Intracellular accumulation and oligosaccharide processing of alkaline phosphatase under disassembly of the Golgi complex caused by brefeldin A.
Electron microscopic observations showed that the fungal metabolite brefeldin A caused disassembly of the Golgi complex in human choriocarcinoma cells and accumulation of alkaline phosphatase (ALP) in the endoplasmic reticulum (ER) and nuclear envelope, where ALP was not apparently detectable in control cells. Pulse/chase experiments with [35S]methionine demonstrated that in the control cells, ALP synthesized as a 63-kDa precursor form was rapidly converted to a 66-kDa form, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type, which was expressed on the cell surface after 30 min of chase. In contrast, in the brefeldin-A-treated cells the precursor was gradually converted to a 65-kDa form, slightly smaller than the control mature form, which was not expressed on the cell surface even after a prolonged time of chase. Kinetics of the ALP processing in the brefeldin-A-treated cells demonstrated that the precursor was initially converted to an intermediate form, partially sensitive to endo-beta-N-acetylglucosaminidase H (endo H), then to an endo-H-resistant 65-kDa form. In addition, this form was found to be sensitive to neuraminidase digestion, though its sialylation was not so complete as that of the control mature form. Taken together, these results suggest that under disassembly of the Golgi complex caused by brefeldin A, oligosaccharide-processing enzymes including sialyltransferase, an enzyme in the trans Golgi cisterna(e) and/or the trans Golgi network, might be redistributed into the ER and involved in processing of the oligosaccharides of ALP accumulating there.
Takami
N
N
Department of Biochemistry, Fukuoka University School of Medicine, Japan.
Oda
K
K
Fujiwara
T
T
Ikehara
Y
Y
D000469:Alkaline Phosphatase / Q000378:metabolism*; D020126:Brefeldin A; D002822:Choriocarcinoma / Q000378:metabolism / Q000648:ultrastructure; D003517:Cyclopentanes / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000187:drug effects*; D006801:Humans; D007525:Isoelectric Focusing; D008854:Microscopy, Electron; D009844:Oligosaccharides / Q000378:metabolism*; D011233:Precipitin Tests; D014407:Tumor Cells, Cultured
D003517:Cyclopentanes; D009844:Oligosaccharides; D020126:Brefeldin A; D000469:Alkaline Phosphatase