Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence

When lipid synthesis is limited in HepG2 cells, apoprotein B100 (apoB100) i s not secreted but rapidly degraded by the ubiquitin-proteasome pathway. To investigate apoB100 biosynthesis and secretion further, the physical and f unctional states of apoB100 destined for either degradation or lipoprotein assembly were studied under conditions in which lipid synthesis, proteasoma l activity, and microsomal triglyceride transfer protein (MTP) lipid-transf er activity were varied. Cells were pretreated with a proteasomal inhibitor (which remained with the cells throughout the experiment) and radiolabeled for 15 min. During the chase period, labeled apoB100 remained associated w ith the microsomes, Furthermore, by crosslinking sec61 beta to apoB100, we showed that apoB100 remained close to the translocon at the same time apoB1 00-ubiquitin conjugates could be detected, When lipid synthesis and lipopro tein assembly/secretion were stimulated by adding oleic acid (OA) to the ch ase medium, apoB100 was deubiquitinated, and its interaction with sec61 bet a was disrupted, signifying completion of translocation concomitant with th e formation of lipoprotein particles. MTP participates in apoB100 transloca tion and lipoprotein assembly. In the presence of OA, when MTP lipid-transf er activity,vas inhibited at the end of pulse labeling, apoB100 secretion w as abolished, In contrast, when the labeled apoB100 was allowed to accumula te in the cell for 60 min before adding OA and the inhibitor, apoB100 lipid ation and secretion were no longer impaired. Overall, the data imply that d uring most of its association with the endoplasmic reticulum, apoB100 is cl ose to or within the translocon and is accessible to both the ubiquitin-pro teasome and lipoprotein-assembly pathways, Furthermore, MTP lipid-transfer activity seems to be necessary only for early translocation and lipidation events.