Glucosylceramide synthesized in vitro from endogenous ceramide is uncoupled from synthesis of lactosylceramide in Golgi membranes from chicken embryo neural retina cells.

It is known that ceramide (Cer), the precursor of sphingoglycolipids and of sphingomyelin, participates in events leading to activation of the apoptotic pathway, and per se or through conversion to glucosylceramide (GlcCer) modulates formation of neuritic processes in developing neurons. To learn about the fate of de novo synthesized Cer and GlcCer we examined, in Golgi membranes from chicken embryo neural retina cells, the metabolic relationships of endogenous Cer, GlcCer and lactosylceramide (LacCer). Incubation of the membranes with UDP-[3H]Glc revealed a pool of endogenous Cer useful for synthesis of GlcCer. Most of the GlcCer synthesized, however, was not used for synthesis of LacCer, indicating that it was functionally uncoupled from LacCer synthase. On the other hand, incubation with UDP-[3H]Gal revealed a pool of endogenous GlcCer that depending of the integrity of the membranes was functionally coupled to LacCer and ganglioside synthesis. These results indicate that most GlcCer formed in vitro from Cer is topologically segregated from the synthesis of LacCer. However, subfractionation in sucrose gradients of Golgi membranes labeled with both precursors failed to separate membranes enriched in [3H]GlcCer from those enriched in [3H]Gal-labeled LacCer. It is concluded that despite both transfer steps co-localize in the Golgi membranes, coupling of GlcCer synthesis to LacCer synthesis requires conditions not present in our in vitro assay. This suggests that a coupling activity exists that could be relevant for regulation of the cytoplasmic levels of Cer and GlcCer.