Reconstitution, identification, and purification of the rat liver golgi membrane GDP-fucose transporter.
Glycosylation of glycoproteins, proteoglycans, and glycolipids occurring in the Golgi apparatus requires the translocation of nucleotide sugars from the cytosol into the lumen of the Golgi. Translocation is mediated by specific nucleotide sugar transporters, integral Golgi membrane proteins that regulate the above glycosylation reactions. A defect in GDP-fucose transport into the lumen of the Golgi apparatus has been recently identified in a patient affected by leukocyte adhesion deficiency type II syndrome (Lubke, T., Marquardt, T., von Figura, K., and Korner, C. (1999) J. Biol. Chem. 274, 25986-25989). We have now identified and purified the rat liver Golgi membrane GDP-fucose transporter, a protein with an apparent molecular mass of 39 kDa, by a combination of column chromatography, native functional size determination on a glycerol gradient, and photoaffinity labeling with 8-azidoguanosine-5'-[alpha-(32)P] triphosphate, an analog of GDP-fucose. The purified transporter appears to exist as a homodimer within the Golgi membrane. When reconstituted into phosphatidylcholine liposomes, it was active in GDP-fucose transport and was specifically photolabeled with 8-azidoguanosine-5'-[alpha-(32)P]triphosphate. Transport was also stimulated 2-3-fold after preloading proteoliposomes with GMP, the putative antiporter.
Puglielli
L
L
Department of Molecular, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118, USA.
Hirschberg
C B
CB
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D051381:Rats
D008081:Liposomes; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; C020485:proteoliposomes; D006154:Guanosine Diphosphate Fucose; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid