Identification and purification of the rat liver Golgi membrane UDP-N-acetylgalactosamine transporter
Identification and purification of the rat liver Golgi membrane UDP-N-acetylgalactosamine transporter
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DOI：
10.1074/jbc.274.7.4474
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发表时间：
1999-02-12
影响因子：
4.8
通讯作者：
Hirschberg, CB
中科院分区：
文献类型：
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作者：
Puglielli, L;Mandon, EC;Hirschberg, CB
糖蛋白、蛋白多糖和鞘糖脂的糖基化主要发生在内质网和高尔基体的管腔内。核苷酸糖是参与高尔基体糖基化反应的所有糖的供体，在细胞质中合成，需要专门的转运体转移到高尔基体的管腔内。通过控制高尔基体管腔内糖核苷酸的供应，这些转运体直接调节高尔基体大分子的糖基化。我们鉴定并纯化了大鼠肝脏高尔基膜UDP-N-乙酰氨基半乳糖转运蛋白。通过常规色层析和染料色层析相结合，将转运蛋白纯化到明显的均一状态。从粗大鼠肝脏高尔基膜出发，获得了类似于63,000倍的纯化(产率为6%)，得到了一种表观分子质量为43 kDa的蛋白质。当转运蛋白被重组为磷脂酰胆碱囊泡时，转运蛋白是活跃的，并且可以被P-3-(4-azidoanilido)-uridine-5‘-[p(1)-P-32]triphosphate，(一种类似于UDP-N-乙酰半乳糖胺的类似物)特异性地光标记。对甘油梯度的天然功能大小的测定表明，转运蛋白以同源二聚体的形式存在于高尔基体膜中。
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 Golgis 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 similar to 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 P-3-(4-azidoanilido)-uridine-5'-[p(1)-P-32]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.