TOPOGRAPHY OF GLYCOSYLATION AND UDP-XYLOSE PRODUCTION

In order to define the location and organization of the numerous react ions involved in polysaccharide assembly during synthesis of proteogly cans and glycoproteins, the topography of some of the glycosylation re actions in chondroitin sulfate synthesis was examined using a relative ly new technique for generating permeable cells. Permeable chondrocyte s were shown to directly take up nucleotide sugar precursors and incor porate them into chondroitin sulfate proteoglycan (CSPG), allowing spe cific labeling at each step in chondroitin sulfate synthesis. Subcellu lar fractionation following labeling with UDP-[C-14]xylose, UDP-[C-14] galactose, UDP-[C-14]glucuronic acid, or [S-35]PAPS localized the labe led CSPG to the compartment where each glycosylation reaction occurred . From these experiments it appears that xylose addition begins in the endoplasmic reticulum and continues in the Golgi apparatus where gala ctose, glucuronic acid, and sulfate are added. This conclusion was con firmed by direct visualization of xylose incorporation using electron microscopic autoradiography (Vertel, B. M., Walters, L. M., Flay, N., Kearns, A. E., and Schwartz, N. B. (1993) J. Biol. Chem. 268, 11105-11 112). Further examination of xylose addition showed that permeable cho ndrocytes can utilize both exogenous UDP-xylose transported into the l umen and UDP-xylose generated from UDP-glucuronic acid within the lume n. The enzyme responsible for this reaction, UDP-glucuronate carboxy-l yase, co-localized with xylosyltransferase activity in subcellular fra ctions. Orientation toward the lumen in subcellular compartments was d etermined by trypsin sensitivity in the permeable chondrocytes. Theref ore, we conclude that UDP-xylose can be produced in the lumen of the c ompartment where it is utilized in CSPG synthesis, obviating the need for a direct transport mechanism for this nucleotide sugar and providi ng close regulation of UDP-xylose and UDP-glucuronic acid levels.