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Notes: Abstract: Brefeldin A (BFA) has been used extensively to study the intracellular transport and processing of proteins and sphingolipids because of its dramatic alteration of the structural and functional organization of the Golgi. We have examined the effect of BFA on the synthesis of galactosylceramide sulfate (SGalCer) and its immediate precursor galactosylceramide (GalCer) in an immortalized Schwann cell line (S16) to determine the intracellular sites of synthesis of these two related glycolipids. During a 6-h labeling period, a dose-dependent inhibition of [35S]sulfate incorporation into SGalCer was observed with 95% inhibition occurring at 0.5 µg/ml BFA. Labeling of newly synthesized galactosphingolipids with [3H]-palmitic acid for 6 h in the presence of BFA resulted in increased incorporation of label into GalCer containing nonhydroxy fatty acids (NFA-GalCer) to 162% of control values, whereas labeling of GalCer containing 2-hydroxy fatty acids (HFA-GalCer) was reduced to 63% of control. After 24 h, these values were at 366 and 91%, respectively. These results indicate that at least some of the HFA-GalCer was initially synthesized at a location distal to the BFA block and separate from the site of NFA-GalCer synthesis. Examination of [3H]palmitic acid incorporation into free ceramides showed an increase of 133 and 161% for hydroxy and nonhydroxy fatty acid ceramides, respectively, in cells treated for 6 h with BFA in comparison with levels found in untreated control cells, indicating that BFA did not block fatty acid 2-hydroxylation or the formation of HFA ceramide. Incorporation of [3H]palmitic acid into glucosylceramide and GM3 was increased over control levels whereas labeling of GM2 was inhibited, consistent with what has been reported previously for the effect of BFA on these glycolipids in other cell types. These results suggest that there are at least two separate intracellular sites for the galactosylation of HFA and NFA ceramide, respectively, which can be distinguished by their sensitivity to BFA. Our results also indicate that the site of GalCer sulfation is not redistributed to the endoplasmic reticulum in the presence of BFA and therefore may be localized to the distal Golgi or trans-Golgi network.