INTRACELLULAR DEGRADATION IN THE REGULATION OF SECRETION OF APOLIPOPROTEIN B-100 BY RABBIT HEPATOCYTES

Isolated rabbit hepatocytes were incubated with [S-35]methionine to la bel intracellular pools of apolipoprotein B (apo-B). The cells were th en reincubated with an excess of unlabelled methionine in the presence of oleate or protease inhibitors and the intracellular sites of accum ulation of radiolabelled apo-B and the mass of apo-B were determined b y isolation and analysis of subcellular fractions. Oleate or inhibitor s of metalloproteases (o-phenanthroline), serine proteases (aprotinin) , serine/cysteine proteases (leupeptin) or cysteine proteases (calpain inhibitor I; ALLN) but not aspartate proteases (pepstatin) resulted i n inhibition of the cellular degradation of apo-B. The effect of o-phe nanthroline was reversed by the addition of zinc ions. Oleate, o-phena nthroline and leupeptin also stimulated secretion of radioIabelIed apo -B; the effects of the inhibitors and oleate were additive, suggesting that they could act via different mechanisms. o-Phenanthroline caused accumulation of apo-B in the rough endoplasmic reticulum (RER) and sm ooth endoplasmic reticulum (SER) membranes; leupeptin caused accumulat ion of apo-B in the SER and cis-Golgi membranes, and ALLN and aprotini n caused accumulation of apo-B in the trans-Golgi membranes. These res ults suggest that intracellular degradation of apo-B occurs in the end oplasmic reticulum and in the trans-Golgi membranes and involves diffe rent proteases. Apo-B that accumulates in the ER membrane can be, dive rted into the lumen for secretion; however, apo-B that accumulates in the trans-Golgi membrane is irretrievably diverted from secretion.