Background: How Golgi phosphoprotein 3 (GOLPH3) regulates Golgi localization of glycosyltransferases in mammalian cells is poorly understood. Results: GOLPH3 mediates incorporation of glycosyltransferases into coatomer-coated vesicles. Conclusion: GOLPH3 regulates localization of glycolsyltransferases by working as a coatomer adaptor. Significance: This study provides further insight into the molecular mechanism underlying regulation of glycosyltransferases localization by GOLPH3.

Mucin type O-glycans with core 2 branches are distinct from nonbranched O-glycans, and the amount of core 2 branched O-glycans changes dramatically during T cell differentiation. This oligosaccharide is synthesized only when core 2 ␤-1,6-N-acetylglucosaminyltransferase (C2GnT) is present, and the expression of this glycosyltransferase is highly regulated. To understand how O-glycan synthesis is regulated by the orderly appearance of glycosyltransferases that form core 2 branched O-glycans, the subcellular localization of C2GnT was determined by using antibodies generated that are specific to C2GnT. The studies using confocal light microscopy demonstrated that C2GnT was localized mainly in cis to medial-cisternae of the Golgi. We then converted C2GnT to a trans-Golgi enzyme by replacing its Golgi retention signal with that of ␣-2,6-sialyltransferase, which resides in trans-Golgi. Chinese hamster ovary cells expressing wild type C2GnT and the chimeric C2GnT were then subjected to oligosaccharide analysis. The results obtained clearly indicate that the conversion of C2GnT into a trans-Golgi enzyme resulted in a substantial decrease of core 2 branched oligosaccharides.These results, taken together, strongly suggest that the predominance of core 2 branched oligosaccharides in those cells expressing C2GnT is due to the fact that C2GnT is located earlier in the Golgi than ␣-2,3-sialyltransferase that competes with C2GnT for the common substrate. Furthermore, alteration of Golgi localization renders the chimeric C2GnT much less efficient in synthesizing core 2 branched oligosaccharides, indicating the critical role of orderly subcellular localization of glycosyltransferases.Leukosialin (CD43) is a major sialoglycoprotein present in leukocytes and heavily glycosylated by mucin-type O-glycans (1-5). This glycoprotein of human origin contains approximately 80 O-linked oligosaccharides in its extracellular domain consisting of 234 amino acids (1, 6). These O-linked oligosaccharides are highly sialylated and have been shown to exhibit an antiadhesive property (7). It has been also shown that the structure of oligosaccharides attached to leukosialin changes significantly during development of T cells. While resting human T lymphocytes express tetrasaccharides, NeuNAc␣233Gal␤133(NeuNAc␣236)GalNAc, activated T lymphocytes almost exclusively express branched hexasaccharides, NeuNAc␣233Gal␤133(NeuNAc␣233Gal␤134GlcNAc ␤136) GalNAc (8). Moreover, such change is associated with T cell development in thymus; while immature thymocytes in cortical thymus express the hexasaccharides, relatively mature medullary thymocytes express the tetrasaccharides (9).The conversion of O-glycan biosynthesis is due to the turning on or off of core 2 ␤-1,6-N-acetylglucosaminyltransferase (C2GnT).1 It has been demonstrated that activated T lymphocytes express a substantial amount of C2GnT activity, while resting T lymphocytes express negligible C2GnT activity (8). By in situ hybridization of the transcript, it has been shown that immature cortical thymo...

Modification of Golgi glycosyltransferases, such as formation of disulfide-bonded dimers and proteolytical release from cells as a soluble form, are important processes to regulate the activity of glycosyltransferases. To better understand these processes, six glycosyltransferases were selected on the basis of the donor sugars, including two N-acetylglucosaminyltransferases, core 1 beta1,3-N-acetylglucosaminyltransferase (C1-beta3GnT) and core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT-I); two fucosyltransferases, alpha1,2-fucosyltransferase-I (FucT-I) and alpha1,3-fucosyltransferase-VII (FucT-VII); and two sialyltransferases, alpha2,3-sialyltransferase-I (ST3Gal-I) and alpha2,6-sialyltransferase-I (ST6Gal-I). These enzymes were fused with enhanced green fluorescence protein and stably expressed in Chinese hamster ovary cells. Spectrofluorimetric detection and immunoblotting analyses showed that all of these glycosyltransferases except FucT-VII were secreted in the medium. By examining dimers formed in cells and culture media, we found that all of the enzymes, except ST3Gal-I, form a combination of monomers and dimers in cells, whereas the molecules released in the media are either exclusively monomers (C2GnT-I and ST6Gal-I), dimers (FucT-I) or a mixture of both (C1-beta3GnT). These results indicate that dimerization does not always lead to Golgi retention. Analysis of the N-glycosylation status of the enzymes revealed that the secreted proteins are generally more heavily N-glycosylated and sialylated than their membrane-associated counterparts, suggesting that the proteolytic cleavage occurs before the glycosylation is completed. Using FucT-I and ST6Gal-I as a model, we also show that these glycosyltransferases are able to perform autoglycosylation in the dimeric forms. These results indicate that different glycosyltranferases differ significantly in dimerization, proteolytic digestion and secretion, and autoglycosylation. These results strongly suggest that disulfide-bonded dimerization and secretion differentially plays a role in the processing and function of different glycosyltransferases in the Golgi apparatus.