GA2/GM2/GD2 synthase localizes to the trans-Golgi network of CHO-K1 cells

UDP-GalNAc :lactosylceramide/GM3/GD3 beta-1,4-N-acetyl-galactosaminyltransf erase (GalNAc-T) transforms its accepters into the gangliosides GA2, GM2 an d GD2, It is well established that it is a Golgi-located glycosyltransferas e, but its sub-Golgi localization is still unclear. We addressed this quest ion in Chinese hamster ovary K1 cell clones stably transfected with a c-myc -tagged version of GalNAc-T which express the enzyme at different levels of activity, In these cell clones we examined the effect of brefeldin A (BFA) on the synthesis of glycolipids (in metabolic-labelling experiments) and o n the sub-Golgi localization of the GalNAc-T (by immunocytochemistry), We f ound that in cell clones expressing moderate levels of activity, GalNAc-T i mmunoreactivity behaved as the trans-Golgi network (TGN) marker mannose-6-P receptor (M6PR) both in BFA-treated and untreated cells, and that BFA comp letely blocked the synthesis of GM2, GM1 and GD1a. On the other hand, in ce ll clones expressing high levels of activity and treated with BFA, most Gal NAc-T immunoreactivity redistributed to the endoplasmic reticulum, as did t he medial-Golgi marker mannosidase II, and the synthesis of GM2, GM1 and: G D1a was not completely blocked. These results indicate that GalNAc-T is a T GN-located enzyme and that the mechanism that localizes it to this compartm ent involves steps that, when saturated, lead to its mislocalization- to th e cis-, medial- or trans-Golgi. Changes of Golgi membrane properties by mod ification of local glycolipid composition due to the activity of the expres sed enzyme were not the main cause of mislocalization, since it persists wh en glycolipid synthesis is inhibited with D,L-threo-1-phenyl-2-hexadecandyl amino-3-pyrrolidino-1-propanol-HCl.