PROTEASOME-MEDIATED DEGRADATION OF APOLIPOPROTEIN-B TARGETS BOTH NASCENT PEPTIDES COTRANSLATIONALLY BEFORE TRANSLOCATION AND FULL-LENGTH APOLIPOPROTEIN-B AFTER TRANSLOCATION INTO THE ENDOPLASMIC-RETICULUM

A major portion of newly synthesized apolipoprotein B (apoB) is degrad ed intracellularly, This degradation has been demonstrated to be media ted largely by the ubiquitin-proteasome pathway. We examined whether n ascent apoB polypeptides or full-length apoB is selectively retrotrans located from the endoplasmic reticulum into the cytosol for degradatio n. Herein, we found that full-length apoB as well as partial-length ap oB peptides are ubiquitinated in HepG2 cells, and ubiquitination is an exclusively cytosolic process. Calnexin, which binds specifically to glycoproteins, has been postulated to promote apoB folding and complet e translocation; me found that ubiquitinated apoB is bound to calnexin , suggesting that ubiquitinated apoB is glycosylated. In addition to c alnexin binding, we have other pieces of evidence that the full-length intracellular ubiquitinated apoB is glycosylated, because (i) it bind s to concanavalin A, and (ii) glycan can be demonstrated in the full-l ength ubiquitinated apoB by a chemical detection method involving oxid ation of adjacent hydroxyl groups in the glycan moiety. Because glycos ylation occurs inside the endoplasmic reticulum, the full-length glyco sylated apoB must have been retrotranslocated into the cytosol for ubi quitination and proteasome-mediated degradation. Next we synchronized translation in HepG2 cells by puromycin treatment. A pulse-chase exper iment using [S-35]methionine labeling of intracellular apoB in these s ynchronized cells demonstrated that nascent partial-length apoB peptid es are also ubiquitinated cotranslationally, We conclude that the ubiq uitin proteasome mediated degradation of apoB targets both nascent pep tides cotranslationally before translocation as well as full-length ap oB after its translocation into the endoplasmic reticulum.