The microsomal triglyceride transfer protein catalyzes the post-translational assembly of apolipoprotein B-100 very low density lipoprotein in McA-RH7777 cells

In cells in which the lipoprotein assembly process had been inactivated by brefeldin A (BFA), membrane-associated apoB-100 disappeared without forming lipoproteins or being secreted, indicating that it was degraded, Reactivation of the assembly process by chasing the cells in the absence of BFA, gave rise to a quantitative recovery of the membrane-associated apoB-100 in the very low density lipoprotein (VLDL) fraction in the medium, These results indicate that the membrane-associated apoB-100 can be converted to VLDL. A new method was developed by which the major amount (88%) of microsomal apoB-100 but not integral membrane proteins could be extracted, The major effect of this method was to increase the recovery of apoB-100 that banded in the LDL and HDL density regions, suggesting that the membrane-associated form of apoB-100 is partially lipidated, We also investigated the role of the microsomal triglyceride transfer protein (MTP) in the assembly of apoB-100 VLDL using a photoactivatable MTP inhibitor (BMS-192951), This compound strongly inhibited the assembly and secretion of apoB-100 VLDL when present during the translation of the protein, To investigate the importance of MTP during the later stages in the assembly process, the cells were preincubated with BFA (to reversibly inhibit the assembly of apoB-100 VLDL) and pulse-labeled (+BFA) and chased (+BFA) for 30 min to obtain full length apoB-100 associated with the microsomal membrane, Inhibition of MTP after the 30-min chase blocked assembly of VLDL, This indicates that MTP is important for the conversion of full-length apoB-100 into VLDL, Results from experiments in which a second chase (-BFA) was introduced before the inactivation of MTP indicated that only early events in this conversion of full-length apoB-100 into VLDL were blocked by the MTP inhibitor, Together these results indicate that there is a MTP-dependent "window" in the VLDL assembly process that occurs after the completion of apoB-100 but before the major amount of lipids is added to the VLDL particle, Thus the assembly of apoB-100 VLDL from membrane-associated apoB-100 involves an early MTP-dependent phase and a late MTP-independent phase, during which the major amount of lipid is added.