COMPARTMENTAL ORGANIZATION OF THE SYNTHESIS OF GM3, GD3, AND GM2 IN GOLGI MEMBRANES FROM NEURAL RETINA CELLS

The relationship among lactosylceramide-(lacCer), GD3- and GM2-synthas es and between the two last transferases and their common GM3 acceptor was investigated in intact Golgi membrane from chick embryo neural re tina cells at early (8-days) and late (14 days) stages of the embryoni c development. [H-3]Gal was incorporated into endogenous glucosylceram ide by incubation of Golgi membranes with UDP-[H-3]Gal. Conversion of the synthesized [H-3]Gal-LacCer into GM3, and of the latter into GD3, GM2 and GD2 was examined after a second incubation step with unlabeled CMP-NeuAc and/or UDP-GalNAc. With CMP-NeuAc, most [H-3]Gal-LacCer was converted into GM3 in either 8- or 14- day membranes. However, while about 90% of GM3 was converted into GD3 in 8-day membranes, only about 25% followed this route in 14-day membranes. With CMP-NeuAc and UDP-G alNAc, about 90% of GM3 was used for synthesis of GM2 in 14-day membra nes, while in 8-day membranes about 80% followed the route to GD3, and a part to GD2. Performing the second incubation step in the presence of increasing detergent concentrations showed that conversion of GM3 t o GM2 was inhibited at concentrations lower than those required for in hibition of LacCer to GM3 conversion. Taken together, results indicate that transfer steps leading to synthesis of GM3, GD3, GM2 and GD2 fro m LacCer are functionally coupled in the Golgi membranes, and that GD3 - and GM2-synthases compete in a common compartment for using a fracti on of GM3 as substrate. In this competition, the relative activities o f the transferases and their relative saturation with the respective d onor sugar nucleotides, are important factors influencing conversion o f GM3 toward either GD3 or GM2.