Intracellular degradation in the regulation of secretion of apolipoprotein B-100 by rabbit hepatocytes.
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.
Cartwright
I J
IJ
Department of Molecular Biology and Biotechnology, University of Sheffield, U.K.
Higgins
J A
JA
D000818:Animals; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000096:biosynthesis / Q000378:metabolism*; D007611:Aprotinin / Q000494:pharmacology; D002478:Cells, Cultured; D002712:Chlorides / Q000494:pharmacology; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism; D018871:Endoplasmic Reticulum, Smooth / Q000378:metabolism; D006023:Glycoproteins / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism; D006706:Homeostasis; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008099:Liver / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011817:Rabbits; D013462:Sulfur Radioisotopes; D017967:Zinc Compounds / Q000494:pharmacology
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D002712:Chlorides; D006023:Glycoproteins; D007976:Leupeptins; D009829:Oleic Acids; D010618:Phenanthrolines; D011480:Protease Inhibitors; D013462:Sulfur Radioisotopes; D017967:Zinc Compounds; C039304:calpain inhibitors; D019301:Oleic Acid; C016837:zinc chloride; D007611:Aprotinin; D008715:Methionine; C032854:leupeptin; C025205:1,10-phenanthroline