Oligomerization and topology of the Golgi membrane protein glucosylceramide synthase

Glucosylceramide synthase (GCS) catalyzes the transfer of glucose from UDP- glucose to ceramide to form glucosylceramide, the precursor of most higher order glycosphingolipids, Recently, we characterized GCS activity in highly enriched fractions from rat liver Golgi membranes (Paul, P., Kamisaka, Y., Marks, D. L., and Pagano, R. E. (1996) J. Biol. Chem. 271, 2287-2293), and human GCS was cloned by others (Ichikawa, S., Sakiyama, H., Suzuki, G., Hi dari, K. I.-P, J., and Hirabayashi, Y, (1996) Proc. Natl. Acad. Sci. U. S, A. 93, 4638-4643), However, the polypeptide responsible for GCS activity ha s never been identified or characterized. In this study, we made polyclonal antibodies against peptides based on the predicted amino acid sequence of human GCS and used these antibodies to characterize the GCS polypeptide in rat liver Gels membranes. Western blotting of rat liver Golgi membranes, hu man cells, and recombinant rat GCS expressed in bacteria showed that GCS mi grates as an similar to 38-kDa protein on SDS-polyacrylamide gels. Trypsini zation and immunoprecipitation studies with Gels membranes showed that both the C terminus and a hydrophilic loop near the N terminus of GCS are acces sible from the cytosolic face of the Golgi membrane, Treatment of Golgi mem branes with N-hydroxysuccinimide ester based cross-linking reagents yielded an similar to 50-kDa polypeptide recognized by anti-GCS antibodies; howeve r, treatment of similar to 10,000-fold purified Golgi GCS with the same rea gents did not yield cross-linked GCS forms. These results suggest that GCS forms a dimer or oligomer with another protein in the Golgi membrane, The m igration of solubilized Golgi GCS in glycerol gradients was also consistent with a predominantly oligomeric organization of GCS.