Studies on Degradative Mechanisms Mediating Post-translational Fragmentation of Apolipoprotein B and the Generation of the 70-kDa Fragment

Abstract

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 post-translational 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 post-translational 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.