Department of Biochemistry, Stanford University School of Medicine, Stanford, CA.
Departament de Biomedicina, Unitat de Biologia Cel·lular, Facultat de Medicina i Ciències de la Salut, Centre de Recerca Biomèdica CELLEX, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Universitat de Barcelona, Barcelona, Spain.
J Cell Biol. 2022 Feb 7;221(2). doi: 10.1083/jcb.202105060. Epub 2021 Dec 22.
We report here two genome-wide CRISPR screens performed to identify genes that, when knocked out, alter levels of lysosomal cholesterol or bis(monoacylglycero)phosphate. In addition, these screens were also performed under conditions of NPC1 inhibition to identify modifiers of NPC1 function in lysosomal cholesterol export. The screens confirm tight coregulation of cholesterol and bis(monoacylglycero)phosphate in cells and reveal an unexpected role for the ER-localized SNX13 protein as a negative regulator of lysosomal cholesterol export and contributor to ER-lysosome membrane contact sites. In the absence of NPC1 function, SNX13 knockdown redistributes lysosomal cholesterol and is accompanied by triacylglycerol-rich lipid droplet accumulation and increased lysosomal bis(monoacylglycero)phosphate. These experiments provide unexpected insight into the regulation of lysosomal lipids and modification of these processes by novel gene products.
我们在此报告了两项全基因组 CRISPR 筛选实验，旨在鉴定那些敲除后会改变溶酶体胆固醇或双（单酰基甘油）磷酸酯水平的基因。此外，这些筛选实验还在 NPC1 抑制条件下进行，以鉴定 NPC1 功能在溶酶体胆固醇外排中的调节剂。这些筛选实验证实了细胞中胆固醇和双（单酰基甘油）磷酸酯的紧密核心调控，并揭示了内质网定位的 SNX13 蛋白作为溶酶体胆固醇外排的负调节剂和内质网-溶酶体膜接触位点的贡献者的意外作用。在没有 NPC1 功能的情况下，SNX13 的敲低会重新分配溶酶体胆固醇，并伴有富含三酰基甘油的脂滴积累和溶酶体双（单酰基甘油）磷酸酯增加。这些实验为溶酶体脂质的调节以及通过新型基因产物对这些过程的修饰提供了意想不到的见解。
