Apolipoprotein B-100 destined for lipoprotein assembly and intracellular degradation undergoes efficient translocation across the endoplasmic reticulum membrane.

It has been proposed that inefficient translocation across the endoplasmic reticulum (ER) membrane gives rise to transmembrane forms of apolipoprotein B-100 (apoB). However, we previously demonstrated that the amino-terminal 50% of apoB (apoB-50) was efficiently translocated across the ER membrane in the nonhepatic cell line COS-1. To determine whether liver-specific factors modulate apoB membrane translocation or topology, hybrid proteins containing 300 amino acid overlapping segments of apoB-48 were transiently expressed in HepG2 cells and their protease sensitivities were examined in membrane vesicles. The hybrid proteins demonstrated the same range of protection from exogenously added protease (75-100%) as a transfected secretory control protein. When endogenous apoB was examined, its protection from trypsin in intact membranes was -80%, a value similar to that of two endogenous secretory control proteins, transferrin and alpha 2-macroglobulin. No discretely sized fragments of apoB were generated by trypsin digestion of membranes unless they were first permeabilized with detergent. In contrast to the behavior of apoB and other control proteins, albumin predominantly resisted degradation by trypsin in both intact and detergent permeabilized membranes. HepG2 cells were treated with ALLN, a protease inhibitor that has been proposed to inhibit the turnover of partially translocated forms of apoB. Although an -6-fold increase in intracellular apoB was observed in ALLN-treated cells, no corresponding increase in protease sensitivity was observed. These results indicate that the efficient translocation of apoB across the ER membrane occurs independently of its ability to undergo assembly into a secretion competent lipoprotein.