LOCALIZATION OF O-GLYCAN INITIATION, SPHINGOMYELIN SYNTHESIS, AND GLUCOSYLCERAMIDE SYNTHESIS IN VERO CELLS WITH RESPECT TO THE ENDOPLASMIC RETICULUM-GOLGI INTERMEDIATE COMPARTMENT

The identification of an endoplasmic reticulum-Golgi intermediate comp artment (ERGIC), defined by the 53-kDa transmembrane marker protein ER GIC-53, has added to the complexity of the exocytic pathway of higher eukaryotic cells. Recently, a subcellular fractionation procedure was established for the isolation of the ERGIC from Vero cells (Schweizer, A., Matter, K., Ketcham, C. M., and Hauri, H.-P. (1991) J. Cell Biol, 113, 45-54) which provides a means to study more precisely the compar tmentalization of the various enzymic functions along the early secret ory pathway. Here, we have investigated if O-glycan initiation and sph ingomyelin synthesis are associated with the ERGIC by analyzing both t he responsible enzyme activities and their corresponding products. Mor eover, the synthesis of glucosylceramide, the precursor of most glycos phingolipids, was also analyzed. In the purified ERGIC fraction UDP. G alNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc transferas e) was only minimally enriched, sphingomyelin synthase was not enriche d, and UDP-glucose:ceramide glucosyl transferase specific activity was lower than in the homogenate. On Percoll gradients all three enzymes cofractionated with Golgi markers rather than ERGIC-53. Accordingly, s phingomyelin concentrations were extremely low in the ERGIC fraction. Double immunofluorescence localization of core N-acetylgalactosamine, the product of GalNAc transferase, by monoclonal antibodies against Ga lNAc-Ser/Thr (Tn antigen) revealed only little apparent overlap with E RGIC-53. This was particularly evident in brefeldin A-treated cells wh ich showed entirely different patterns of Tn antigens and ERGIC-53. Th e results suggest that in the secretory pathway of Vero cells O-glycan initiation and sphingomyelin as well as glucosylceramide synthesis ma inly occur beyond the ERGIC in the Golgi apparatus.