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.

UDP-sugar pyrophosphatase of rat retina: subcellular localization and topography.

Rat retinal tissue possesses as a developmentally regulated, highly active pyrophosphatase activity that hydrolyzes UDP-GalNAc and UDP-Gal but not CMP-NeuAc (Martina et al.: J Neurochem 62:1274-1280, 1995). We show here that this activity, measured with UDP-[3H]GalNAc as substrate, is associated to the membrane fraction of rat retinal homogenates and, upon subfractionation by isopycnic centrifugation in sucrose density gradients, is concentrated in fractions enriched in light Golgi membranes. We examined also the topographic disposition of the catalytic site of the enzyme in the transverse plane of the membranes by measuring the effect of protease treatment and of added EDTA on its activity. Pronase inhibited 50% of the translocation of UDP-[3H]GalNAc to the lumen of the Golgi vesicles but did not affect the enzyme activity either in the absence or in the presence of detergent. EDTA, a membrane-impermeant molecule, inhibited 90% of the activity of the enzyme but did not affect translocation of UDP-[3H]GalNAc and inhibited only 25% the incorporation of [3H]GalNAc into endogenous glycoconjugates. These results indicate that the translocation of UDP-[3H]GalNAc was not necessary for hydrolysis to occur and strongly suggest that the catalytic site of the UDP-sugar pyrophosphatase is oriented toward the cytosolic side of the Golgi vesicles. We speculate that this activity limits the availability of UDP-GalNAc to its specific translocator and, consequently, the luminal concentration of the nucleotide in the Golgi vesicles. In this way, by limiting the availability of UDP-GalNAc for the conversion of GM3 to GM2 by the GM3:N-acetyl-galactosaminyl transferase, it would contribute to the preferential use of GM3 for synthesis of GD3 and other "b" pathway gangliosides that are characteristic of the rat retina.

Functional coupling of glycosyl transfer steps for synthesis of gangliosides in Golgi membranes from neural retina cells.

The synthesis of the oligosaccharide of gangliosides is carried out in the Golgi complex by successive sugar transfers to proper glycolipid acceptors. To examine how the product of one glycosylation step couples with the next transfer step, the endogenous gangliosides of Golgi membranes from 14-day-old chick embryo retina were labeled from CMP-[3H]NeuAc or UDP-[3H]GalNAc or UDP-[3H]Gal in conditions which do not allow vesicular intercompartmental transport. After saturation of the endogenous acceptor capacity, labeling was mostly in the immediate acceptors of the corresponding labeled sugars. However, some labeled intermediates progressed to more glycosylated gangliosides if the membranes were incubated in a second step in the presence of the necessary unlabeled sugar nucleotides. This was particularly evident in the case of membranes incubated with UDP-[3H]Gal, in which most of the [3H]Gal-labeled lactosylceramide synthesized in the first step was converted to GM3 and GD3, or to GM2 or to GD1a in a second incubation step in the presence of unlabeled CMP-NeuAc alone, or together with UDP-GalNAc, or together with UDP-Gal plus UDP-GalNAc, respectively. Conversion was time dependent and dilution-independent. Since prior reports using brefeldin A indicate that transfer steps catalyzed by GalNAc-T, Gal-T2, and Sial-T4 localize in the trans-Golgi network (TGN), our results lead to the following major conclusions: (a) transfer steps catalyzed by GalNAc-T, Gal-T2, and Sial-T4 colocalize and are functionally coupled in the TGN; (b) proximal Golgi Gal-T1, Sial-T1, and Sial-T2, and their corresponding glycolipid acceptors, extend their presence to the TGN, and (c), GalNAc-T and Sial-T2 compete for a common pool of acceptor GM3 in the synthesis of GM2 and GD3.