Identification and purification of the rat liver Golgi membrane UDP-N-acetylgalactosamine transporter

Glycosylation of glycoproteins, proteoglycans, and glycosphingolipids occur s mainly in the lumen of the endoplasmic reticulum and the Golgi apparatus. Nucleotide sugars, donors of all the sugars involved in Gels glycosylation reactions, are synthesized in the cytoplasm and require specialized transp orters to be translocated into the lumen of the Golgi apparatus. By control ling the supply of sugar nucleotides in the lumen of the Golgi apparatus, t hese transporters directly regulate the glycosylation of macromolecules tra nsiting the Golgi, We have identified and purified the rat liver Golgi memb rane UDP-N-acetylgalactosamine transporter. The transporter was purified to apparent homogeneity by a combination of conventional and dye color chroma tography, An similar to 63,000-fold purification (6% yield) was achieved st arting from crude rat liver Golgi membranes and resulting in a protein with an apparent molecular mass of 43 kDa, The transporter was active when reco nstituted into phosphatidylcholine vesicles and could be specifically photo labeled with P-3-(4-azidoanilido)-uridine-5'-[p(1)-P-32]triphosphate, an an alog of UDP-N-acetylgalactosamine. Native functional size determination on a glycerol gradient suggested that the transporter exists as a homodimer wi thin the Golgi membrane.