Yang Tong, Espenshade Peter J, Wright Michael E, Yabe Daisuke, Gong Yi, Aebersold Ruedi, Goldstein Joseph L, Brown Michael S
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75390, USA.
Cell. 2002 Aug 23;110(4):489-500. doi: 10.1016/s0092-8674(02)00872-3.
Using coimmunoprecipitation and tandem mass spectrometry, we identify INSIG-1 as an ER protein that binds the sterol-sensing domain of SREBP cleavage-activating protein (SCAP) and facilitates retention of the SCAP/SREBP complex in the ER. In sterol-depleted cells, SCAP escorts SREBPs from ER to Golgi for proteolytic processing, thereby allowing SREBPs to stimulate cholesterol synthesis. Sterols induce binding of SCAP to INSIG-1, as determined by blue native-PAGE, and this is correlated with the inhibition of SCAP exit from the ER. Overexpression of INSIG-1 increases the sensitivity of cells to sterol-mediated inhibition of SREBP processing. Mutant SCAP(Y298C) fails to bind INSIG-1 and is resistant to sterol-mediated inhibition of ER exit. By facilitating sterol-dependent ER retention of SCAP, INSIG-1 plays a central role in cholesterol homeostasis.
利用免疫共沉淀和串联质谱技术，我们鉴定出胰岛素诱导基因1（INSIG-1）是一种内质网（ER）蛋白，它能结合固醇调节元件结合蛋白裂解激活蛋白（SCAP）的固醇感应结构域，并促进SCAP/SREBP复合物滞留在内质网中。在固醇缺乏的细胞中，SCAP将SREBPs从内质网转运至高尔基体进行蛋白水解加工，从而使SREBPs能够刺激胆固醇合成。通过蓝色非变性聚丙烯酰胺凝胶电泳（blue native-PAGE）测定，固醇可诱导SCAP与INSIG-1结合，这与抑制SCAP从内质网输出相关。INSIG-1的过表达增加了细胞对固醇介导的SREBP加工抑制的敏感性。突变型SCAP（Y298C）无法结合INSIG-1，且对固醇介导的内质网输出抑制具有抗性。通过促进固醇依赖性的SCAP在内质网中的滞留，INSIG-1在胆固醇稳态中发挥核心作用。
