Format

Send to

Choose Destination
Nat Cell Biol. 2013 Jan;15(1):50-60.

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

Author information

1
Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK. pk275@cam.ac.uk

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.

PMID:
23263279
PMCID:
PMC3588604
DOI:
10.1038/ncb2652
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center