Abstract
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.
Highlights
Glycosylation of glycoproteins, proteoglycans, and glycolipids occurring in the lumen of the Golgi apparatus requires the simultaneous presence of glycosyltransferases and nucleotide sugars in this compartment
Less than 10% of the initial Golgi membrane protein was recovered in this fraction, together with ϳ80% of the total GDP-fucose transport activity
We have identified, purified, and characterized the GDP-fucose transport activity from rat liver Golgi membranes
Summary
Glycosylation of glycoproteins, proteoglycans, and glycolipids occurring in the lumen of the Golgi apparatus requires the simultaneous presence of glycosyltransferases and nucleotide sugars in this compartment. While LAD I patients carry mutations in the genes that encode members of the integrin family, LAD II patients are thought to have a general enzymatic defect in the pathway that synthesizes GDPfucose from GDP-mannose (reviewed in Ref. 14). Even if this is true for some of these patients (reviewed in Ref. 14), most of them do not show an evident defect in sequence, expression levels, or enzymatic activity of the different enzymes involved in the GDP-fucose biosynthetic pathway. This paper is available on line at http://www.jbc.org cific and selective techniques in order to generate transfectant cells and the possibility of finding a nonstructural gene
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