title: A human genome-wide screen for regulators of clathrin-coated vesicle formation reveals an unexpected role for the V-ATPase.
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.
authors.affiliation: Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK. pk275@cam.ac.uk
authors.forename: Patrycja
authors.initials: P
authors.lastname: Kozik
authors.forename: Nicola A
authors.initials: NA
authors.lastname: Hodson
authors.forename: Daniela A
authors.initials: DA
authors.lastname: Sahlender
authors.forename: Nikol
authors.initials: N
authors.lastname: Simecek
authors.forename: Christina
authors.initials: C
authors.lastname: Soromani
authors.forename: Jiahua
authors.initials: J
authors.lastname: Wu
authors.forename: Lucy M
authors.initials: LM
authors.lastname: Collinson
authors.forename: Margaret S
authors.initials: MS
authors.lastname: Robinson
mesh_terms: D002784:Cholesterol / Q000502:physiology; D022163:Clathrin-Coated Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis / Q000235:genetics*; D005372:Filipin / Q000378:metabolism; D055785:Gene Knockdown Techniques; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018942:Macrolides / Q000494:pharmacology; D034622:RNA Interference; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D018942:Macrolides; D005372:Filipin; C040929:bafilomycin A1; D002784:Cholesterol; D025262:Vacuolar Proton-Translocating ATPases