PROTEOLYSIS COUPLED SECRETION OF THE N-TERMINUS OF APOLIPOPROTEIN-B -CHARACTERIZATION OF A TRANSIENT, TRANSLOCATION ARRESTED INTERMEDIATE

We have shown that non-hepatic Chinese hamster ovary cells (CHO) have a specific inability to translocate and secrete apolipoprotein B (apoB ), leading to its complete degradation in the endoplasmic reticulum (T hrift, It. N., Drisko, J., Dueland, S., Trawick, J. D., and Davis, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9161-9165). To gain an u nderstanding why a protein having no predictable trans-membrane sequen ces can be stably integrated into the endoplasmic reticulum, we determ ined the topography and metabolic fate of apoB in both CHO cells and h uman hepatoma cells (HepG2). Using epitope-specific antibodies, we sho w that in microsomes from both cell types, apoB assumes a trans-membra ne orientation having 69 kDa of its N terminus in the lumen and the re maining portion of the C terminus residing on the cytoplasmic surface. In both cell types, proteolytic cleavage of the translocation arreste d apoB by a process that can be blocked by acetyl-leucine, leucine, no rleucal, produces an 85-kDa N-terminal fragment that resumes transloca tion and is secreted. This same N-terminal 85-kDa fragment is also fou nd in human plasma. These results show that sequences residing outside of the membrane spanning domain can block translocation. Moreover, ou r data provide compelling evidence showing that apoB undergoes an unus ual transient, translocation arrest, that serves as an entrance into t he intracellular degradative pathway regulating its secretion.