Reevaluating the effect of Brefeldin A (BFA) on ganglioside synthesis: the location of GM2 synthase cannot be deduced from the inhibition of GM2 synthesis by BFA.
Brefeldin A reversibly disassembles the Golgi complex, causing mixing of the Golgi cisternae with the ER while the trans Golgi network persists as part of a separate endosomal membrane system. Because of this compartmental separation, Brefeldin A treatment has been used to map the sub-Golgi locations of several Golgi enzymes including GM2 synthase. We previously proposed that GM2 synthase might be located in a distal portion of the Golgi complex which in the presence of Brefeldin A would be separated from the substrate ganglioside GM3 present in the mixed ER-Golgi membrane system. In the present study we show using GM2 synthase chimeras that GM2 synthesis was blocked by Brefeldin A when GM2 synthase was distributed throughout all Golgi subcompartments or even when it was restricted to the medial Golgi. Because these findings opposed our speculation regarding a distal location of this enzyme, we sought an alternative explanation for the inhibition of ganglioside synthesis by Brefeldin A. However, Brefeldin A did not degrade GM2 synthase, prevent its homodimerization, or inhibit its in vitro activity. Brefeldin A did result in the conversion of a portion of membrane bound GM2 synthase into a soluble form which has minimal capability to produce GM2 in whole cells. However, this conversion was not sufficient to explain the nearly total loss of GM2 production in intact cells in the presence of Brefeldin A. Nevertheless, the results of this study indicate that Brefeldin A-induced inhibition of ganglioside synthesis cannot be used to deduce the location of GM2 synthase.
Young
W W
WW
Departments of Biological and Biophysical Sciences and Biochemistry and Molecular Biology, Schools of Dentistry and Medicine, University of Louisville, Louisville, KY 40292, USA.
Allende
M L
ML
Jaskiewicz
E
E
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology*; D016466:CHO Cells; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005678:G(M2) Ganglioside / Q000096:biosynthesis*; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014162:Transfection; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D020126:Brefeldin A; D005678:G(M2) Ganglioside; D017350:N-Acetylgalactosaminyltransferases; D011993:Recombinant Fusion Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase