A NOVEL METHOD TO CO-LOCALIZE GLYCOSAMINOGLYCAN-CORE OLIGOSACCHARIDE GLYCOSYLTRANSFERASES IN RAT-LIVER GOLGI - COLOCALIZATION OF GALACTOSYLTRANSFERASE-I WITH A SIALYLTRANSFERASE

4-Methylumbelliferyl-beta-xyloside (Xyl beta MU) primes glycosaminogly can synthesis by first serving as an acceptor for the addition of 2 ga lactoses and 1 glucuronic acid residue to make the typical core struct ure, GlcUA beta 1, 3Gal beta 1,3Gal beta 1,4Xyl beta MU. To investigat e the relative localization of these biosynthetic enzymes, intact and properly oriented rat liver Golgi preparations were incubated with Xyl beta MU and 1 mu M UDP-[H-3]Gal and then chased with 5 mu M of unlabe led UDP-Gal, UDP-GlcUA, UDP-GlcNAc, UDP-GalNAc, and CMP-Neu5Ac. Under these conditions, no intervesicular transport occurs and acceptor labe ling depends entirely upon transporter-mediated delivery of the labele d sugar nucleotides into the lumen of a vesicle and co-localization of the appropriate glycosyltransferases. The labeled products were isola ted from the incubation medium and from within the Golgi and their str uctures analyzed by C18, anion-exchange, and amine adsorption high per formance liquid chromatography in combination with glycosidase digesti ons, Surprisingly, the major products within the Golgi mere two sialyl ated xylosides (Sia alpha 2,3Gal beta 1,4Xyl beta MU and Sia alpha 2,8 Sia alpha 2,3Gal beta 1,4Xyl beta MU) rather than the expected group o f partially completed GAG core structures, Less than 10% of the produc ts within the Golgi are the expected core structures containing a seco nd Gal residue or, in addition, GlcUA. The amount of the sialylated pr oducts is only partially decreased if the chase is omitted or if the c hase is done in the absence of added CMP-Sia, suggesting a pool of pre viously transported CMP-Sia drives synthesis of the major products, Co nversely, when detergent permeabilized vesicles are provided with high concentration of the same sugar nucleotides, the ratio of sialylated products is reduced and replaced by an increase in GAG-like products. These results argue that GAG core-specific Gal transferase I and II ar e not extensively co-localized within the same Golgi compartment. By c ontrast, glycosaminoglycan core Gal transferase I is substantially co- localized with an alpha-2,3-sialyltransferase and an alpha-2,8-sialylt ransferase. Incubating intact Golgi vesicles with exogenous diffusible accepters offers a novel method to assess the functional co-localizat ion of glycosyltransferases of multiple pathways within the Golgi comp artments.