A human genome-wide screen for regulators of clathrin-coated vesicle formation reveals an unexpected role for the V-ATPase.

Patrycja Kozik, Nicola A Hodson, Daniela A Sahlender, Nikol Simecek, Christina Soromani, Jiahua Wu, Lucy M Collinson, Margaret S Robinson

Index: Nat. Cell Biol. 15(1) , 50-60, (2013)

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Abstract

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.