Intracellular degradation in the regulation of secretion of apolipoprotein B-100 by rabbit hepatocytes

• Published in: Biochemical journal 1996-03, Vol.314 ( Pt 3) (3), p.977-984
• Main Authors: Cartwright, I J, Higgins, J A
• Format: Article
• Language: English
• Subjects: Animals; Apolipoprotein B-100; Apolipoproteins B - biosynthesis; Apolipoproteins B - metabolism; Aprotinin - pharmacology; Cells, Cultured; Chlorides - pharmacology; Endoplasmic Reticulum, Rough - metabolism; Endoplasmic Reticulum, Smooth - metabolism; Glycoproteins - pharmacology; Golgi Apparatus - metabolism; Homeostasis; Kinetics; Leupeptins - pharmacology; Liver - metabolism; Methionine - metabolism; Oleic Acid; Oleic Acids - pharmacology; Phenanthrolines - pharmacology; Protease Inhibitors - pharmacology; Rabbits; Sulfur Radioisotopes; Zinc Compounds - pharmacology
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3140977

Isolated rabbit hepatocytes were incubated with [35S]methionine to label intracellular pools of apolipoprotein B (apo-B). The cells were then reincubated with an excess of unlabelled methionine in the presence of oleate or protease inhibitors and the intracellular sites of accumulation of radiolabelled apo-B and the mass of apo-B were determined by isolation and analysis of subcellular fractions. Oleate or inhibitors of metalloproteases (o-phenanthroline), serine proteases (aprotinin), serine/cysteine proteases (leupeptin) or cysteins proteases (calpain inhibitor I; ALLN) but not aspartate proteases (pepstatin) resulted in inhibition of the cellular degradation of apo-B. The effect of o-phenanthroline was reversed by the addition of zinc ions. Oleate, o-phenanthroline and leupeptin also stimulated secretion of radiolabelled 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 smooth endoplasmic reticulum (SER) membranes; leupeptin caused accumulation of apo-B in the SER and cis-Golgi membranes, and ALLN and aprotinin caused accumulation of apo-B in the trans-Golgi membranes. These results suggest that intracellular degradation of apo-B occurs in the endoplasmic reticulum and in the trans-Golgi membranes and involves different proteases. Apo-B that accumulates in the ER membrane can be diverted into the lumen for secretion; however, apo-B that accumulates in the trans-Golgi membrane is irretrievably diverted from secretion.