Janero D R, Lane M D
J Biol Chem. 1983 Dec 10;258(23):14496-504.
Primary cultures of estrogen-induced chick parenchymal liver cells have been used to study the assembly of the apolipoprotein and glycerolipid constituents of hepatic very low density lipoprotein (VLDL). Liver cell monolayers in serum-free medium were pulse-labeled for 2.5 min with either [3H]leucine or [3H]glycerol and were then chased for 4 h. The kinetics of secretion of VLDL 3H-apolipoproteins (labeled from [3H]leucine) and [3H]triacylglycerol and 3H-phosphoglycerides (labeled from [3H]glycerol) was determined through the chase. Maximal rates of VLDL [3H]triacylglycerol secretion were reached by 20-25 min into the chase, preceding the initial appearance of 3H-apolipoproteins in the medium at 30-35 min of chase time. The secretion of VLDL 3H-phosphoglycerides was maximal much earlier, between 5 and 15 min into the chase, plateaued for 15 min, and then began again at 30 min, thereby displaying a distinctive biphasic pattern. In the presence of cycloheximide or puromycin, such that apopeptide synthesis was halted from the start of the chase, the secretion of VLDL 3H-glycerolipid was depressed after 30 min of chase, without having influenced the temporal pattern of the newly synthesized VLDL 3H-apolipoprotein and 3H-glycerolipid secretion. As compared to the cycloheximide-treated cells in which apolipoprotein B nascent chains were arrested intracellularly, the puromycin-treated cells secreted discharged apolipoprotein B nascent chains as VLDL constituents assembled with triacylglycerol. The differential kinetics of 3H-apolipoprotein, [3H]triacylglycerol, and 3H-phosphoglyceride secretion as VLDL and the timing of the effects of protein synthesis inhibitors on their secretion indicate that VLDL constituents are assembled sequentially in the intact liver cell. Some VLDL phosphoglyceride and the VLDL triacylglycerol are assembled with apolipoprotein early in the secretory pathway, forming a triacylglycerol-rich lipid-protein particle into which further phosphoglyceride is introduced just prior to its secretion as mature VLDL.
