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

Nature Cell Biology volume 15, pages 50–60 (2013)Cite this article

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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.

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Figure 1: Summary of the multi-step screening strategy.
Figure 2: Genome-wide primary screen for surface accumulation of CCV cargo.
Figure 3: Secondary screen for efficiency of CME.
Figure 4: Secondary screen to analyse the morphology of CCSs at the PM.
Figure 5: Functional clusters within the hits.
Figure 6: siRNA-mediated depletion of V-ATPase or prolonged inhibition with BafA1 leads to accumulation of large, non-constricted CCSs at the PM.
Figure 7: Inhibition of V-ATPase does not lock clathrin in microcages.
Figure 8: Soluble cholesterol rescues function and morphology of the PM CCPs in the V-ATPase-depleted cells.

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Acknowledgements

We would like to thank M. Boutros (DKFZ) for providing the siRNA reannotation data, S. Arden (CIMR) for mRNA isolation, J. Bauer (Centre for Microarray Resources, Department of Pathology, Cambridge) for analysis of the microarray data, S. Grinstein (University of Toronto) for the pH sensor plasmids and helpful suggestions, J. Skepper (Multi-Imaging Centre, Department of Anatomy, University of Cambridge) for help with the critical-point drying, all members of the Robinson laboratory, in particular G. Borner, for invaluable discussions, and P. Luzio and J. Kilmartin for reading the manuscript and helpful suggestions. This work was financially supported by the Wellcome Trust.

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  1. Daniela A. Sahlender, Christina Soromani & Jiahua Wu

    Present address: Present addresses: Department for Fundamental Neurosciences, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland (D.A.S.); Department of Clinical Biochemistry, University College London Hospitals, 60 Whitfield Street, London W1T 4EU, UK (C.S.); Medimmune, Granta Park, Cambridge CB21 6GH, UK (J.W.),

Authors and Affiliations

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

    Patrycja Kozik, Nicola A. Hodson, Daniela A. Sahlender, Nikol Simecek, Christina Soromani, Jiahua Wu & Margaret S. Robinson

  2. London Research Institute, Lincoln’s Inn Fields Laboratories, London WC2A 3LY, 44 Lincoln’s Inn Fields, UK

    Lucy M. Collinson

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Contributions

P.K. and M.S.R. designed the research; P.K. and C.S. carried out the genome-wide screen; P.K. and N.A.H. carried out the secondary screens; D.A.S. performed the thin-section analysis; D.A.S. and L.M.C. performed the unroofing; P.K. and N.A.H. performed the V-ATPase experiments; P.K., N.A.H., D.A.S., N.S. and J.W. analysed data; P.K. and N.S. designed the web page; P.K. and M.S.R. supervised the project; and P.K. and M.S.R. wrote the manuscript.

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Correspondence to Patrycja Kozik, Daniela A. Sahlender, Christina Soromani, Jiahua Wu or Margaret S. Robinson.

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Kozik, P., Hodson, N., Sahlender, D. et al. A human genome-wide screen for regulators of clathrin-coated vesicle formation reveals an unexpected role for the V-ATPase. Nat Cell Biol 15, 50–60 (2013). https://doi.org/10.1038/ncb2652

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