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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References
Behnia, R. & Munro, S. Organelle identity and the signposts for membrane traffic. Nature 438, 597–604 (2005).
Bonifacino, J. S. & Glick, B. S. The mechanisms of vesicle budding and fusion. Cell 116, 153–166 (2004).
Lee, M. C. & Miller, E. A. Molecular mechanisms of COPII vesicle formation. Semin. Cell Dev. Biol. 18, 424–434 (2007).
Beck, R., Rawet, M., Wieland, F. T. & Cassel, D. The COPI system: molecular mechanisms and function. FEBS Lett. 583, 2701–2709 (2009).
Simpson, J. C. Screening the secretion machinery: high throughput imaging approaches to elucidate the secretory pathway. Semin. Cell Dev. Biol. 20, 903–909 (2009).
Gilchrist, A. et al. Quantitative proteomics analysis of the secretory pathway. Cell 127, 1265–1281 (2006).
Bard, F. et al. Functional genomics reveals genes involved in protein secretion and Golgi organization. Nature 439, 604–607 (2006).
Wendler, F. et al. A genome-wide RNA interference screen identifiestwo novel components of the metazoan secretory pathway. EMBO J. 29, 304–314 (2010).
Farhan, H. et al. MAPK signaling to the early secretory pathway revealed by kinase/phosphatase functional screening. J. Cell Biol. 189, 997–1011 (2010).
Gordon, D. E., Bond, L. M., Sahlender, D. A. & Peden, A. A. A targeted siRNA screen to identify SNAREs required for constitutive secretion in mammalian cells. Traffic 11, 1191–1204 (2010).
Simpson, J. C. et al. An RNAi screening platform to identify secretion machinery in mammalian cells. J. Biotechnol. 129, 352–365 (2007).
Neumann, B. et al. Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes. Nature 464, 721–727 (2010).
Zilberstein, A., Snider, M. D., Porter, M. & Lodish, H. F. Mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein. Cell 21, 417–427 (1980).
Sato, K. & Nakano, A. Mechanisms of COPII vesicle formation and protein sorting. FEBS Lett. 581, 2076–2082 (2007).
Wendeler, M. W., Paccaud, J. P. & Hauri, H. P. Role of Sec24 isoforms in selective export of membrane proteins from the endoplasmic reticulum. EMBO Rep. 8, 258–264 (2007).
Scales, S. J., Pepperkok, R. & Kreis, T. E. Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI. Cell 90, 1137–1148 (1997).
Stephens, D. J., Lin-Marq, N., Pagano, A., Pepperkok, R. & Paccaud, J. P. COPI-coated ER-to-Golgi transport complexes segregate from COPII in close proximity to ER exit sites. J. Cell Sci. 113, 2177–2185 (2000).
Gurkan, C. et al. Large-scale profiling of Rab GTPase trafficking networks: the membrome. Mol. Biol. Cell 16, 3847–3864 (2005).
Dejgaard, S.Y. et al. Rab18 and Rab43 have key roles in ER–Golgi trafficking. J. Cell Sci. 121, 2768–2781 (2008).
Nuoffer, C., Davidson, H. W., Matteson, J., Meinkoth, J. & Balch, W. E. A GDP-bound of rab1 inhibits protein export from the endoplasmic reticulum and transport between Golgi compartments. J. Cell Biol. 125, 225–237 (1994).
Yang, C., Slepnev, V. I. & Goud, B. Rab proteins form in vivo complexes with two isoforms of the GDP-dissociation inhibitor protein (GDI). J. Biol. Chem. 269, 31891–31899 (1994).
Plutner, H. et al. Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments. J. Cell Biol. 115, 31–43 (1991).
Iinuma, T. et al. Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains. J. Cell Sci. 122, 1680–1690 (2009).
Belgareh-Touzé, N. et al. Yeast functional analysis: identification of two essential genes involved in ER to Golgi trafficking. Traffic 4, 607–617 (2003).
Uemura, T. et al. p31 deficiency influences endoplasmic reticulum tubular morphology and cell survival. Mol. Cell. Biol. 29, 1869–1881 (2009).
Kim, J. et al. Functional genomic screen for modulators of ciliogenesis and cilium length. Nature 464, 1048–1051 (2010).
Guo, Y. et al. Functional genomic screen reveals genes involved in lipid-droplet formation and utilization. Nature 453, 657–661 (2008).
Jacquier, N. et al. Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae. J. Cell Sci. 124, 2424–2437 (2011).
Pepperkok, R., Simpson, J. C. & Wiemann, S. Being in the right location at the right time. Genome Biol. 2, 1024.1–1024.4 (2001).
Schnell, U., Dijk, F., Sjollema, K. A. & Giepmans, B. N. G. Immunolabeling artifacts and the need for live-cell imaging. Nat. Methods 9, 152–158 (2012).
Simpson, J. C., Wellenreuther, R., Poustka, A., Pepperkok, R. & Wiemann, S. Systematic subcellular localisation of novel proteins identified by large-scale cDNA sequencing. EMBO Rep. 1, 287–292 (2000).
Meng, X. et al. CLIC2-RyR1 interaction and structural characterization by cryo-electron microscopy. J. Mol. Biol. 387, 320–334 (2009).
Singh, H., Cousin, M. A. & Ashley, R. H. Functional reconstitution of mammalian ‘chloride intracellular channels’ CLIC1, CLIC4 and CLIC5 reveals differential regulation by cytoskeletal actin. FEBS J. 274, 6306–6316 (2007).
Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).
Matsuyama, A. et al. ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nat. Biotech. 24, 841–847 (2006).
Simpson, J. C. & Pepperkok, R. Localizing the proteome. Genome Biol. 4, 240 (2003).
Szklarczyk, D. et al. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res. 39, D561–D568 (2011).
Hutt, D. M., Da-Silva, L. F., Chang, L. H., Prosser, D. C. & Ngsee, J. K. PRA1 inhibits the extraction of membrane-bound rab GTPase by GDI1. J. Biol. Chem. 275, 18511–18519 (2000).
Sivars, U., Aivazian, D. & Pfeffer, S. R. Yip3 catalyses the dissociation of endosomal Rab–GDI complexes. Nature 425, 856–859 (2003).
Liu, H. P., Wu, C. C. & Chang, Y. S. PRA1 promotes the intracellular trafficking and NF-kappaB signaling of EBV latent membrane protein 1. EMBO J. 25, 4120–4130 (2006).
Gougeon, P. Y., Prosser, D. C., Da-Silva, L. F. & Ngsee, J. K. Disruption of Golgi morphology and trafficking in cells expressing mutant prenylated rab acceptor-1. J. Biol. Chem. 277, 36408–36414 (2002).
Berg, T. J. et al. Splice variants of SmgGDS control small GTPase prenylation and membrane localization. J. Biol. Chem. 285, 35255–35266 (2010).
Wright, L. P. & Philips, M. R. Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras. J. Lipid Res. 47, 883–891 (2006).
Rojas, J. M., Oliva, J. L. & Santos, E. Mammalian son of sevenless Guanine nucleotide exchange factors: old concepts and new perspectives. Genes Cancer 2, 298–305 (2011).
Chuang, Y. Y., Valster, A., Coniglio, S. J., Backer, J. M. & Symons, M. The atypical Rho family GTPase Wrch-1 regulates focal adhesion formation and cell migration. J. Cell Sci. 120, 1927–1934 (2007).
Rollason, R., Korolchuk, V., Hamilton, C., Jepson, M. & Banting, G. A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells. J. Cell Biol. 184, 721–736 (2009).
Farhan, H. & Rabouille, C. Signalling to and from the secretory pathway. J. Cell Sci. 124, 171–180 (2011).
Erfle, H. et al. Reverse transfection on cell arrays for high content screening microscopy. Nat. Protoc. 2, 392–399 (2007).
Lowe, M. & Kreis, T. E. In vitro assembly and disassembly of coatomer. J. Biol. Chem. 270, 31364–31371 (1995).
Mahalanobis, P. On the generalized distance in statistics. Proc. Natl Inst. Sci. India 2, 49–55 (1936).
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.
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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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DOI: https://doi.org/10.1038/ncb2510
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