Brefeldin A enables synthesis of active lipoprotein lipase in cld/cld and castanospermine-treated mouse brown adipocytes via translocation of Golgi components to endoplasmic reticulum.
Brown adipocytes cultured from newborn combined-lipase-deficient (cld/cld) mice and castanospermine (CST)-treated 3T3-L1 adipocytes synthesize lipoprotein lipase (LPL) which is inactive and retained in the endoplasmic reticulum (ER) [Masuno, Blanchette-Mackie, Chernick and Scow (1990) J.Biol. Chem. 265, 1628-1638; Masuno, Blanchette-Mackie, Schultz, Spaeth, Scow and Okuda (1992) J. Lipid Res.33, 1343-1349]. Brefeldin A (BFA), which is known to block protein transport from ER and translocate Golgi components to ER, was used here to study the effect of translocated Golgi enzymes on LPL retained in ER of cld/cld and CST-treated mouse brown adipocytes. Brown adipocytes cultured from newborn normal mice contained 3000-5000 m-units of LPL activity/mg of DNA and secreted 35 m-units of LPL activity/mg of DNA per h. BFA at 10 micrograms/ml doubled LPL activity in normal cells within 2 h as it stopped completely secretion of active LPL. LPL in mouse cells has two N-oligosaccharide chains per subunit. Analyses with SDS/PAGE and immunoblotting showed that about one-third of LPL subunits in untreated normal cells were totally endo-beta-N-acetylglucosaminidase (endo H)-resistant, one-third were partially endo H-resistant, and one-third were totally endo H-sensitive. BFA decreased to zero the proportion of subunits which were totally endo H-resistant, while it increased the proportion which were partially endo H-resistant. Thus, BFA blocked processing of one oligosaccharide chain per subunit to endo H-resistance. Sucrose-gradient centrifugation studies showed that BFA increased the proportion of LPL subunits in normal cells which were present as active dimers. LPL activity in cld/cld adipocytes was 120 m-units/mg of DNA and that in normal adipocytes treated with CST was 430 m-units/mg of DNA. Most LPL subunits in such cells were totally endo H-sensitive and some were partially endo H-resistant, but none were totally endo H-resistant. Some of the subunits, in both cld/cld and CST-treated cells, were present as inactive LPL dimers. BFA increased LPL activity in cld/cld cells to 2100 m-units/mg of DNA and that in CST-treated cells to 2600 m-units/mg of DNA within 2 h. BFA increased in both groups the proportion of LPL subunits which were partially endo H-resistant. BFA also increased the proportion which were present as active dimers. Immunofluorescence studies in normal and cld/cld adipocytes showed that BFA caused retention of LPL in large tubular and spherical structures and in ER, but not in Golgi. When BFA was withdrawn and protein synthesis was blocked with cycloheximide, LPL in normal cells was transferred to Golgi within 30 min and disappeared within 60 min, whereas LPL in cld/cld cells was retained in large vesicles and ER. The findings indicate that BFA enabled synthesis of active LPL in cld/cld and CST-treated cells via translocation of Golgi components to ER. Also, cld/cld cells synthesized LPL which could be processed to active lipase and the enzymes needed for activation of the lipase were present in Golgi of such cells. Production of inactive LPL in cld/cld adipocytes probably results from their inability to transport LPL from ER to Golgi.
Park
J W
JW
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Blanchette-Mackie
E J
EJ
Scow
R O
RO
D017667:Adipocytes / Q000187:drug effects / Q000201:enzymology*; D002001:Adipose Tissue, Brown / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D000818:Animals; D000831:Animals, Newborn; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A; D002451:Cell Compartmentation; D003517:Cyclopentanes / Q000494:pharmacology*; D004721:Endoplasmic Reticulum; D006031:Glycosylation / Q000187:drug effects; D006056:Golgi Apparatus; D007212:Indolizines / Q000494:pharmacology; D008049:Lipase / Q000235:genetics; D008071:Lipoprotein Lipase / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008817:Mice, Mutant Strains; D015388:Organelles / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D054836:Indolizidines
D020126:Brefeldin A; D003517:Cyclopentanes; D007212:Indolizines; D008049:Lipase; D008071:Lipoprotein Lipase; C037806:castanospermine; D054836:Indolizidines