Studies on degradative mechanisms mediating post-translational fragmentation of apolipoprotein B and the generation of the 70-kDa fragment

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 lactacystin-inhibitable 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 beta-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.