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Apolipoprotein B-100 (apoB-100) appears in three forms in the endoplasmic reticulum of Hep G2 cells: (1) tightly bound to the membrane, ie, not extractable by sodium carbonate. This form is glycosylated but protease sensitive when present in intact microsomes, suggesting that it is only partially translocated to the microsomal lumen; (2) extractable by sodium carbonate and present on low-density lipoprotein-verylow-density lipoprotein (LDL-VLDL)-like particles. This form is glycosylated and secreted into the medium; and (3) extractable by sodium carbonate but having a higher density than the LDL-VLDL-like particles. This form, referred to as Fraction I, is glycosylated and protected against proteases when present in intact microsomal vesicles, indicating that it is completely translocated to the luminal side of the microsomal membrane. Fraction I is not secreted into the medium, but it disappears with time from the cell, suggesting that it is degraded. Oleic acid induced a 2.7-fold increase in the rate of the biosynthesis of triacylglycerol but not of phosphatidylcholine in Hep G2 cells. Incubation of the cells with oleic acid had no significant effect on the rate of initiation of the apoB-100-containing lipoproteins, nor did it influence the amount of apoB-100 that was associated with the membrane or the turnover of apoB-100 in the membrane. Instead, it increased the proportion of the nascent apoB polypeptides on initiated lipoproteins that was converted into full-length apoB-100 on LDL-VLDL-like particles, giving rise to an increased amount of these particles in the lumen of the secretory pathway. Pulse-chase experiments showed that incubation with oleic acid gave rise to an increased formation of LDL-VLDL-like particles on behalf of the formation of Fraction I. This effect of oleic acid could partially explain the protective effect of the fatty acid on apoB-100, preventing it from undergoing posttranslational degradation. (Arterioscler Thromb. 1993;13:1743-1754 KEY WORDS • apoB-100 • Hep G2 cells • triacylglycerol biosynthesis A polipoprotein (apo) B-100 occurs mainly on the / \ liver-derived lipoproteins, ie, very-low-density li-JL A . poproteins (VLDLs), intermediate-density lipoproteins, and low-density lipoproteins (LDLs). ApoB-100 is essential for the assembly of these lipoproteins.The interaction between apoB-100 and the lipids can occur cotranslationally and simultaneously with the translocation of the protein to the lumen of the secretory pathway.1 ApoB-100 could also be bound to the endoplasmic reticulum (ER) membrane to be consigned to posttranslational degradation. 12 The incorporation of apoB into lipoprotein particles is first detected when the nascent polypeptide has reached a size of 80 to 100 kDa. These nascent polypeptides form particles with a diameter and density of a high-density lipoprotein (HDL) particle. As the nascent polypeptide increases in length, the size and lipid load of the particle that is being formed increase. Thus, the size of the nascent polypep-

Permeabilized Hep G2 cells have been used to investigate the turnover of apolipoprotein B-100 (apoB-100). When such cells were chased in the presence of buffer, there was no biosynthesis of apoB-100, nor was the protein secreted from the cells. Thus the turnover of apoB-100 in these cells reflected the posttranslational degradation of the protein. Pulse-chase studies indicated that apoB-100 was degraded both when associated with the membrane and when present as lipoproteins in the secretory pathway. Neither albumin nor ␣ 1 -antitrypsin showed any significant posttranslational intracellular degradation under the same condition. The kinetics for the turnover of apoB-100 in the luminal content differed from that of apoB-100 that was associated with the microsomal membrane. Moreover, while the degradation of the luminal apoB-100 was inhibited by N-acetylleucyl-leucyl-norleucinal (ALLN), this was not the case for the membrane-associated protein. Together these results suggest the existence of different pathways for the degradation of luminal apoB-100 and membrane-associated apoB-100. This was further supported by results from pulse-chase studies in intact cells, showing that ALLN increased the amount of radioactive apoB-100 that associated with the microsomal membrane during the pulse-labeling of the cells. However, ALLN did not influence the rate of turnover of the membrane-associated apoB-100.The presence of an ATP-generating system during the chase of the permeabilized cells prevented the disappearance of pulse-labeled apoB-100 from the luminal lipoprotein-associated pool. The ATP-generating system combined with cytosol protected the total apoB-100 in the system from being degraded. The cells cultured in the presence of oleic acid and chased after permeabilization in the presence of cytosol and the ATP-generating system showed an increase in the amount of apoB-100 present on dense ("high density lipoprotein-like") particles. This increase was linear during the time investigated (i.e. from 0 to 2 h chase) and independent of protein biosynthesis. Our results indicate that the dense particle was generated by a redistribution of apoB-100 within the secretory pathway and that it most likely was assembled from the membrane-associated form of apoB-100. These results indicate that the release of apoB-100 from this membrane-associated form to the microsomal lumen is dependent on cytosolic factors and a source of metabolic energy.

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We have used pulse-chase methodology to study the synthesis of apolipoprotein B in a human hepatomaderived cell line, the Hep G2 cells. A 2-min pulse with [35S]methionine was followed by a chase period varying from 5-90 min. A protein of large molecular mass (estimated molecular mass: 312 f 41 kDa, mean & SD, n = 8) could be immunoprecipitated from the cells at all chase periods between 5 min and 60 min with both monoclonal antibodies to a narrow density cut of the low density lipoprotein LDL-2 (density: 1.030-1.055 g/ ml) and polyclonal antibodies to the apolipoprotein B apoB 100 or to a narrow density cut of LDL-2 (density: 1.030 -1.055 g/ml). In addition to this large molecular mass protein, nascent polypeptides could be precipitated after 5, 10 and 15 min of chase.The apolipoprotein B molecules that had been labelled during the pulse disappeared from the cells after 60 -90 min of chase, while they started to appear in the medium after 30 -35 rnin of chase.The results obtained indicate (a) that apolipoprotein B is synthesized as one polypeptide with a large molecular mass, (b) that newly synthesized apolipoprotein B molecules are secreted after a delay of 30 -35 min, (c) that no intracellular accumulation of apolipoprotein B occurs, and (d) that apolipoprotein B is recovered in the density fraction < 1.21 g/ml of the medium suggesting that it is secreted in lipoprotein form.