Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK.
Nat Cell Biol. 2013 Jan;15(1):50-60. doi: 10.1038/ncb2652.
Clathrin-mediated endocytosis is essential for a wide range of cellular functions. We used a multi-step siRNA-based screening strategy to identify regulators of the first step in clathrin-mediated endocytosis, formation of clathrin-coated vesicles (CCVs) at the plasma membrane. A primary genome-wide screen identified 334 hits that caused accumulation of CCV cargo on the cell surface. A secondary screen identified 92 hits that inhibited cargo uptake and/or altered the morphology of clathrin-coated structures. The hits include components of four functional complexes: coat proteins, V-ATPase subunits, spliceosome-associated proteins and acetyltransferase subunits. Electron microscopy revealed that V-ATPase depletion caused the cell to form aberrant non-constricted clathrin-coated structures at the plasma membrane. The V-ATPase-knockdown phenotype was rescued by addition of exogenous cholesterol, indicating that the knockdown blocks clathrin-mediated endocytosis by preventing cholesterol from recycling from endosomes back to the plasma membrane.
网格蛋白介导的内吞作用对于广泛的细胞功能至关重要。我们使用基于多步 siRNA 的筛选策略来鉴定网格蛋白介导的内吞作用第一步（在质膜处形成网格蛋白包被的囊泡 (CCV)）的调节剂。全基因组的初步筛选确定了 334 个导致 CCV 货物在细胞表面积累的命中点。二次筛选确定了 92 个抑制货物摄取和/或改变网格蛋白包被结构形态的命中点。这些命中点包括四个功能复合物的组成部分：包膜蛋白、V-ATPase 亚基、剪接体相关蛋白和乙酰转移酶亚基。电子显微镜显示，V-ATPase 耗竭导致细胞在质膜处形成异常的非收缩网格蛋白包被结构。通过添加外源性胆固醇可以挽救 V-ATPase 敲低表型，表明敲低通过阻止胆固醇从内体再循环回质膜来阻止网格蛋白介导的内吞作用。
