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 th e protein secreted from the cells, Thus the turnover of apoB-100 in th ese cells reflected the posttranslational degradation of the protein, Pulse-chase studies indicated that apoB-100 was degraded both when ass ociated with the membrane and when present as lipoproteins in the secr etory pathway, Neither albumin nor alpha(1)-antitrypsin showed any sig nificant posttranslational intracellular degradation under the same co ndition, The kinetics for the turnover of apoB-100 in the luminal cont ent differed from that of apoB-100 that was associated with the micros omal 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 resul ts suggest the existence of different pathways for the degradation of luminal apoB-100 and membrane-associated apoB-100, This was further su pported by results from pulse-chase studies in intact cells, showing t hat ALLN increased the amount of radioactive apoB-100 that associated with the microsomal membrane during the pulse-labeling of the cells. H owever, ALLN did not influence the rate of turnover of the membrane-as sociated 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 AT P-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 cy tosol and the ATP-generating system showed an increase in the amount o f apoB-100 present on dense (''high density lipoprotein-like'') partic les, This increase was linear during the time investigated (i.e. from 0 to 2 h chase) and independent of protein biosynthesis, Our results i ndicate that the dense particle was generated by a redistribution of a poB-100 within the secretory pathway and that it most likely was assem bled from the membrane-associated form of apoB-100, These results indi cate that the release of apoB-100 from this membrane-associated form t o the microsomal lumen is dependent on cytosolic factors and a source of metabolic energy.