Microsomal membrane-associated apoB is the direct precursor of secreted VLDL in primary cultures of rat hepatocytes

Brefeldin A (BFA) added to primary cultures of rat hepatocytes, at a concen tration of 0.2 mug/ml, prevented the assembly of newly synthesized apolipop rotein B (apoB) into mature, secretory VLDL but did not prevent the secreti on of apoB as denser particles (HDL apoB), or of albumin. The unassembled a poB remained associated with the membranes of the cellular microsomal fract ion. There was no effect of BFA on the removal of apoB from the lumen of th ese vesicles. VLDL apoB formed only a minor component of the total apoB in the microsomal lumen. Higher (5 mug/ml) concentrations of BFA were required to prevent the secretion of HDL apoB and albumin. Under these conditions a poB accumulated in the microsomal lumen, as well as in the membranes of the se vesicles. Again, apoB VLDL formed only a minor proportion of the total l umenal apoB. ApoB-48 VLDL and apoB-100 VLDL assembly could be restored by r emoving BFA from the medium. This reactivation of VLDL assembly was accompa nied by an increased removal of apoB from the microsomal membranes, but the re was no detectable increase in the small quantity of VLDL apoB that was r ecovered from the microsomal lumen. In the absence of BFA, during pulse-cha se experiments the pattern of change in the specific radioactivity of micro somal membrane apoB was similar to that of the secreted VLDL apoB whereas t hat of the lumenal apoB resembled that of the secreted HDL apoB. jlr The re sults suggest that membrane-associated apoB is the main direct precursor of secreted VLDL apoB in primary cultures of rat hepatocytes and that VLDL as sembly does not involve primarily microsomal lumenal apoB as an intermediat e.