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 concentration of 0.2 microg/ml, prevented the assembly of newly synthesized apolipoprotein B (apoB) into mature, secretory VLDL but did not prevent the secretion of apoB as denser particles (HDL apoB), or of albumin. The unassembled apoB remained associated with the membranes of the cellular microsomal fraction. There was no effect of BFA on the removal of apoB from the lumen of these vesicles. VLDL apoB formed only a minor component of the total apoB in the microsomal lumen. Higher (5 microg/ml) concentrations of BFA were required to prevent the secretion of HDL apoB and albumin. Under these conditions apoB accumulated in the microsomal lumen, as well as in the membranes of these vesicles. Again, apoB VLDL formed only a minor proportion of the total lumenal apoB. ApoB-48 VLDL and apoB-100 VLDL assembly could be restored by removing BFA from the medium. This reactivation of VLDL assembly was accompanied by an increased removal of apoB from the microsomal membranes, but there was no detectable increase in the small quantity of VLDL apoB that was recovered from the microsomal lumen. In the absence of BFA, during pulse-chase experiments the pattern of change in the specific radioactivity of microsomal membrane apoB was similar to that of the secreted VLDL apoB whereas that of the lumenal apoB resembled that of the secreted HDL apoB. The results suggest that membrane-associated apoB is the main direct precursor of secreted VLDL apoB in primary cultures of rat hepatocytes and that VLDL assembly does not involve primarily microsomal lumenal apoB as an intermediate.
Hebbachi
A M
AM
Oxford Lipid Metabolism Group, Metabolic Research Laboratory, Nuffield Department of Clinical Medicine, University of Oxford, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, UK.
Gibbons
G F
GF
D000818:Animals; D001055:Apolipoproteins B / Q000378:metabolism*; D001692:Biological Transport; D020126:Brefeldin A / Q000494:pharmacology; D002478:Cells, Cultured; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008075:Lipoproteins, HDL / Q000378:metabolism; D008079:Lipoproteins, VLDL / Q000378:metabolism*; D008861:Microsomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011868:Radioisotopes; D051381:Rats; D013997:Time Factors
D001055:Apolipoproteins B; D008075:Lipoproteins, HDL; D008079:Lipoproteins, VLDL; D011498:Protein Precursors; D011868:Radioisotopes; D020126:Brefeldin A