Co-translational interactions of apoprotein B with the ribosome and translocon during lipoprotein assembly or targeting to the proteasome

Hepatic lipoprotein assembly and secretion can be regulated by proteasomal degradation of newly synthesized apoB, especially if lipid synthesis or lip id transfer is low. Our previous studies in HepG2 cells showed that, under these conditions, newly synthesized apoB remains stably associated with the endoplasmic reticulum (ER) membrane (Mitchell, D. M., Zhou, M., Pariyarath , R., Wang, H., Aitchison, J. D., Ginsberg, T-I. N., and Fisher, E. A (1998 ) Proc. Natl. Acad. Sci. U.S.A. 95, 14733-14738). We now show that independ ent of lipid synthesis, apoB chains that appear full-length are, in fact, i ncompletely translated polypeptides still engaged by the ribosome and assoc iated with the ER translocon. In the presence of active lipid synthesis and transfer, translation and lipoprotein assembly are completed, and the comp lexes exit the ER. Upon omitting fatty acids from, or adding a microsomal t riglyceride transfer protein inhibitor to, culture media to reduce lipid sy nthesis or transfer, respectively, apoB was degraded while it remained asso ciated with the ER and complexed with cytosolic hsp70 and proteasomes. Thus , unlike other ER substrates of the proteasome, such as major histocompatib ility complex class I molecules, apoB does not fully retrotranslocate to th e cytosol before entering the ubiquitin-proteasome pathway. Although, upon immunofluorescence, apoB in proteasome-inhibited cells accumulated in punct ate structures similar in appearance to aggresomes (cytosolic structures co ntaining molecules irreversibly lost from the secretory pathway), these apo B molecules could be secreted when lipid synthesis was stimulated. The resu lts suggest a model in which 1) apoB translation does not complete until li poprotein assembly terminates, and 2) assembly with lipids or entry into th e ubiquitin-proteasome pathway occurs while apoB polypeptides remain associ ated with the translocon and attached to the ribosome.