Cytosolic components are required for proteasomal degradation of newly synthesized apolipoprotein B in permeabilized HepG2 cells

Recent studies have proposed that post-translational degradation of apolipo protein B100 (apoB) involves the cytosolic ubiquitin-proteasome pathway. In this study, immunocytochemistry indicated that endoplasmic reticulum (ER)- associated proteasome molecules were concentrated in perinuclear regions of digitonin-permeabilized HepG2 cells. Signals produced by antibodies that r ecognize both alpha- and beta-subunits of the proteasome co-localized in th e ER with specific domains of apoB, The mechanism of apoB degradation in th e ER by the ubiquitin-proteasome pathway was studied using pulse-chase labe ling and digitonin-permeabilized cells. ApoB in permeabilized cells incubat ed at 37 degrees C in buffer alone was relatively stable. When permeabilize d cells were incubated with both exogenous ATP and rabbit reticulocyte lysa te (RRL) as a source of ubiquitin-proteasome factors, >50% of [H-3]apoB was degraded in 30 min. The degradation of apoB in the intact ER of permeabili zed cells was much more rapid than that of extracted [H-3]apoB incubated wi th RRL and ATP in vitro, The degradation of apoB was reduced by clasto-lact acystin beta-lactone, a potent proteasome inhibitor, and by ubiquitin K48R mutant protein, an inhibitor of polyubiquitination. ApoB in HepG2 cells was ubiquitinated, and polyubiquitination of apoB was stimulated by incubation of permeabilized cells with RRL. These results suggest that newly synthesi zed apoB in the ER is accessible to the cytoplasmic ubiquitin-proteasome pa thway and that factors in RRL stimulate polyubiquitination of apoB, leading to rapid degradation of apoB in permeabilized cells.