LOCALIZATION OF 3 HUMAN POLYPEPTIDE GALNAC-TRANSFERASES IN HELA-CELLSSUGGESTS INITIATION OF O-LINKED GLYCOSYLATION THROUGHOUT THE GOLGI-APPARATUS

O-glycosylation of proteins is initiated by a family of UDP-N-acetylga lactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-T), In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcell ular fractionation, immunofluorescence and immunoelectron microscopy, We show that all three GalNAc-transferases are concentrated about tenf old in Golgi stacks over Golgi associated tubular-vesicular membrane s tructures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are prese nt throughout the Golgi stack suggesting that initiation of O-glycosyl ation may not be restricted to the cis Golgi, but occur at multiple si tes within the Golgi apparatus, GalNAc-T1 distributes evenly across th e Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the t rans side and in the medial part of the Golgi stack, respectively, Mor eover, we have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER, We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neith er by subcellular fractionation nor by situ glycosylation of an ER-ret ained form of CD8 (CD8/E19). However, upon relocation of chimeric GalN Ac-T1 or -T2 to the ER, CD8/E19 is glycosylated with different efficie ncies indicating that all components required for initiation of O-glyc osylation are present in the ER except for polypeptide GalNAc-transfer ases.