Analysis of SCAP N-glycosylation and Trafficking in Human Cells
Analysis of SCAP N-glycosylation and Trafficking in Human Cells
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DOI：
10.3791/54709
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发表时间：
2016-11-01
影响因子：
1.2
通讯作者：
Guo, Deliang
中科院分区：
文献类型：
--
作者：
Cheng, Chunming;Guo, Jeffrey Yunhua;Guo, Deliang
脂肪生成增加是癌症和代谢性疾病的共同特征。固醇调节元件结合蛋白(SREBPs)是一个膜结合转录因子家族，控制胆固醇、脂肪酸和磷脂合成的重要基因的表达，在这些疾病中经常上调。在SREBP核转位过程中，SREBP裂解激活蛋白(SCAP)在SREBP从内质网(ER)转运到高尔基体以及随后的蛋白水解性激活中起着核心作用。最近，我们发现葡萄糖介导的SCAP的N-糖基化是SCAP/SREBP离开内质网并向高尔基体移动的先决条件。N-糖基化稳定SCAP，引导SCAP/SREBP转运。在这里，我们描述了一种分离人细胞膜组分的方法，并制备了用免疫印迹法检测SCAP N-糖基化和总蛋白的样品。我们进一步提供了一种利用共聚焦显微镜监测SCAP贩运的方法。该方法适用于哺乳动物细胞SCAP N-糖基化和转运的研究。
Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.