Reconstitution of ATP- and cytosol-dependent transport of de Novo synthesized ceramide to the site of sphingomyelin synthesis in semi-intact cells
Reconstitution of ATP- and cytosol-dependent transport of de Novo synthesized ceramide to the site of sphingomyelin synthesis in semi-intact cells
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DOI：
10.1074/jbc.m004470200
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发表时间：
2000-09-29
影响因子：
4.8
通讯作者：
Hanada, K
中科院分区：
文献类型：
--
作者：
Funakoshi, T;Yasuda, S;Hanada, K
在半完整的中国仓鼠卵巢细胞中，重建了内质网合成的神经酰胺向存在鞘磷脂（SM）合成酶的高尔基室的运输。当穿孔细胞在15℃下由[H-3]鞘氨醇产生的[H-3]神经酰胺在37℃下进行处理时，[H-3]神经酰胺以依赖细胞溶胶的方式转化为-[H-3]SM。在动力学、ATP依赖性和温度依赖性等方面，穿孔细胞中[H-3]神经酰胺到-[H-3]SM的转化与完整细胞中一致。中国仓鼠卵巢细胞突变株LY-A的细胞质溶胶在穿孔的野生型细胞中不支持[H-3]神经酰胺向-[H-3] SM的转化，而野生型细胞质溶胶则恢复了穿孔的LY-A细胞的转化。Brefeldin a处理的细胞，内质网和高尔基体合并，不再需要细胞质将[H-3]神经酰胺转化为[H-3]SM。这些结果表明，在半完整细胞中[H-3]神经酰胺转化为[H-3]SM的实验是对神经酰胺依赖的细胞溶胶转运活性的可靠的体外实验，并且LY-A细胞在参与神经酰胺转运的胞质因子中存在缺陷。此外，在半完整细胞中，[H-3]神经酰胺向[H-3]葡萄糖神经酰胺的转化对细胞质的依赖性很小，这表明神经酰胺通过细胞质独立（或较少依赖）的途径到达葡萄糖神经酰胺合成位点。
Transport of ceramide synthesized at the endoplasmic reticulum to the Golgi compartment, where sphingomyelin (SM) synthase exists, was reconstituted within semi-intact Chinese hamster ovary cells. When [H-3]ceramide that had been produced from [H-3]sphingosine at 15 degrees C in perforated cells was chased at 37 degrees C, [H-3]ceramide-to-[H-3]SM conversion occurred in a cytosol-dependent manner. In various aspects (i.e. kinetics, ATP dependence, and temperature dependence), [H-3]ceramide-to-[H-3]SM conversion in perforated cells was consistent with that in intact cells. The cytosol from LY-A strain, a Chinese hamster ovary cell mutant defective in endoplasmic reticulum-to-Golgi transport of ceramide, did not support [H-3]ceramide-to-[H-3] SM conversion in perforated wild-type cells, whereas the wild-type cytosol rescued the conversion in perforated LY-A cells. Brefeldin A-treated cells, in which the endoplasmic reticulum and the Golgis apparatus were merged, no longer required cytosol for conversion of [H-3]ceramide to [H-3]SM. These results indicated that the assay of [H-3]ceramide-to-[H-3]SM conversion in semi-intact cells is a faithful in vitro assay for the activity of cytosol-dependent transport of ceramide and that LY-A cells are defective in a cytosolic factor involved in ceramide transport. In addition, conversion of [H-3]ceramide to [H-3]glucosylceramide in semi-intact cells was little dependent on cytosol, suggesting that ceramide reached the site of glucosylceramide synthesis by a cytosol-independent (or less dependent) pathway.