Use of brefeldin A to define sites of glycosphingolipid synthesis: GA2/GM2/GD2 synthase is trans to the brefeldin A block.
Brefeldin A (BFA) induces the rapid redistribution of the Golgi complex into the endoplasmic reticulum (ER), causing the glycoproteins that are retained in the ER to be processed by Golgi enzymes. We have examined the effects of BFA on the synthesis of glycosphingolipids (GSL) to map the intracellular sites of GSL synthesis. In several cultured cell types, BFA inhibited the synthesis of the neutral GSL gangliotriaosylceramide (GA2) and monosialoganglioside GM2 and disialoganglioside GD2, where GD2 is GalNAc(beta 1----4)- [NeuAc(alpha 2----8)NeuAc(alpha 2----3)]Gal(beta 1----4)GlcCer, GM2 lacks the NeuAc(alpha 2----8) unit, and GA2 lacks both NeuAc(alpha 2----8) and NeuAc(alpha 2----3) units. The observed decrease in labeling of GA2, GM2, and GD2 in the presence of BFA was not due either to enhanced degradation of these glycolipids or to shedding of these glycolipids from the cells. In rat liver all three of these glycolipids have been shown by others to be synthesized by the same enzyme, GA2/GM2/GD2 synthase, which catalyzes the addition of N-acetylgalactosamine to lactosylceramide (Lac-Cer), GM3 [NeuAc(alpha 2----3)Gal(beta 1----4)GlcCer], and GD3 [NeuAc(alpha 2----8)NeuAc-(alpha 2----3)Gal(beta 1----4)GlcCer], respectively. Studies with a fluorescent glycolipid analog indicated that BFA redistributed the trans-Golgi stacks into a reticular pattern characteristic of the ER. These studies localize GA2/GM2/GD2 synthase, a key enzyme involved in the synthesis of complex gangliosides, to a compartment late in the intracellular trafficking pathway, which remains functionally distinct from the ER in the presence of BFA.
Young
W W
WW
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.
Lutz
M S
MS
Mills
S E
SE
Lechler-Osborn
S
S
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D020126:Brefeldin A; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000648:ultrastructure; D005700:Galactosyltransferases / Q000378:metabolism*; D006028:Glycosphingolipids / Q000096:biosynthesis*; D006056:Golgi Apparatus / Q000187:drug effects / Q000648:ultrastructure; D006801:Humans; D008545:Melanoma; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D000900:Anti-Bacterial Agents; D020126:Brefeldin A; D003517:Cyclopentanes; D005700:Galactosyltransferases; D006028:Glycosphingolipids; D017350:N-Acetylgalactosaminyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase