Hepatitis C virus nonstructural proteins inhibit apolipoprotein B100 secretion

Host genes involved in lipid metabolism are differentially regulated during the early stages of hepatitis C virus (HCV) infection. The majority of lipids synthesized in the liver are exported to other tissues in the form of lipoproteins. The formation of these lipoproteins is dependent upon the association of triglycerides with apolipoprotein B100. Using the HCV subgenomic replicon expression system, we show that secretion of apoB100 is significantly reduced. Inhibition of apoB100 degradation by ALLN did not improve secretion. Triglyceride levels as well as microsomal triglyceride transfer protein mRNA and activity levels were reduced in replicon-expressing cells, indicating potential reasons for the observed decrease. Further evidence is presented for the interaction between the HCV nonstructural protein 5A and apoB100. These results provide further insight into the alteration of lipid metabolism by HCV.     

Hyperexpression of N-acetylglucosaminyltransferase-III in liver tissues of transgenic mice causes fatty body and obesity through severe accumulation of Apo A-I and Apo B

N-Acetylglucosaminyltransferase (GnT)-III catalyzes the attachment of an N-acetylglucosamine (GlcNAc) residue to mannose in beta(1-4) configuration in the region of N-glycans and forms a bisecting GlcNAc. To investigate the pathophysiological role of dysregulated glycosylation mediated by aberrantly expressed GnT-III, we generated transgenic mice hyperexpressing the human GnT-III in the liver by introducing human GnT-III cDNA under the control of mouse albumin enhancer/promoter. Total five transgenic founder mice (pGnTSVTpA-10, -14, -20, -25, and -51) expressed the human GnT-III in their livers and were characterized by molecular genetic means. The copy number of transgene integrated into the genome of these mice ranged between 1 and 3 copies per haploid genome. Northern and Western blot analyses showed that the transgene is specifically expressed in the liver but not in any other tissues tested. The triglyceride level in GnT-III transgenic mice was significantly decreased, however, no significant differences in the levels of glucose, cholesterol, or albumin were observed between transgenic and nontransgenic mice. Although glutamate oxaloacetic transaminase and glutamic pyruvic transaminase activities of transgenic mice were also higher than those of nontransgenic mice, no differences in total bililubin and total protein were observed between the two animal lines. Large amounts of apolipoprotein (Apo) A-I and Apo B were specifically detected in the intracellular liver of transgenic mice. The accumulation of Apo A-I in hepatocytes may be due to aberrant glycosylation, since glycosylated Apo A-I was not observed in transgenic mice. However, the accumulated Apo B was severely glycosylated. Therefore, it is suggested that highly expressed transgenic GnT-III allowed unknown target proteins to be glycosylated in large amounts, and the resulting target protein(s) disrupted in assembly formation of Apo A-I in the hepatocytes and cause a decrease in the release of lipoproteins and accumulations of Apo A-I and Apo B in the liver. The transgenic mice showed aberrant glycosylation by GnT-III, resulting in numerous lipid droplets in liver tissues and the obesity. These mice showed microvesicular fatty changes with abnormal lipid accumulation in the hepatocytes. Our study provides the basis for future analysis of the role of glycosylation in hepatic pathogenesis. In the transgenic mice, Apo A-I and Apo B were significantly increased compared with levels in nontransgenic liver tissues.     

Overexpression of human diacylglycerol acyltransferase 1, acyl-coa:cholesterol acyltransferase 1, or acyl-CoA:cholesterol acyltransferase 2 stimulates secretion of apolipoprotein B-containing lipoproteins in McA-RH7777 cells

The relative importance of each core lipid in the assembly and secretion of very low density lipoproteins (VLDL) has been of interest over the past decade. The isolation of genes encoding diacylglycerol acyltransferase (DGAT) and acyl-CoA:cholesterol acyltransferases (ACAT1 and ACAT2) provided the opportunity to investigate the effects of isolated increases in triglycerides (TG) or cholesteryl esters (CE) on apolipoprotein B (apoB) lipoprotein biogenesis. Overexpression of human DGAT1 in rat hepatoma McA-RH7777 cells resulted in increased synthesis, cellular accumulation, and secretion of TG. These effects were associated with decreased intracellular degradation and increased secretion of newly synthesized apoB as VLDL. Similarly, overexpression of human ACAT1 or ACAT2 in McA-RH7777 cells resulted in increased synthesis, cellular accumulation, and secretion of CE. This led to decreased intracellular degradation and increased secretion of VLDL apoB. Overexpression of ACAT2 had a significantly greater impact upon assembly and secretion of VLDL from liver cells than did overexpression of ACAT1. The addition of oleic acid (OA) to media resulted in a further increase in VLDL secretion from cells expressing DGAT1, ACAT1, or ACAT2. VLDL secreted from DGAT1-expressing cells incubated in OA had a higher TG:CE ratio than VLDL secreted from ACAT1- and ACAT2-expressing cells treated with OA. These studies indicate that increasing DGAT1, ACAT1, or ACAT2 expression in McA-RH7777 cells stimulates the assembly and secretion of VLDL from liver cells and that the core composition of the secreted VLDL reflects the enzymatic activity that is elevated.     

Disulfide bonds are required for folding and secretion of apolipoprotein B regardless of its lipidation state

Apolipoprotein (apo) B-100, an essential protein for the assembly and secretion of very low density lipoproteins depends on lipid binding (lipidation) for its secretion. Seven of its 8 disulfides are clustered within the N-terminal 21%. The role of these disulfides in the secretion of lipidated or unlipidated truncated forms of apoB was studied in C127 cells expressing apoB-17, apoB-29, or apoB-41. These cells do not express microsomal triglyceride transfer protein yet secrete apoB-41 on triacylglycerol-rich lipoproteins while apoB-29 and apoB-17 are secreted with little or no lipid, respectively. Dithiothreitol utilized in pulse-chase studies prevented the cotranslational formation of disulfides and when added posttranslationally reduced native disulfides. As a result, the secretion of reduced apoB forms was blocked and they were retained in the cells. Reduced apoB polypeptides were rescued following removal of dithiothreitol, as they underwent post-translational disulfide bonding, attained their mature form, and were subsequently secreted. Together the data suggest that in C127 cells the formation of native disulfides is critical for the folding and secretion of apoB independent of its length, its requirement for lipidation or microsomal triglyceride transfer protein expression. Therefore, these cells provide an appropriate model to study the folding of apoB in great detail.     

Overexpression of apolipoprotein B in the heart impedes cardiac triglyceride accumulation and development of cardiac dysfunction in diabetic mice

The heart secretes apolipoprotein B (apoB) containing lipoproteins. Herein, we examined whether the overexpression of a human apoB transgene in the heart affects triglyceride accumulation and development of cardiac dysfunction in streptozotocin-treated diabetic mice. Blood glucose, plasma free fatty acids, and plasma triglycerides were similarly affected in diabetic wild type mice and diabetic apoB transgenic mice as compared with non-diabetic mice of the same genotype. After 12 weeks, heart triglycerides were increased by 48% in diabetic wild type mice. These mice displayed an increased expression of brain natriuretic peptide and deterioration of heart function on echocardiography. In diabetic apoB transgenic mice, heart triglyceride levels were identical to those in non-diabetic wild type and apoB transgenic mice, and brain natriuretic peptide expression as well as echocardiographic indexes of heart function were only marginally affected or unaffected. The findings suggest that triglyceride accumulation in the heart is important for development of diabetic cardiomyopathy in mice, and that lipoprotein formation by cardiomyocytes plays an integrated role in cardiac lipid metabolism.     

Chinese hamster ovary cells require the coexpression of microsomal triglyceride transfer protein and cholesterol 7alpha-hydroxylase for the assembly and secretion of apolipoprotein B-containing lipoproteins

Due to the absence of microsomal triglyceride transfer protein (MTP), Chinese hamster ovary (CHO) cells lack the ability to translocate apoB into the lumen of the endoplasmic reticulum, causing apoB to be rapidly degraded by an N-acetyl-leucyl-leucyl-norleucinal-inhibitable process. The goal of this study was to examine if expression of MTP, whose genetic deletion is responsible for the human recessive disorder abetalipoproteinemia, would recapitulate the lipoprotein assembly pathway in CHO cells. Unexpectedly, expression of MTP mRNA and protein in CHO cells did not allow apoB-containing lipoproteins to be assembled and secreted by CHO cells expressing apoB53. Although expression of MTP in cells allowed apoB to completely enter the endoplasmic reticulum, it was degraded by a proteolytic process that was inhibited by dithiothreitol (1 mM) and chloroquine (100 microM), but resistant to N-acetyl-leucyl-leucyl-norleucinal. In marked contrast, coexpression of the liver-specific gene product cholesterol 7alpha-hydroxylase with MTP resulted in levels of MTP lipid transfer activity that were similar to those in mouse liver and allowed intact apoB53 to be secreted as a lipoprotein particle. These data suggest that, although MTP-facilitated lipid transport is not required for apoB translocation, it is required for the secretion of apoB-containing lipoproteins. We propose that, in CHO cells, MTP plays two roles in the assembly and secretion of apoB-containing lipoproteins: 1) it acts as a chaperone that facilitates apoB53 translocation, and 2) its lipid transfer activity allows apoB-containing lipoproteins to be assembled and secreted. Our results suggest that the phenotype of the cell (e.g. expression of cholesterol 7alpha-hydroxylase by the liver) may profoundly influence the metabolic relationships determining how apoB is processed into lipoproteins and/or degraded.     

Generation of hepatoma cell lines deficient in microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apolipoprotein B (apoB)48- and apoB100-containing lipoproteins in the intestine and liver, respectively. Loss of function mutations in MTP cause abetalipoproteinemia. Heterologous cells are used to evaluate the function of MTP in apoB secretion to avoid background MTP activity in liver and intestine-derived cells. However, these systems are not suitable to study the role of MTP in the secretion of apoB100-containing lipoproteins, as expression of a large apoB100 peptide using plasmids is difficult. Here, we report a new cell culture model amenable for studying the role of different MTP mutations on apoB100 secretion. The endogenous MTTP gene was ablated in human hepatoma Huh-7 cells using single guide RNA and RNA-guided clustered regularly interspaced short palindromic repeats-associated sequence 9 ribonucleoprotein complexes. We successfully established three different clones that did not express any detectable MTTP mRNA or MTP protein or activity. These cells were defective in secreting apoB-containing lipoproteins and accumulated lipids. Furthermore, we show that transfection of these cells with plasmids expressing human MTTP cDNA resulted in the expression of MTP protein, restoration of triglyceride transfer activity, and secretion of apoB100. Thus, these new cells can be valuable tools for studying structure-function of MTP, roles of different missense mutations in various lipid transfer activities of MTP, and their ability to support apoB100 secretion, compensatory changes associated with loss of MTP, and in the identification of novel proteins that may require MTP for their synthesis and secretion.     

Lipid micelles stimulate the secretion of triglyceride-enriched apolipoprotein B48-containing lipoproteins by Caco-2 cells

Intestinal triglyceride-rich lipoproteins (TRL) are synthesized from dietary lipids. This study was designed to evaluate the effects of lipid micelles, mimicking post-digestive duodenal micelles, on the fate of apolipoprotein B (apoB)48-containing lipoproteins by Caco-2 cells. Such micelles, consisting of oleic acid (OA), taurocholate, 2-monooleoylglycerol (2-MO), cholesterol (Chol), and L-alpha-lysophospatidylcholine, were the most efficient inducers of OA uptake and esterification. The efficiency of TG and apoB48 secretion increased specifically as a function of cell differentiation. PAGE analysis of secreted lipoproteins separated by sequential ultracentrifugation after [35S] labeling revealed differences in the secretion of apoB100- and apoB48-containing lipoproteins. In absence of micelles, apoB48 was secreted mostly in "HDL-like" particles, as observed in enterocytes in vivo. Micelle application increased 2.7-fold the secretion of apoB, resulting in 53 times more apoB48 being recovered as TG-enriched lipoproteins at d < 1.006 g/ml. Electron microscopy revealed the presence of lipid droplets in the secretory pathway and the accumulation of newly synthesized TG in cytoplasmic lipid droplets, as in enterocytes in vivo. We showed that these droplets could be used for secretion. However, apoB48 preferentially bound to newly synthesized TG in the presence of micelles, accounting in part for the functional advantage of apoB editing in the intestine. While Caco-2 cells express both apoB isoforms, our results show that the apical supply of complex lipid micelles favors the physiological route of apoB48-containing TG-enriched lipoproteins.     

乙肝病毒X 蛋白通过CREB 上调HBx结合蛋白促进肝癌细胞迁移

乙型肝炎病毒X蛋白（hepatitis B virus X protein,HBx）对肝癌的发生发展具有十分重要的作用. HBx 具有促进肝癌迁移的作用,但其作用的分子机制不清. 本研究对 HBx 促进肝癌细胞迁移的分子机制进行了探讨. 伤口愈合和 Boyden’s chamber结果表明,HBx 可明显促进肝癌 HepG2 细胞迁移. 在稳定转染 HBx 的 HepG2（HepG2-X）细胞中转染 HBx 结合蛋白（hepatitis B X-interacting protein,HBXIP）的 RNA 干扰片段,可明显抑制 HBx 的促迁移作用. 免疫组化和实时定量 PCR 结果表明,HBXIP 在肝癌组织中显著高表达,并且与 HBx 表达成正相关. 荧光素酶报告基因和免疫印迹结果表明,HBx 显著增强 HBXIP 的启动子活性和蛋白质表达水平. 应用 HBx 的 RNA 干扰处理 HepG2-X 细胞,HBXIP 的启动子活性和蛋白质表达水平明显下降.将 HBXIP 启动子区的cAMP效应元件结合因子（CREB）结合位点突变后,HBx 上调 HBXIP 的作用消失. 应用 CREB 的 RNA 干扰处理肝癌细胞,在启动子水平和蛋白质水平上, HBx 对 HBXIP 的上调作用被显著抑制. 染色质免疫共沉淀结果表明,HBx 能够通过 CREB 结合到 HBXIP 的启动子上,进而发挥激活 HBXIP 的功能. 本研究结果表明,HBx 促进肝癌细胞迁移的作用是通过 CREB 上调 HBXIP 实现的. 这一发现对进一步揭示 HBx 促进肝癌细胞迁移的分子机制具有重要意义.     

Upregulated microRNA-29a by hepatitis B virus X protein enhances hepatoma cell migration by targeting PTEN in cell culture model

Hepatitis B virus X protein (HBx) plays important roles in the development of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) contribute to cancer development by acting as oncogenes or tumor suppressors. Previously, we reported that HBx was able to promote the migration of hepatoma HepG2 cells. However, the regulation of miRNAs in the development of HBV-related HCC is poorly understood. In the present study, we reported that miR-29a was a novel regulator of migration of hepatoma cells mediated by HBx. Our data showed that the expression of miR-29a was dramatically increased in p21-HBx transgenic mice, HBx-transfected hepatoma HepG2-X (or H7402-X) cells and HepG2.2.15 cells that constitutively replicate HBV. However, our data showed that miR-29a was upregulated in 4 of the 11 clinical HCC samples. We found that the overexpression of miR-29a promoted the migration of HepG2 cells, while a specific miR-29a inhibitor could partially abolish the enhanced migration of HepG2-X cells. Moreover, we identified PTEN was one of the target genes of miR-29a in HepG2 cells. The deletion of the miR-29a-binding site was able to abolish the role of miR-29a in suppression of luciferase activity of the PTEN 3'UTR reporter. Meanwhile, the overexpression of PTEN was able to reverse the promoted migration of HepG2 cells mediated by miR-29a. Moreover, our data showed that the modulation of Akt phosphorylation, a downstream factor of PTEN, was involved in the cell migration enhanced by miR-29a, suggesting that miR-29a is responsible for the cell migration through its target gene PTEN. Thus, we conclude that miR-29a is involved in the regulation of migration of hepatoma cells mediated by HBx through PTEN in cell culture model.     

Regulation of apoB secretion from HepG2 cells: evidence for a critical role for cholesteryl ester synthesis in the response to a fatty acid challenge

Secretion of hepatic apoB lipoproteins removes excess triglyceride from the liver. However, the mechanism by which synthesis of apoB, which occurs on the rough endoplasmic reticulum, is coordinated with synthesis of triglyceride, which takes place in the smooth endoplasmic reticulum, is not known. To examine this question, we have manipulated intracellular synthesis of triglyceride and cholesteryl ester in HepG2 cells and determined the impact of these maneuvers on apoB secretion. Since cholesteryl ester is the only major lipid class synthesized in the rough endoplasmic reticulum, our hypothesis was that, in response to a fatty acid challenge, synthesis of cholesteryl ester rather than synthesis of triglyceride would be the immediate trigger to apoB secretion. Oleate complexed to bovine serum albumin caused intracellular triglyceride synthesis to increase 6-fold and cholesteryl ester synthesis to increase almost 3-fold, while apoB secretion into the medium increased by 2.5-fold (P less than 0.0125) at all time points between 4 and 24 h. Addition of acylation stimulating protein to the medium further stimulated both triglyceride and cholesteryl ester synthesis (58% and 108%, respectively) above oleate alone and this resulted in a 50% increase in apoB secretion (P less than 0.0025). By contrast, both progesterone and 2-bromooctanoate inhibited triglyceride and cholesteryl ester synthesis and these effects were associated with reduced apoB secretion. Lovastatin inhibited cholesteryl ester synthesis (45%, P less than 0.0025); however, at the doses used, triglyceride formation was unaffected. Under these circumstances, apoB secretion was reduced by 25% (P less than 0.05). Similarly, 58-035 (an inhibitor of acyl CoA:cholesterol acyltransferase) on the one hand reduced cholesteryl ester synthesis markedly (59%, P less than 0.005), but on the other increased triglyceride synthesis though not statistically significantly (65%, P NS), and again this resulted in decreased apoB secretion (25%, P less than 0.005). Control experiments established that changes in low density lipoprotein catabolism did not contribute importantly to the quantity of apoB in the medium. Taken together, the data indicate that, at least in HepG2 cells, there are parallel changes in cholesteryl ester synthesis and apoB secretion and suggest that it is cholesteryl ester synthesis, not triglyceride synthesis, that is the immediate regulator of apoB secretion when these cells are exposed to an increased influx of fatty acids. However, alternative or additional regulatory mechanisms, such as, for example, a role for acylation of apoB, are not excluded by these studies.     

Direct effects of growth hormone on production and secretion of apolipoprotein B from rat hepatocytes

The aim of this study was to investigate the direct effects of growth hormone (GH) on production and secretion of apolipoprotein B (apoB)-containing lipoproteins from hepatocytes. Bovine GH (5-500 ng/ml) was given for 1 or 3 days to rat hepatocytes cultured on laminin-rich matrigel in serum-free medium. The effects of GH were compared with those of 3 nM insulin and 500 microM oleic acid. GH increased the editing of apoB mRNA, and the proportion of newly synthesized apoB-48 (of total apoB) in the cells and secreted into the medium changed in parallel. GH increased total secretion of apoB-48 (+30%) and apoB-48 in very low density lipoproteins (VLDL) more than twofold. Total apoB-100 secretion decreased 63%, but apoB-100-VLDL secretion was unaffected by GH. Pulse-chase studies indicated that GH increased intracellular early degradation of apoB-100 but not apoB-48. GH had no effect on apoB mRNA or LDL receptor mRNA levels. The triglyceride synthesis, the mass of triglycerides in the cells, and the VLDL fraction of the medium increased after GH incubation. Three days of insulin incubation had effects similar to those of GH. Combined incubation with oleic acid and GH had additive effects on apoB mRNA editing and apoB-48-VLDL secretion. In summary, GH has direct effects on production and secretion of apoB-containing lipoproteins, which may add to the effects of hyperinsulinemia and increased flux of fatty acids to the liver during GH treatment in vivo.     

Apolipoprotein B genetic polymorphisms in several human hepatoma derived liver cell lines.

The genetic polymorphism of apoB EcoRI and XbaI restriction sites and the 3' VNTR hypervariable region was examined in nine human hepatoma derived liver cell lines and related to the cells' ability to secrete lipids and apoB. EcoRI and XbaI genotypes appeared to be unrelated to triglyceride, cholesterol and apoB accumulating in the medium. The VNTR consisted of alleles with 47 to 67 repeats; however, these repeats were not associated with elevated concentrations of lipid or apoB. Data suggest that in the hepatoma cell lines, apoB polymorphisms in EcoRI, XbaI and the VNTR hypervariable region are not sufficient in themselves to account for triglyceride, cholesterol and apoB in the medium. It is possible that intracellular apoB synthesis and/or degradation as well as postsecretory apoB binding and uptake are responsible for the variability of apoB and lipid accumulation in the culture medium.     

Loss and gain of function of Grp75 or mitofusin 2 distinctly alter cholesterol metabolism,but all promote triglyceride accumulation in hepatocytes

In the liver, contact sites between the endoplasmic reticulum (ER) and mitochondria (named MAMs) may be crucial hubs for the regulation of lipid metabolism, thus contributing to the exacerbation or prevention of fatty liver. We hypothesized that tether proteins located at MAMs could play a key role in preventing triglyceride accumulation in hepatocytes and nonalcoholic fatty liver disease (NAFLD) occurrence. To test this, we explored the role of two key partners in building MAM integrity and functionality, the glucose-regulated protein 75 (Grp75) and mitofusin 2 (Mfn2), which liver contents are altered in obesity and NAFLD. Grp75 or Mfn2 expression was either silenced using siRNA or overexpressed with adenoviruses in Huh7 cells.Silencing of Grp75 and Mfn2 resulted in decreased ER-mitochondria interactions, mitochondrial network fusion state and mitochondrial oxidative capacity, while overexpression of the two proteins induced mirror impacts on these parameters. Furthermore, Grp75 or Mfn2 silencing decreased cellular cholesterol content and enhanced triglyceride secretion in ApoB100 lipoproteins, while their overexpression led to reverse effects. Cellular phosphatidylcholine/phosphatidylethanolamine ratio was decreased only upon overexpression of the proteins, potentially contributing to altered ApoB100 assembly and secretion. Despite the opposite differences, both silencing and overexpression of Grp75 or Mfn2 induced triglyceride storage, although a fatty acid challenge was required to express the alteration upon protein silencing. Among the mechanisms potentially involved in this phenotype, ER stress was closely associated with altered triglyceride metabolism after Grp75 or Mfn2 overexpression, while blunted mitochondrial FA oxidation capacity may be the main defect causing triglyceride accumulation upon Grp75 or Mfn2 silencing. Further studies are required to decipher the link between modulation of Grp75 or Mfn2 expression, change in MAM integrity and alteration of cholesterol content of the cell.In conclusion, Grp75 or Mfn2 silencing and overexpression in Huh7 cells contribute to altering MAM integrity and cholesterol storage in opposite directions, but all promote triglyceride accumulation through distinct cellular pathways. This study also highlights that besides Mfn2, Grp75 could play a central role in hepatic lipid and cholesterol metabolism in obesity and NAFLD.     

A Drosophila microsomal triglyceride transfer protein homolog promotes the assembly and secretion of human apolipoprotein B. Implications for human and insect transport and metabolism

The assembly and secretion of triglyceride-rich lipoproteins in vertebrates requires apolipoprotein B (apoB) and the endoplasmic reticulum-localized cofactor, microsomal triglyceride transfer protein (MTP). Invertebrates, particularly insects, transport the majority of their neutral and polar lipids in lipophorins; however, the assembly of lipophorin precursor particles was presumed to be MTP-independent. A Drosophila melanogaster expressed gene sequence (CG9342), displaying 23% identity with human MTP, was recently identified. When coexpressed in COS cells, CG9342 promoted the assembly and secretion of apoB34 and apoB41 (N-terminal 34 and 41% of human apoB). The apoB34-containing particles assembled by human MTP and CG9342 displayed similar peak densities of approximately 1.169 g/ml and similar lipid compositions. However, CG9342 displayed differential sensitivities to two inhibitors of human MTP and low vesicle-based lipid transfer activity, in vitro. In addition, important predicted structural distinctions exist between the human and Drosophila proteins suggesting overlapping but not identical functional roles. We conclude that CG9342 and human MTP are orthologs that share only a subset of functions, consistent with known differences in intracellular and extracellular aspects of vertebrate and invertebrate lipid transport and metabolism.     

Efficient glycosylation site utilization by intracellular apolipoprotein B. Implications for proteasomal degradation

The balance between the hepatic assembly of apolipoprotein B (apoB) and its presecretory degradation at the level of the endoplasmic reticulum (ER) may control the secretion of apoB-containing lipoproteins. In one model, apoB that fails to assemble with lipid undergoes translocation arrest, exposing the protein to the cytosolic proteasome. To examine apoB's translocation behavior under various metabolic conditions, glycosylation site utilization studies were performed. A 70-amino acid peptide containing three sites for N-linked glycosylation was appended to the C-terminus of apoB-50 (amino-terminal 50% of apoB) and expressed in both hepatic and nonhepatic cell lines. When the C-terminal reporter peptide was released by cyanogen bromide cleavage, all of the sites were glycosylated irrespective of cell type, labeling time, or assembly status. Similar peptide mapping of endogenous apoB-100 expressed in HepG2 cells was performed to monitor glycosylation at Asn residues 2752 (apoB-61), 2955 (apoB-65), and 3074 (apoB-68). N-linked glycosylation occurred at a minimum of two of the three sites, a frequency identical to that observed in apoB-100 recovered from cell media. Treatment of cells with proteasome inhibitors produced a 2. 5-fold increase in intracellular apoB but failed to cause accumulation of an unglycosylated form. These results indicate that 1) the efficient translocation of apoB into the ER occurs independently of microsomal triglyceride transfer protein and its assembly with lipid and 2) despite its large size and affinity for lipid, delivery of misassembled apoB to the proteasome requires retrograde translocation from the ER lumen to cytosol.