Brefeldin A induces endoplasmic reticulum-associated O-glycosylation of galactosyltransferase

Recent data from several laboratories show that Brefeldin A (BFA) induces a microtubule-dependent back-flow of Golgi components to the endoplasmic reticulum (ER) thereby causing disassembly of the Golgi apparatus and its fusion with ER membranes. In order to delineate the effect of BFA on resident Golgi proteins, we investigated its effect on biosynthesis, maturation and intracellular transport of galactosyltransferase (gal-T), an established trans-Golgi enzyme. Using a protocol of metabolic labeling/immunoprecipitation followed by electrophoretic/fluorographic analysis, we show that in the presence of BFA, gal-T matures to a molecular form of 48.5 kD, a size intermediate between the 2 precursor forms of 44 and 47 and the mature form of 54 kD (Strous and Berger: J. Biol. Chem., 257:7623-28, 1982). Little mature form was detectable in the presence of BFA even after prolonged chase times of up to 28 hr. The intermediate form was sensitive to O-glycanase and endoglycosidase H, indicating early O-glycosylation without sialylation and lack of complex N-glycosylation, respectively. In order to define the compartment responsible for O-glycosylation in the presence of BFA, a temperature block of 25 degrees C was applied which inhibited recovery of Golgi elements from BFA-induced fusion with ER. At this temperature and in absence of BFA, biosynthesis of gal-T was not appreciably affected, while maturation was completely inhibited as indicated by the presence of unmodified precursor forms of gal-T. After 60 min preincubation with BFA, a time period sufficient to demonstrate complete fusion of Golgi with ER, subsequent biosynthesis of gal-T at 25 degrees C in absence of BFA led to the intermediate form, while precursor forms were not detectable. These data provide direct evidence for BFA-induced redistribution to the EF of Golgi enzymes involved in O-glycosylation and their early functional involvement in biosynthesis of newly synthesized gal-T.