TRANSPORT OF UDP-GALACTOSE INTO THE GOLGI LUMEN REGULATES THE BIOSYNTHESIS OF PROTEOGLYCANS

The lumen of the Golgi apparatus is the subcellular site where galacto se is transferred, from UDP-galactose, to the oligosaccharide chains o f glycoproteins, glycolipids, and proteoglycans. The nucleotide sugar, which is synthesized in the cytosol, must first be transported into t he Golgi lumen by a specific UDP-galactose transporter, Previously, a mutant polarized epithelial cell (MDCKII-RCA(r)) with a 2% residual ra te of transport of UDP-galactose into the lumen of Golgi vesicles was described (Brandli, A. W., Hansson, G. C., Rodriquez-Boulan, E., and S imons, K. (1988) J. Biol. Chem. 263, 16283-16290). The mutant has an e nrichment in glucosyl ceramide and cell surface glycoconjugates bearin g terminal N-acetylglucosamine, as well as a 75% reduction in sialylat ion of cell surface glycoproteins and glycosphingolipids. We have now studied the biosynthesis of galactose containing proteoglycans in this mutant and the corresponding parental cell line. Wild-type Madin-Darb y canine kidney cells synthesize significant amounts of chondroitin su lfate, heparan sulfate, and keratan sulfate, while the above mutant sy nthesizes chondroitin sulfate and heparan sulfate but not keratan sulf ate, the only proteoglycan containing galactose in its glycosaminoglyc an polymer, The mutant also synthesizes chondroitin 6-sulfate rather t han only chondroitin 4-sulfate as wild-type cells. Together, the above results demonstrate that the Golgi membrane UDP-galactose transporter is rate-limiting in the supply of UDP-galactose into the Golgi lumen; this in turn results in selective galactosylation of macromolecules. Apparently, the K-m for galactosyltransferases involved in the synthes is of Linkage regions of heparan sulfate and chondroitin sulfate are s ignificantly lower than those participating in the synthesis of kerata n sulfate polymer, glycoproteins, and glycolipids. The results also su ggest that the 6-O-sulfotransferases, in the absence of their natural substrates (keratan sulfate) may catalyze the sulfation of chondroitin 4-sulfate as alternative substrate.