Association of the Golgi UDP-galactose transporter with UDP-galactose: Ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum
Association of the Golgi UDP-galactose transporter with UDP-galactose: Ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum
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DOI：
10.1091/mbc.e03-03-0130
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发表时间：
2003-08-01
影响因子：
3.3
通讯作者：
van Meer, G
中科院分区：
文献类型：
--
作者：
Sprong, H;Degroote, S;van Meer, G
UDP-半乳糖通过UDP-半乳糖转运蛋白（UGT）到达高尔基体腔，用于蛋白质和脂质的半乳糖基化。神经酰胺和甘油二酯在内质网内通过UDP-半乳糖：神经酰胺半乳糖基转移酶进行半乳糖基化。UDP-半乳糖是如何从胞质转运到内质网的尚不清楚。我们将神经酰胺半乳糖基转移酶cDNA转染到CHOlec 8细胞中，该细胞具有缺陷的UGT并且没有内源性神经酰胺半乳糖基转移酶。与人UGT 1共转染极大地刺激了高尔基体中乳糖神经酰胺和内质网中半乳糖神经酰胺的合成。UDP-半乳糖直接输入到内质网，因为转染UGT显着增强半乳糖神经酰胺在内质网膜的合成。亚细胞分级和双标记免疫荧光显微镜显示，异位表达的UGT和神经酰胺半乳糖基转移酶的一个相当大的部分驻留在内质网的CHOlec 8细胞。当UGT在具有内源性神经酰胺半乳糖基转移酶的人肠细胞中表达时，观察到相同的结果。与此相反，在CHOlec 8单独转染UGT 1，转运定位专门的高尔基复合体。UGT和神经酰胺半乳糖基转移酶是完全洗涤剂可溶性的，并形成复合物，因为它们可以共免疫沉淀。我们的结论是，神经酰胺半乳糖基转移酶确保供应UDP-半乳糖在内质网腔中保留UGT的分子复合物。
UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex.