TRANSPORT OF NEWLY SYNTHESIZED GLUCOSYLCERAMIDE TO THE PLASMA-MEMBRANE BY A NON-GOLGI PATHWAY

High-gradient magnetic affinity chromatography (HIMAC) has been used t o obtain highly enriched plasma membranes, free of intracellular membr ane contaminants, from cultured Chinese hamster ovary (CHO) cells in y ields of greater-than-or-equal-to 80%. Using this procedure we have ch aracterized the transport of glucosylceramide (GlcCer) and the ganglio side GM3 to the plasma membrane. Newly synthesized GlcCer reaches the plasma membrane in 7.2 min, whereas GM3 requires 21.5 min to reach the plasma membrane. Brefeldin A prevents transport of newly synthesized GM3 and sphingomyelin to the plasma membrane but has no effect on the transport of GlcCer. Similarly, incubation of CHO cells at 15-degrees- C blocks transport of GM3 and sphingomyelin to the plasma membrane but has no effect on GlcCer movement. We propose that carrier-mediated tr ansport accounts for a major fraction of the plasma membrane GlcCer. P ulse-chase studies with either [H-3]glucose or [H-3]palmitate indicate that newly synthesized GlcCer which has reached the plasma membrane i s not utilized for the synthesis of GM3 but is instead rapidly either degraded or converted into an as yet unidentified product. Our results indicate that in addition to serving as a precursor for higher glycos ylation in the Golgi, a major fraction of newly synthesized GlcCer is rapidly transported to the plasma membrane by a non-Golgi pathway and then rapidly turned over.