Abstract
Glycosylation of glycoproteins, proteoglycans, and glycosphingolipids occurs mainly in the lumen of the endoplasmic reticulum and the Golgi apparatus. Nucleotide sugars, donors of all the sugars involved in Golgi glycosylation reactions, are synthesized in the cytoplasm and require specialized transporters to be translocated into the lumen of the Golgi apparatus. By controlling the supply of sugar nucleotides in the lumen of the Golgi apparatus, these transporters directly regulate the glycosylation of macromolecules transiting the Golgi. We have identified and purified the rat liver Golgi membrane UDP-N-acetylgalactosamine transporter. The transporter was purified to apparent homogeneity by a combination of conventional and dye color chromatography. An approximately 63,000-fold purification (6% yield) was achieved starting from crude rat liver Golgi membranes and resulting in a protein with an apparent molecular mass of 43 kDa. The transporter was active when reconstituted into phosphatidylcholine vesicles and could be specifically photolabeled with P3-(4-azidoanilido)-uridine-5'-[P1-32P]triphosphate, an analog of UDP-N-acetylgalactosamine. Native functional size determination on a glycerol gradient suggested that the transporter exists as a homodimer within the Golgi membrane.
Highlights
Glycoproteins, proteoglycans, and glycosphingolipids play fundamental roles in physiologic and pathologic processes, including cell growth, development, modulation of growth factors and transmembrane signals, oncogenesis, immune response, control of permeability in kidney basement membrane, and coagulation
Nucleotide sugars, donors of all of the sugars involved in Golgi glycosylation reactions, are synthesized in the cytoplasm and require specialized transporters to be translocated into the Golgi apparatus to participate in these biosynthetic events
We began the purification with a crude Golgi membrane preparation; the UDP-GalNAc transport activity was extracted after a two-step solubilization with Triton X-100
Summary
Glycoproteins, proteoglycans, and glycosphingolipids play fundamental roles in physiologic and pathologic processes, including cell growth, development, modulation of growth factors and transmembrane signals, oncogenesis, immune response, control of permeability in kidney basement membrane, and coagulation These glycoconjugates act as receptors for hormones, viruses, and bacterial toxins Nucleotide sugar transporters are of physiologic importance because mutant mammalian, yeast, and Leishmania cells deficient in these transport activities have a defect in the biosynthesis of the corresponding macromolecules (9 –12). This defect seems to be quantitative and qualitative. No cells have been identified that display a defect in the transport
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