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Papers In Press, published online ahead of print June 13, 2002
Lipoprotein & Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4W7
Corresponding Author: zyao{at}ottawaheart.ca
Previous studies with McA-RH7777 cells showed a 15-20 min temporal delay in the oleate-induced VLDL assembly after apoB100 translation. Here, we determined whether the post-translational assembly of apoB100-VLDL occurred within the endoplasmic reticulum (ER) or in post-ER compartments. At steady state, apoB100 distributed throughout subcellular compartments (distal Golgi, cis/medial Golgi and ER). Pulse-chase studies showed that it took ~20 min for newly synthesized apoB100 to exit ER and accumulate in cis/medial Golgi. At the end of subsequent 20-min chase, a small fraction of apoB100 accumulated in distal Golgi and a large amount of apoB100 was secreted into the medium as VLDL. VLDL was not detected either in the lumen of ER or that of cis/medial Golgi where apoB100 was membrane-associated and sensitive to endoglycosidase (Endo) H treatment. In contrast, VLDL particles were found in the lumen of distal Golgi where apoB100 was resistant to Endo H digestion. Formation of lumenal VLDL almost coincided with the appearance of VLDL in the medium, suggesting the site of VLDL assembly being proximal to the site of secretion. When microsomal triglyceride transfer protein activity was inactivated after apoB had exited the ER, formation of VLDL and their subsequent secretion was unaffected. Lipid analysis by tandem mass spectrometry showed that oleate treatment increased the mass of membrane phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by 68% and 27%, respectively. The increase in membrane PC was attributed to increase in 18:1 species in the ER and distal Golgi. In contrast, the increase in membrane PE was restricted in the ER with the elevation of almost every detected species. Taken together, these results suggest that VLDL assembly in McA-RH7777 cells takes place in compartments at the distal end of the secretory pathway.
J. Biol. Chem, 10.1074/jbc.M200249200
Submitted on January 9, 2002
Revised on June 10, 2002
Accepted on June 13, 2002
Intracellular assembly of very low density lipoproteins containing apolipoprotein B100 in rat hepatoma McA-RH7777 Cells
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