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Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway

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Abstract

The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.

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Figure 1: Primary and validation secretion screens in HeLa cells.
Figure 2: Analysis of well-known membrane traffic regulators in the secretion screens.
Figure 3: Comparison with data from other large-scale phenotypic screens.
Figure 4: Analysis of the subcellular localization of candidates identified in the secretion screens.
Figure 5: Secondary screening analysis of the hit genes identified in the secretion screens.
Figure 6: Characterization of selected networks of genes identified as being important for regulation of secretion.
Figure 7: Functional link between EGF stimulation and secretory pathway function.

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Acknowledgements

We thank OSIS, Olympus Europe and the ALMF team at EMBL, Heidelberg for support. In addition, we acknowledge technical and bioinformatic help from J. Bulkescher, C. Conrad, U. Liebel, P. Rogers, C. Tischer and T. Walter. This project was financially supported by grants to J.E. within the MitoCheck consortium by the European Commission (FP6-503464) as well as by the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN) (NGFN-2 SMP-RNAi, FKZ01GR0403); and R.P. and S.W. (BMBF NGFN2 SMP-Cell and NGFN-Plus IG-CSG); and R.P. (Baden Württemberg Stiftung, Germany, Programme ‘siRNA’). The R.P. laboratory is also supported by the EU-funded network of excellence ‘Systems Microscopy’. A.M. was supported by a fellowship of the programme of Becas de Especialización en Organismos Internacionales of the Spanish Ministry of Education and Science (MEC). The J.C.S. laboratory is supported by a Principal Investigator (PI) award (09/IN.1/B2604) from Science Foundation Ireland (SFI).

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J.C.S. and R.P. designed the study and prepared the manuscript. J.C.S., B.J., V.L., F.V., H.E., M.G.B., J.B. and S.B. performed experiments; J.C.S., C.C., V.R.S., J-K.H., B.N., A.M. and R.P. analysed data; and V.B., S.W. and J.E. provided critical advice. All authors commented on the manuscript at the preparation stages.

Corresponding authors

Correspondence to Jeremy C. Simpson or Rainer Pepperkok.

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The authors declare no competing financial interests.

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Simpson, J., Joggerst, B., Laketa, V. et al. Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway. Nat Cell Biol 14, 764–774 (2012). https://doi.org/10.1038/ncb2510

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