Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. II. Processing, subunit assembly, and intracellular transport.
The biosynthesis and turnover of lipoprotein lipase (LPL) have been investigated in adipose 3T3-F442A cells labeled with [35S]methionine. Pulse-chase experiments, endo-beta-N-acetylglucosaminidase H treatment, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have indicated that LPL is synthesized in the endoplasmic reticulum as a glycoprotein of Mr = 55,500 bearing two N-oligosaccharide side chains of the high mannose-type. This precursor form of LPL is transported within 10 min to the Golgi apparatus, and this event is accompanied by the formation of a mature species of Mr = 58,000. Treatment of the Mr = 58,000 species with glycopeptidase F yielded a Mr = 51,000 protein similar to that observed after treatment of the Mr = 55,500 precursor form or after inhibition of N-glycosylation in tunicamycin-treated cells. The precursor form of LPL of Mr = 55,500 does not accumulate in the cells since, after a labeling period of 2 h, only the Mr = 58,000 species is detected. It is shown that only 20% of the newly synthesized molecules of Mr = 58,000 are constitutively secreted, whereas 80% are degraded, most likely in lysosomes, as indicated by the inhibitory effect of leupeptin upon the degradation process. Under heparin stimulation, quantitative secretion of the mature form of LPL takes place whereas the intracellular degradation is arrested. Heparin is able to mobilize intracellular LPL without changing the rate of LPL export from the endoplasmic reticulum to the cell surface. Sucrose gradient centrifugation of the material from intracellular cisternae shows that the Mr = 55,500 precursor form is present as a monomer (s = 4.1 S), whereas the Mr = 58,000 mature form is present as a homodimer (s = 6.8 S) to which LPL activity is associated. The results are interpreted as LPL being transiently stored under a dimeric form before its degradation. A sorting process of LPL in the Golgi apparatus, followed by its entry either mainly in a regulated pathway or in a constitutive pathway, is proposed.
Vannier
C
C
Centre de Biochimie, Centre National de la Recherche Scientifique (UPR 7300), Nice, France.
Ailhaud
G
G
D000818:Animals; D001692:Biological Transport; D002236:Carbohydrate Conformation; D002384:Catalysis; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000096:biosynthesis* / Q000378:metabolism; D051379:Mice; D009844:Oligosaccharides / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship
D009844:Oligosaccharides; D008071:Lipoprotein Lipase