The assembly and secretion of apoB 100 containing lipoproteins in Hep G2 cells. Evidence for different sites for protein synthesis and lipoprotein assembly.
Pulse-chase studies combined with subcellular fractionation indicated that LpB 100 (i.e. the apoprotein B (apoB) 100 containing lipoproteins) was released to the lumen of the secretory pathway in a subcellular fraction enriched in smooth vesicles, and referred to as SMF (the smooth membrane fraction). The migration of SMF during gradient ultracentrifugation as well as kinetic studies indicated that the fraction was derived from a pre-Golgi compartment, probably the smooth endoplasmic reticulum (ER). Only small amounts of LpB 100 could be detected during these pulse-chase experiments in the subcellular fractions derived from the rough endoplasmatic reticulum (RER). SMF contained the major amount of the diacylglycerol acyltransferase activity present in the ER, while the major amount of membrane bound apoB 100 was present in the RER. Pulse-chase studies of the intracellular transfer of apoB 100 demonstrated the formation of a large membrane-bound preassembly pool in the ER, while no significant amount of apoB 100 radioactivity was present in the membrane of the Golgi apparatus. The maximal radioactivity of LpB 100, recovered from the ER or the Golgi lumen, was small compared with the radioactivity recovered from the ER membrane, indicating that the assembled LpB 100 rapidly leaves the cells. This in turn indicates that the rate-limiting step in the secretion of apoB 100 was the transfer of the protein from the ER membrane to the LpB 100 in the lumen. A portion of the intracellular pool of apoB 100 was not secreted but underwent posttranslational degradation.
Borén
J
J
Department of Medical Biochemistry, University of Göteborg, Sweden.
Wettesten
M
M
Sjöberg
A
A
Thorlin
T
T
Bondjers
G
G
Wiklund
O
O
Olofsson
S O
SO
D000118:Acetylglucosaminidase; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000096:biosynthesis* / Q000378:metabolism; D006528:Carcinoma, Hepatocellular; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient / Q000379:methods; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000096:biosynthesis / Q000302:isolation & purification; D008113:Liver Neoplasms; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008861:Microsomes / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure; D014407:Tumor Cells, Cultured / Q000378:metabolism*
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D008074:Lipoproteins; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase