Intracellular assembly and degradation of apolipoprotein B-100-containing lipoproteins in digitonin-permeabilized HEP G2 cells

Permeabilized Hep G2 cells have been used to investigate the turnover of apolipoprotein B-100 (apoB-100). When such cells were chased in the presence of buffer, there was no biosynthesis of apoB-100, nor was the protein secreted from the cells, Thus the turnover of apoB-100 in these cells reflected the posttranslational degradation of the protein, Pulse-chase studies indicated that apoB-100 was degraded both when associated with the membrane and when present as lipoproteins in the secretory pathway, Neither albumin nor alpha(1)-antitrypsin showed any significant posttranslational intracellular degradation under the same condition, The kinetics for the turnover of apoB-100 in the luminal content differed from that of apoB-100 that was associated with the microsomal membrane. Moreover, while the degradation of the luminal apoB-100 was inhibited by N-acetyl-leucyl-leucyl-norleucinal (ALLN), this was not the case for the membrane-associated protein, Together these results suggest the existence of different pathways for the degradation of luminal apoB-100 and membrane-associated apoB-100, This was further supported by results from pulse-chase studies in intact cells, showing that ALLN increased the amount of radioactive apoB-100 that associated with the microsomal membrane during the pulse-labeling of the cells. However, ALLN did not influence the rate of turnover of the membrane-associated apoB-100. The presence of an ATP-generating system during the chase of the permeabilized cells prevented the disappearance of pulse labeled apoB-100 from the luminal lipoprotein associated pool. The ATP-generating system combined with cytosol protected the total apoB-100 in the system from being degraded, The cells cultured in the presence of oleic acid and chased after permeabilization in the presence of cytosol and the ATP-generating system showed an increase in the amount of apoB-100 present on dense (''high density lipoprotein-like'') particles, This increase was linear during the time investigated (i.e. from 0 to 2 h chase) and independent of protein biosynthesis, Our results indicate that the dense particle was generated by a redistribution of apoB-100 within the secretory pathway and that it most likely was assembled from the membrane-associated form of apoB-100, These results indicate that the release of apoB-100 from this membrane-associated form to the microsomal lumen is dependent on cytosolic factors and a source of metabolic energy.