Assembly of very low density lipoproteins in mouse liver: evidence of heterogeneity of particle density in the Golgi apparatus

The assembly of very low density lipoproteins (VLDL) by hepatocytes is beli eved to occur via a two-step process. The first step is the formation of a dense phospholipid and protein-rich particle that is believed to be convert ed to VLDL by the addition of bulk triglyceride in a second step. Previous studies in our laboratory led us to hypothesize a third assembly step that occurs in route to or in the Golgi apparatus. To investigate this hypothesi s, nascent lipoproteins were recovered from Golgi apparatus-rich fractions isolated from mouse liver. The Golgi fractions were enriched 125-fold in ga lactosyltransferase and contained lipoprotein particles averaging approxima tely 35 nm in diameter. These lipoproteins were separated by ultracentrifug ation into two fractions: d < 1.006 g/ml and d1.006-1.210 g/ml. The d < 1.0 06 g/ml fraction contained apolipoprotein B-100 (apoB-100), apoB-48, and ap oE, while the d1.006-1.210 g/ml fraction contained these three apoproteins as well as apoA-I and apoA-IV. Both fractions contained a 21-kDa protein th at was isolated and sequenced and identified as major urinary protein. Appr oximately 50% of the apoB was recovered with the denser fraction. To determ ine if these small, dense lipoproteins were secreted without further additi on of lipid, mice were injected with Triton WR1339 and [H-3]leucine, and th e secretion of apoB-100 and apoB-48 into serum VLDL (d < 1.006 g/ml) and d1 .006-1.210 g/ml fractions was monitored over a 2-h period. More than 80% of the newly synthesized apoB-48 and nearly 100% of the apoB-100 were secrete d with VLDL. These studies provide the first characterization of nascent li poproteins recovered from the Golgi apparatus of mouse liver. We conclude t hat these nascent hepatic Golgi lipoproteins represent a heterogeneous popu lation of particles including VLDL as well as a population of small, dense lipoproteins. The finding of the latter particles, coupled with the demonst ration that the primary secretory product of mouse liver is VLDL, suggests that lipid may be added to nascent lipoproteins within the Golgi apparatus.