Sprong Hein, Degroote Sophie, Nilsson Tommy, Kawakita Masao, Ishida Nobuhiro, van der Sluijs Peter, van Meer Gerrit
Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands.
Mol Biol Cell. 2003 Aug;14(8):3482-93. doi: 10.1091/mbc.e03-03-0130. Epub 2003 May 3.
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.
UDP-半乳糖通过UDP-半乳糖转运体（UGT）进入高尔基体腔，并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中由UDP-半乳糖：神经酰胺半乳糖基转移酶进行半乳糖基化。目前尚不清楚UDP-半乳糖是如何从细胞质转运到内质网中的。我们将神经酰胺半乳糖基转移酶cDNA转染到CHOlec8细胞中，该细胞的UGT有缺陷且没有内源性神经酰胺半乳糖基转移酶。与人UGT1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-半乳糖直接导入内质网，因为用UGT转染显著增强了内质网膜中半乳糖神经酰胺的合成。亚细胞分级分离和双标记免疫荧光显微镜显示，相当一部分异位表达的UGT和神经酰胺半乳糖基转移酶存在于CHOlec8细胞的内质网中。当UGT在具有内源性神经酰胺半乳糖基转移酶的人肠道细胞中表达时也观察到同样的情况。相反，在单独转染UGT 1的CHOlec8细胞中，转运体仅定位于高尔基体复合体。UGT和神经酰胺半乳糖基转移酶完全可溶于去污剂并形成复合物，因为它们可以被共免疫沉淀。我们得出结论，神经酰胺半乳糖基转移酶通过将UGT保留在分子复合物中来确保内质网腔中UDP-半乳糖的供应。
