POSTTRANSLATIONAL MODIFICATIONS DISTINGUISH CELL-SURFACE FROM GOLGI-RETAINED BETA-1,4 GALACTOSYLTRANSFERASE MOLECULES - GOLGI LOCALIZATION INVOLVES ACTIVE RETENTION

beta 1,4 Galactosyltransferase (GalT) is a membrane-bound enzyme local ized predominantly to the trans-Golgi cisternae. Our previous studies have shown that the transmembrane domain of bovine GalT plays a critic al role in Golgi localization (Teasdale, R.D., D'Agostaro, G. and Glee son, P.A., J. Biol. Chem., 267, 4084-4096, 1992), Here we have compare d the localization and post-translational modifications of full-length bovine GalT with a GalT/hybrid molecule where the transmembrane domai n of GalT was replaced with that of the transferrin receptor. GalT/hyb rid molecules were expressed on the surface of transfected cells; howe ver, differences were observed in the distribution of the hybrid molec ules between transfected COS and murine L cells. In transfected COS ce lls, the GalT/hybrid protein was expressed efficiently at the cell sur face, with little Golgi-localized material, whereas in stable murine L cells, which expressed lower levels of the construct, hybrid molecule s were detected both at the cell surface and within the Golgi apparatu s. Expression of the GalT constructs in either COS or L cells produced two glycoprotein products which differed in molecular mass by 7 kDa. The difference in size between the two products is due to post-transla tional modifications which are inhibited by brefeldin A and are theref ore likely to occur in the trans-Golgi network (TGN). Very little of t he high-molecular-weight species was detected for full-length GalT, wh ereas it was a major product for the GalT/hybrid protein. Only the hig her molecular weight species was expressed at the cell surface. Thus, this additional 7 kDa post-translational modification distinguishes mo lecules retained within the Golgi apparatus (lower M(r) species) from those transported through the TGN to the cell surface. These studies i ndicate that (i) the level of expression influences the intracellular distribution of GalT/hybrid molecules and (ii) the localization of ful l-length GalT involves active retention within the Golgi stack, and no t retrieval from later compartments. After treatment of membrane prepa rations from stable L cell clones with a heterobifunctional cross-link ing agent, full-length bovine GalT molecules were found almost exclusi vely as high-molecular-weight aggregates, suggesting that GalT exists as an oligomer or aggregate. This ability to oligomerize may be a requ irement for Golgi retention.