In order to investigate the nature of the protease activities involved in apoB-100 degradation, the effects of agents known to inactivate proteases were examined. Inhibitors of lysosomal function, leupeptin, pepstatin, chymostatin, and chloroquine, had no effect on the degradation of apoB-100 (Table I). On the other hand, these agents inhibited (36.8-64.2%) lysosomal cell protein degradation, which was monitored by the increase in release of acid-soluble radioactivity during serum withdrawal from the medium. These results suggest that lysosomal proteases were not involved in intracellular degradation of apoB-100.

Effect of LDL on Synthesis and Secretion of ApoB-loo--In
order to investigate the regulation of apoB-100 degradation, we examined the effect of exogenous LDL on the synthesis and secretion of apoB-100. We conducted pulse-chase studies after 18 h preincubation in the presence or absence of LDL, in the absence of BFA. Secretion of radiolabeled apoB-100 from the cells which were cultured in the presence of LDL, was reduced throughout the chase period (30-150 min) (Fig.  5B). After 105 min into the chase, the total radioactivities in both cells and medium were lower in the LDL-treated cells as compared with the nontreated cells (Fig. 5, A and B). This  suggests that LDL stimulates the degradation of apoB-100. Under these conditions, cells were fully loaded with LDL, since the incorporation of [Wlacetate into cholesterol was reduced by more than 95% as compared with that in the absence of LDL (data not shown). The synthesis and secretion of apoA-1, however, was not affected by LDL treatment (Fig.  5, C and D).

Effect of LDL on the Intracellular
Degradation of ApoB-100-In order to confirm the effect of exogenous LDL on the intracellular degradation of apoB-100, cells were preincubated in serum-free medium in the presence or absence of LDL for 48 h and pulse-chase studies were conducted in the presence of BFA. There were no significant differences in cell growth and protein synthesis rate between control and LDL-treated cells. LDL induced a statistically significant increase in the intracellular degradation of apoB-100 throughout the chase period, whereas apoA-1 retained in the cells was not degraded either in the presence or absence of LDL (Fig. 6). These results suggest that the increase in intracellular degradation of apoB-100 by LDL treatment results in a decrease in its secretion.

DISCUSSION
It is widely accepted that apoB-loo-containing lipoproteins are assembled by the sequential association of their components during transport from the ER to the Golgi apparatus (4-10). However, the regulation of synthesis and secretion of lipoproteins remains unclear. In the present study, we have shown that a proportion of apoB-100 is degraded in a pre-Golgi compartment during its intracellular transit, and that this degradation may control lipoprotein secretion. Since the total radioactivities recovered in the cells and medium decreased during the second 30 min (30-60 min into the chase) and did not decrease thereafter (Fig. l), this would suggest the occurrence of intracellular degradation of apoB-100. To further investigate whether the degradation of apoB-100 occurs in a pre-or post-Golgi compartment, we examined the fate of apoB-100 retained in the ER by BFA. Under these conditions, a significant amount of apoB-100 retained in the ER was degraded in a temperature-dependent manner and the fate of apoB-100 was different from that of apoA-1. Additionally we confirmed that neither al-antitrypsin ( Fig. 2) nor apoE (data not shown) was degraded intracellularly.
It has been reported that over 50% of apoB-100 in the ER is associated with the membrane, whereas a large portion of apoA-1, albumin and macroglobulin is recovered from the luminal contents (57,s). These results support the possibility that membrane-associated apoB-100 is susceptible to degra- dation in a pre-Golgi compartment. Using inhibitors of lysosomal function we found that lysosomal proteases were not responsible for the degradation of apoB-100, but that this occurs in a pre-Golgi compartment which is either part of, or closely related to, the ER. It is likely that as yet unidentified proteases, which are insensitive to these agents, participate in the degradation of apoB-100 in a pre-Golgi compartment.
Analogous studies have shown that unassembled T cell antigen receptor subunits and inactive acetylcholinesterase, which does not form oligomer in muscle cells, are degraded in a pre-Golgi compartment or the Golgi cisternae, but not in lysosome (23-26). In addition, T cell antigen receptor was also degraded in a temperature-dependent manner (23, 24). These observations suggest that unassembled apoB-100, rather than that assembled in the lipoprotein particles, is also degraded in the same region. Thus, a common nonlysosomal degradation pathway may exist in a pre-Golgi compartment.
The regulation of apoB-100 degradation is of great interest. We selected LDL for these studies, since LDL is known to regulate 3-hydroxy-3-methylglutaryl coenzyme A reductase activity (27) and the expression of LDL receptors (28). Secretion of apoB-100 in the absence of BFA was reduced by LDL treatment, which seems to result from acceleration of intracellular apoB-100 degradation. In separate experiments we found neither mevalonate (5 mM) nor a hydroxymethylglutaryl-CoA reductase inhibitor (CS-514, 500 pg/ml) affected the degradation of apoB-100 retained in Hep G2 cells by BFA (data not shown). These results suggest that the degradation of apoB-100 was enhanced by exogenous LDL, but was not affected by changes in intracellular cholesterol content. Further studies would be necessary to elucidate how LDL stimulates the degradation of apoB-100 and which components in LDL are responsible for the acceleration of the degradation. From another aspect of regulation of apoB-100 secretion, Davis et al. (29) demonstrated that apoB secretion is regulated via degradation and not by changes in mRNA. In addition, several lines of evidence suggest that abetalipoproteinemia, a recessive disease associated with the lack of detectable plasma apoB, is caused by a posttranslational defect in apoB-100 processing or secretion (30).
In this study we found that apoB-100 is degraded in a pre-Golgi compartment, in a temperature-dependent manner, and that this degradation is enhanced by exogenous LDL. Modulation of apoB-100 degradation rate may be a novel mechanism for acute regulation of secretion of apoB-loo-containing lipoproteins.