A NEW APPROACH TO MAPPING COLOCALIZATION OF MULTIPLE GLYCOSYL TRANSFERASES IN FUNCTIONAL GOLGI PREPARATIONS

We have developed a new method to co-localize multiple glycosyl transf erases in different Golgi compartments, The approach relies on the pro ven ability of intact sealed rat liver Golgi preparations to concentra te exogenous labeled sugar nucleotides into the lumen where they glyco sylate either endogenous or artificial accepters, The premise is that if two glycosyl transferases are co-localized within the same compartm ent, they will compete for the limited amount of transported donor, If the donor is consumed in glycosylating al permeable artificial glycos ide within a Golgi compartment, it will be unavailable to glycosylate endogenous products within that same compartment. The greater the degr ee of transferase co-localization, the greater the potential deacrease in glycosylation of endogenous accepters, We provide an example consi stent with these predictions, Adding 1 mu M UDP[H-3]Gal to Golgi prepa rations followed by a chase with a cocktail of unlabeled sugar nucleot ides labels mostly endogenous N-linked oligosaccharides containing bot h beta 1,3- and beta 1,4[H-3]Gal residues with and without sialic acid , Addition of increasing amounts of 4-methylumbelliferyl-beta-xyloside (Xyl beta MU) produces [H-3]Gal1 beta,4Xyl beta MU and leads to a rec iprocal decrease in labeling of a restricted set of the endogenous acc epters. This decrease is preferential for [H-3]Gal beta 1 --> 3GlcNAc beta 1 --> R and, to a lesser extent, [H-3]Gal beta 1 --> 4GlcNAc beta 1 --> R structures in neutral and mono-sialylated oligosaccharides; s ynthesis of these structures in di- and tri-sialylated oligosaccharide s was unaffected, These preferential decreases are not seen in deterge nt permeabilized, sugar nucleotide transport-independent Golgi incubat ions, and are not due to inhibition by the Gal beta 1,4Xyl beta MU pro duct. These results argue that there is significant overlap in the fun ctional co-localization of sialyl and galactosyltransferases in rat li ver Golgi preparations and that GAG chain core specific Galactosyltran sferase I is co-localized with subsets of N-glycan Gal beta 1,3 and Ga l beta 1,4 transferases. This approach can be used with other glycosid es and sugar nucleotides to nap and co-localize other glycosyl transfe rases, The functional compartments defined by this approach may or may not correspond entirely with morphologically defined Golgi domains.