Two ALLN (N-acetyl-leucyl-leucyl-norleucinal)-sensitive endoplasmic reticulum (ER)-localized proteases (ER-60 and ER-72) were recently purified from rat liver. We used an antibody to rat ER-60 to investigate the possible role of this protease in apolipoprotein B (apoB) degradation. First, immunoprecipitation and immunoblotting experiments with the anti-rat ER-60 antibody suggested that HepG2 cells contain a homologue of ER-60 with an approximate molecular mass of 58 -60 kDa. The ER-60 homologue was mostly associated with the luminal contents of HepG2 microsomes. Evidence from co-immunoprecipitation and cross-linking experiments appear to suggest that the ER-60 homologue in HepG2 cells is associated with apoB intracellularly. A small pool of apoB was recovered when HepG2 lysates were subjected to immunoprecipitation with anti-rat ER-60 antibody followed by a second immunoprecipitation with anti-apoB antibody. Furthermore, cross-linking of permeabilized cells with dithiobis(succinimidylpropionate) further demonstrated association of apoB with the ER-60 homologue in HepG2 cells. Three polypeptides with molecular masses of 78, 66, and 50 kDa were consistently found to be associated with apoB as well as the 58-kDa ER-60 homologue. The 78-kDa protein associated with both apoB and ER-60 appeared to represent immunoglobulin heavy chain-binding protein (BiP) based on immunoprecipitation with a monoclonal antibody. Cross-linking and immunoblotting experiments suggested the association of the 78-kDa BiP with both the 58-kDa ER-60 homologue as well as the 550-kDa apoB.In summary, the data suggests that HepG2 cells contain a 58-kDa protein which is homologous to the rat liver ER-60 in size, antigenecity, and intracellular localization. The ER-60 homologue in HepG2 cells appears to be closely associated with apoB, as well as other proteins possibly representing ER chaperones such as BiP. We hypothesize that the ER-60 homologue may be involved in the degradation of apoB in the ER lumen of HepG2 cells.Post-translational degradation of apoB has been shown to modulate the intracellular levels of newly-synthesized apoB molecules (1-19). Recent evidence suggest that apoB degradation may occur in the cytosol by the proteasome (19) as well as in the ER 1 lumen by an unidentified ER protease(s) (17, 18). The identity of the ER-associated protease involved in apoB degradation has remained elusive, however, some characteristic features of this degradative system have recently been documented. The ER-associated protease appears to be responsible for fragmentation of apoB into a number of distinct degradation intermediates including an abundant 70-kDa fragment (12, 16). The activity of the ER-associated protease is also inhibitable by ALLN in a dose-dependent manner (12, 16). Intraluminal degradation of secretion-competent apoB associated with nascent HDL-like and LDL-like lipoprotein particles in the secretory pathway is also ALLN-sensitive and may be mediated by a putative ER-localized protease (17). Work by Ginsberg and co-workers (18)...

We have studied the relationship between the length of apolipoprotein B (apoB) and its intracellular translocation and stability using McArdle RH7777 (McA-RH7777) cells expressing recombinant human apoB variants, ranging in size from B15 to B100. The translocational status of apoB was assessed based on trypsin sensitivity of apoB using isolated microsomes as well as permeabilized cells. In isolated microsomes, shorter apoB variants (

It has been well established that the biogenesis of apoB is mediated co-translationally by the cytosolic proteasome. Here, however, we investigated the role of both the cytosolic proteasome as well as non-proteasome-mediated degradation systems in the post-translational degradation of apoB. In pulse-chase labeling experiments, co-translational (0-h chase) apoB degradation in both intact and permeabilized cells was sensitive to proteasome inhibitors. Interestingly, turnover of apoB in intact cells over a 2-h chase was partially inhibitable by lactacystin, thus suggesting a role for the cytosolic proteasome in the post-translational degradation of apoB. In permeabilized cells, however, there was no posttranslational protection of apoB by lactacystin. Further investigations of proteasomal activity in HepG2 cells revealed that, following permeabilization, there was a dramatic loss of the 20 S proteasomal subunits, and consequently the cells exhibited no detectable lactacystininhibitable activity. Thus, apoB fragmentation and the generation of the 70-kDa apoB degradation fragment, characteristic of permeabilized cells, continued to occur in these cells despite the absence of functional cytosolic proteasome. Similar results were observed when we used a derivative of lactacystin, clastolactacystin ␤-lactone, which represents the active species of the inhibitor. Interestingly, however, the abundance of the 70-kDa fragment could be modulated by the microsomal triglyceride transfer protein inhibitor, BMS-197636, as well as by pretreatment of the permeabilized cells with dithiothreitol. These data thus suggest that although the cytosolic proteasome appears to be involved in the posttranslational turnover of apoB in intact cells, the specific post-translational fragmentation of apoB generating the 70-kDa fragment observed in permeabilized cells occurs independent of the cytosolic proteasome.Hepatic apolipoprotein B100 (apoB) 1 secretion appears to be regulated post-transcriptionally (1-4). More specifically, efficient translocation of newly synthesized apoB molecules across the membrane of the endoplasmic reticulum (ER) is believed to be an important event that contributes to the formation of secretion competent apoB-containing lipoproteins (5-8). Inefficient translocation has been suggested to lead to the formation of a pool of membrane-associated apoB that becomes ubiquitinated (12-20, 22) and ultimately destined for intracellular degradation (6, 8 -11). It is evident that co-translational degradation of membrane-associated apoB is mediated by the cytosolic proteasome based on its sensitivity to proteasome inhibitors such as ALLN, lactacystin, and MG132 (9,[12][13][14][15][16][17][18][19][20][21][22].The involvement of the proteasome in degradation of apoB raises a number of intriguing questions. Clearly, the proteasome is involved in co-translational degradation of membraneassociated apoB, which is expected to have cytosolic exposure. However, recent evidence has suggested that the proteasome may also be involved in the ...

We studied the biogenesis of apolipoprotein B (apoB) in primary hepatocytes isolated from hamster liver, an animal model with striking resemblance to humans in lipoprotein metabolism. Hamster hepatocytes were found to assemble and secrete apoB-containing lipoproteins at a density of VLDL. Intracellular mechanisms of apoB biogenesis were investigated in both intact and permeabilized hamster hepatocytes. Translocational status of hamster apoB-100 was examined using trypsin protection assays in permeabilized cells as well as isolated microsomes which revealed that 27-42% of newly synthesized apoB was trypsin accessible as opposed to a control protein, transferrin, which was found to be essentially insensitive to exogenous trypsin. Subcellular fractionation of membrane and lumenal apoB pools indicated, however, that only a minor fraction of hamster apoB was associated with the microsomal membrane. Approximately 40% of newly synthesized apoB was found to be degraded post-translationally in a process sensitive to MG132. Immunoblotting analysis of apoB immunoprecipitates revealed ubiquitination of hamster apoB suggesting the involvement of the proteasome in its intracellular turnover. In addition to MG132, o -phenanthroline, a metalloprotease inhibitor, was also effective in stabilizing hamster apoB. Experiments in permeabilized hamster hepatocytes further confirmed post-translational instability of hamster apoB which was degraded over a 3-h chase generating proteolytic fragments including 167, 70, 57, and 46 kDa intermediates. Of these only the 70 kDa fragment was ALLN sensitive. Oleate treatment of hamster hepatocytes provided protection against intracellular apoB degradation, but did not stimulate its extracellular secretion. ApoB was assembled in the microsomal lumen into lipoprotein particles with densities of LDL and VLDL which were subsequently secreted as VLDL with a minor fraction forming HDL-like particles. In summary, hamster hepatocytes appear to efficiently assemble and secrete apoB-containing VLDL, although a significant pool of newly synthesized apoB is retained intracellularly and becomes sensitive to proteasome-mediated degradation as well as other proteases in the secretory pathway, generating specific degradative intermediates. -Taghibiglou, C.