The amino-terminal domain of apolipoprotein B does not undergo retrograde translocation from the endoplasmic reticulum to the cytosol - Proteasomal degradation of nascent apolipoprotein B begins at the carboxyl terminus of the protein, while apolipoprotein B is still in its original, translocon

We studied the sequential topology of the NH, and COOH termini of apoB duri ng translocation by expressing, in Chinese hamster ovary (CHO) and HepG2 ce lls, an apoB42 construct with c-Myc and hemagglutinin (HA) tags at 2 and 41 % (relative to apoB100) of its amino acid sequence. We conducted similar st udies using monoclonal antibodies against the NH, and COOH termini of apoB1 00 in HepG2 cells. After radiolabeling, microsomes were immunoisolated from transfected CHO cells using anti-c-Mye or anti-HA antibodies. Throughout a 60-min chase in the presence of N-acetyl-leucylnorleucinal, more than 90% of microsomes were isolated by anti-HA antibodies, whereas less than 10% we re isolated by anti-c-Mye antibodies. Proteinase K digestion of total micro somes consistently generated two fragments (similar to 70 and similar to 12 0 kDa) of apoB42 containing the NH, terminus throughout the chase; no fragm ents containing the COOH terminus were detected. Immunofluorescent studies of transfected CHO cells were consistent with results from the labeling stu dies. Essentially identical results were obtained from pulse-chase studies in both native and apoB42-transfected HepG2 cells. The present studies supp ort a model in which, in the absence of adequate core lipid synthesis, ther e is partial translocation of apoB leading to cytosolic exposure, ubiquitin ation, and proteasomal degradation directly from the original translocation channel.