Reevaluating the effect of Brefeldin A (BFA) on ganglioside synthesis: thelocation of GM2 synthase cannot be deduced from the inhibition of GM2 synthesis by BFA

Brefeldin A reversibly disassembles the Golgi complex, causing mixing of th e Golgi cisternae with the ER while the trans Golgi network persists as par t of a separate endosomal membrane system. Because of this compartmental se paration, Brefeldin A treatment has been used to map the sub-Golgi location s of several Golgi enzymes including GM2 synthase, We previously proposed t hat 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 prese nt study we show using GM2 synthase chimeras that GM2 synthesis was blocked by Brefeldin A when GM2 synthase was distributed throughout all Golgi subc ompartments or even when it was restricted to the medial Golgi, Because the se findings opposed our speculation regarding a distal location of this enz yme, we sought an alternative explanation for the inhibition of ganglioside synthesis by Brefeldin A. However, Brefeldin A did not degrade GM2 synthas e, prevent its homodimerization, or inhibit its in vitro activity, Brefeldi n A did result in the conversion of a portion of membrane bound GM2 synthas e into a soluble form which has minimal capability to produce GM2 in whole cells, However this conversion was not sufficient to explain the nearly tot al loss of GM2 production in intact cells in the presence of Brefeldin A, N evertheless, the results of this study indicate that Brefeldin A-induced in hibition of ganglioside synthesis cannot be used to deduce the location of GM2 synthase.