The assembly of very low density lipoproteins in hepatocytes requires the microsomal triacylglycerol transfer protein (MTP). This microsomal lumenal protein transfers lipids, particularly triacylglycerols (TG), between membranes in vitro and has been proposed to transfer TG to nascent apolipoprotein (apo) B in vivo. We examined the role of MTP in the assembly of apoB-containing lipoproteins in cultured murine primary hepatocytes using an inhibitor of MTP. The MTP inhibitor reduced TG secretion from hepatocytes by 85% and decreased the amount of apoB100 in the microsomal lumen, as well as that secreted into the medium, by 70 and 90%, respectively, whereas the secretion of apoB48 was only slightly decreased and the amount of lumenal apoB48 was unaffected. However, apoB48-containing particles formed in the presence of inhibitor were lipid-poor compared with those produced in the absence of inhibitor. We also isolated a pool of apoB-free TG from the microsomal lumen and showed that inhibition of MTP decreased the amount of TG in this pool by ϳ45%. The pool of TG associated with apoB was similarly reduced. However, inhibition of MTP did not directly block TG transfer from the apoB-independent TG pool to partially lipidated apoB in the microsomal lumen. We conclude that MTP is required for TG accumulation in the microsomal lumen and as a source of TG for assembly with apoB, but normal levels of MTP are not required for transferring the bulk of TG to apoB during VLDL assembly in murine hepatocytes.During assembly of very low density lipoproteins (VLDLs) 1 in the liver, triacylglycerol (TG) is concentrated within the hydrophobic core of apolipoprotein (apo) B-containing lipoprotein particles. A microsomal lumenal protein, the microsomal triacylglycerol transfer protein (MTP), has been implicated in the acquisition of TG by nascent apoB for assembly and secretion of VLDLs (reviewed in Refs. 1-4). Individuals with the rare inherited disease abetalipoproteinemia have a defect in the MTP gene and lack detectable MTP protein and MTP lipid transfer activity (5). Despite a normal apoB gene, plasma apoB is barely detectable in these patients. MTP has the ability to transfer TG and other lipids, including cholesteryl esters, diacylglycerols, and phospholipids, between membranes in vitro (6) and has been proposed to transfer TG to nascent apoBcontaining lipoproteins in vivo. This idea is consistent with immunoprecipitation studies showing that apoB and MTP interact physically at early stages of VLDL assembly (7-9). MTP is expressed primarily in the liver and intestine as a soluble heterodimer with protein-disulfide isomerase (55 kDa) (10), a ubiquitous protein of the endoplasmic reticulum (ER) lumen that catalyzes disulfide bond formation during protein folding (11). The 97-kDa MTP subunit confers all lipid transfer activity to the heterodimer (12). In in vitro assays, the lipid transfer activity of MTP displays Ping Pong Bi Bi kinetics implying that MTP transfers lipids between membranes via a "shuttle" mechanism (13). The tissu...

We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [ 3 H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 ؎ 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in the d < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum.

In cells in which the lipoprotein assembly process had been inactivated by brefeldin A (BFA), membrane-associated apoB-100 disappeared without forming lipoproteins or being secreted, indicating that it was degraded. Reactivation of the assembly process by chasing the cells in the absence of BFA, gave rise to a quantitative recovery of the membrane-associated apoB-100 in the very low density lipoprotein (VLDL) fraction in the medium. These results indicate that the membrane-associated apoB-100 can be converted to VLDL.A new method was developed by which the major amount (88%) of microsomal apoB-100 but not integral membrane proteins could be extracted. The major effect of this method was to increase the recovery of apoB-100 that banded in the LDL and HDL density regions, suggesting that the membrane-associated form of apoB-100 is partially lipidated. We also investigated the role of the microsomal triglyceride transfer protein (MTP) in the assembly of apoB-100 VLDL using a photoactivatable MTP inhibitor (BMS-192951). This compound strongly inhibited the assembly and secretion of apoB-100 VLDL when present during the translation of the protein. To investigate the importance of MTP during the later stages in the assembly process, the cells were preincubated with BFA (to reversibly inhibit the assembly of apoB-100 VLDL) and pulse-labeled (؉BFA) and chased (؉BFA) for 30 min to obtain full-length apoB-100 associated with the microsomal membrane. Inhibition of MTP after the 30-min chase blocked assembly of VLDL. This indicates that MTP is important for the conversion of full-length apoB-100 into VLDL. Results from experiments in which a second chase (؊BFA) was introduced before the inactivation of MTP indicated that only early events in this conversion of full-length apoB-100 into VLDL were blocked by the MTP inhibitor. Together these results indicate that there is a MTP-dependent "window" in the VLDL assembly process that occurs after the completion of apoB-100 but before the major amount of lipids is added to the VLDL particle. Thus the assembly of apoB-100 VLDL from membrane-associated apoB-100 involves an early MTP-dependent phase and a late MTP-independent phase, during which the major amount of lipid is added.

BFA inhibited in a dose dependent way the assembly of apoB-48 very low density lipoprotein (VLDL) but allowed a normal rate of biosynthesis of the apolipoprotein and of the assembly of the dense ("high density lipoprotein (HDL)-like") apoB-48 particle (apoB-48 HDL). The inhibition of the assembly of apoB-48 VLDL occurred at BFA levels that allowed a major secretion of both transferrin and apoB-48 HDL. The assembly of apoB-100 containing lipoproteins was also inhibited by BFA but could be reactivated by a 30 -60 min chase in the absence of BFA, which agreed with the time that was estimated to be needed to restore the secretory pathway (approximately 60 min). Also the assembly of apoB-48 VLDL was reversible. Both apoB-48 and apoB-100 that was labeled in the presence of BFA assembled VLDL after removal of the BFA.Both apoB-100 and apoB-48 were associated with the membrane pellet of the microsomes. Virtually all (122 ؎ 30%) of the membrane associated pulse-labeled apoB-48 remained in the membrane after a 180-min chase in the presence of BFA, compared to only 21 ؎ 2% in normal cells (mean ؎ S.D., n ‫؍‬ 4). The corresponding figures for apoB-100 was 40 ؎ 7% in BFA-treated cells and 9 ؎ 7% in normal cells (mean ؎ S.D., n ‫؍‬ 4).Pulse-chase experiments with BFA offered conditions to selectively follow the turnover of membrane-associated apoB-100. Such experiments indicated that this apoB-100 pool is a precursor to VLDL. Apolipoprotein B (apoB)1 is the major protein component of the triacylglycerol-and cholesterol ester-rich plasma lipoproteins: chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins, and low density lipoproteins (LDL).Two forms of apoB exist, referred to as apoB-100 and apoB-48 (1). ApoB-100 consists of 4536 amino acids, while apoB-48 corresponds to the N-terminal 48% of apoB-100. Both proteins are coded for by the same gene (2). The apoB-100 mRNA is converted to mRNA for apoB-48 by an enzymedependent deamination of cytidine in codon 2153, which converts this codon into the signal for translational stop (3-5).In humans, apoB-48 is expressed mainly in the intestine, where it assembles chylomicrons, while apoB-100 is synthesized in the liver and is necessary for the assembly of VLDL particles (6). In contrast, the rat hepatocytes, as well as the rat hepatoma cells McA-RH7777 (7-9) express substantial amounts of both apoB-100 and apoB-48 (7, 10 -12).Recent results (7) from studies in McA-RH7777 cells indicate that during translation/translocation apoB-48 forms a small dense particle (100 Å in diameter), floating as HDL. This small particle appears to acquire the major amount of lipids in a second step and is in this way converted into a VLDL particle. The second step is dependent upon ongoing biosynthesis of proteins other than apoB; moreover, it is stimulated by oleic acid (7).The assembly of apoB-100 VLDL is more complicated (7), and our results indicate that the VLDL particles are assembled at least to a certain extent in relatively close connection with the translation/tra...