Genetic evidence for ATP-dependent endoplasmic reticulum-to-Golgi apparatus trafficking of ceramide for sphingomyelin synthesis in Chinese hamster ovary cells.
Genetic evidence for ATP-dependent endoplasmic reticulum-to-Golgi apparatus trafficking of ceramide for sphingomyelin synthesis in Chinese hamster ovary cells.
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中国仓鼠卵巢细胞中鞘胺的内质性网状设备的遗传学证据，用于鞘胺合成神经酰胺的遗传学证据。
DOI：
10.1083/jcb.144.4.673
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发表时间：
1999-02-22
影响因子：
7.8
通讯作者：
Hanada, K
中科院分区：
文献类型：
--
作者：
Fukasawa, M;Nishijima, M;Hanada, K
LY-A株是一种对鞘磷脂（SM）定向溶细胞素具有抗性的中国仓鼠卵巢细胞突变体，并且在SM从头合成方面存在缺陷。放射性丝氨酸，鞘氨醇和胆碱的代谢标记实验表明，LY-A细胞有缺陷的合成SM从这些前体，但不是神经酰胺（Cer），鞘糖脂，或磷脂酰胆碱的合成，表明一个特定的缺陷，在转换Cer SM在LY-A细胞。体外实验表明，在LY-A细胞中SM形成的特定缺陷不是由于负责SM合成或降解的酶活性的改变。当用布雷菲德菌素A处理细胞时，其导致高尔基体与内质网（ER）融合，LY-A细胞中的SM从头合成恢复到野生型水平。用荧光Cer类似物N-（4，4-二氟-5，7-二甲基-4-硼杂-3a，4a-二氮杂-s-引达省-3-戊酰基）-d-溴-鞘氨醇（C5-DMB-Cer），揭示了在野生型细胞中，C5-DMB-Cer以细胞内ATP依赖性方式从细胞内膜重新分布到高尔基体，LY-A细胞在C5-DMB-Cer的能量依赖性再分布中存在缺陷。在ATP耗竭条件下，野生型细胞中C5-DMB-Cer向C5-DMB-SM和[3 H]鞘氨醇向[3 H]SM的转化降低至LY-A细胞中的水平，其不受ATP耗竭的影响。糖基磷脂酰肌醇锚定或跨膜蛋白在LY-A细胞的ER到高尔基体的运输似乎是正常的。这些结果表明，ER的高尔基体运输Cer从头SM合成的主要途径是ATP依赖性的，这一途径几乎完全受损LY-A细胞。此外，在LY-A细胞中SM合成的特定缺陷表明鞘糖脂与SM合成的Cer转运途径不同。
LY-A strain is a Chinese hamster ovary cell mutant resistant to sphingomyelin (SM)-directed cytolysin and has a defect in de novo SM synthesis. Metabolic labeling experiments with radioactive serine, sphingosine, and choline showed that LY-A cells were defective in synthesis of SM from these precursors, but not syntheses of ceramide (Cer), glycosphingolipids, or phosphatidylcholine, indicating a specific defect in the conversion of Cer to SM in LY-A cells. In vitro experiments showed that the specific defect of SM formation in LY-A cells was not due to alterations in enzymatic activities responsible for SM synthesis or degradation. When cells were treated with brefeldin A, which causes fusion of the Golgi apparatus with the endoplasmic reticulum (ER), de novo SM synthesis in LY-A cells was restored to the wild-type level. Pulse–chase experiments with a fluorescent Cer analogue, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C5-DMB-Cer), revealed that in wild-type cells C5-DMB-Cer was redistributed from intracellular membranes to the Golgi apparatus in an intracellular ATP-dependent manner, and that LY-A cells were defective in the energy-dependent redistribution of C5-DMB-Cer. Under ATP-depleted conditions, conversion of C5-DMB-Cer to C5-DMB-SM and of [3H]sphingosine to [3H]SM in wild-type cells decreased to the levels in LY-A cells, which were not affected by ATP depletion. ER-to-Golgi apparatus trafficking of glycosylphosphatidylinositol-anchored or membrane-spanning proteins in LY-A cells appeared to be normal. These results indicate that the predominant pathway of ER-to-Golgi apparatus trafficking of Cer for de novo SM synthesis is ATP dependent and that this pathway is almost completely impaired in LY-A cells. In addition, the specific defect of SM synthesis in LY-A cells suggests different pathways of Cer transport for glycosphingolipids versus SM synthesis.