Reconstitution into proteoliposomes and partial purification of the Golgi apparatus membrane UDP-galactose, UDP-xylose, and UDP-glucuronic acid transport activities.
Previous studies in vitro on proteoglycan biosynthesis from our laboratory have shown that nucleotide sugar precursors of all the sugars of the linkage oligosaccharides (xylose, galactose, and glucuronic acid) and of the glycosaminoglycans (N-acetylglucosamine, N-galactosamine, and glucuronic acid) are transported by specific carriers into the lumen of Golgi vesicles. More recently, we also reported the reconstitution in phosphatidylcholine liposomes of detergent-solubilized Golgi membrane proteins containing transport activities of CMP-sialic acid and adenosine-3'-phosphate-5'-phosphosulfate. We have now completed the successful reconstitution into liposomes of the Golgi membrane transport activities of UDP-galactose, UDP-xylose, and UDP-glucuronic acid. Transport of these nucleotide sugars into Golgi protein proteoliposomes occurred with the same affinity, temperature dependence, and sensitivity to inhibitors as observed with intact Golgi vesicles. Preloading of proteoliposomes with UMP, the putative antiporter for Golgi vesicle transport of these nucleotide sugars, stimulated transport of the nucleotide sugars by 2-3-fold. Transport of UDP-xylose into Golgi protein proteoliposomes was dependent on the presence of endogenous Golgi membrane lipids while that of UDP-galactose and UDP-glucuronic acid was not. This suggests a possible stabilizing or regulatory role for Golgi lipids on the UDP-xylose translocator. Finally, we have also shown that detergent-solubilized Golgi membrane translocator proteins can be partially purified by an ion-exchange chromatographic step before successful reconstitution into liposomes, demonstrating that this reconstitution approach can be used for the biochemical purification of these transporters.
Milla
M E
ME
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester 01655.
Clairmont
C A
CA
Hirschberg
C B
CB
D000818:Animals; D001692:Biological Transport; D006056:Golgi Apparatus / Q000378:metabolism*; D066298:In Vitro Techniques; D050356:Lipid Metabolism; D008297:Male; D011510:Proteolipids / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013696:Temperature; D014531:Uridine Diphosphate Galactose / Q000302:isolation & purification / Q000378:metabolism*; D014535:Uridine Diphosphate Glucuronic Acid / Q000302:isolation & purification / Q000378:metabolism*; D014540:Uridine Diphosphate Xylose / Q000302:isolation & purification / Q000378:metabolism*
D011510:Proteolipids; C020485:proteoliposomes; D014535:Uridine Diphosphate Glucuronic Acid; D014531:Uridine Diphosphate Galactose; D014540:Uridine Diphosphate Xylose