RESOURCES Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway Jeremy C. Simpson1,7, Brigitte Joggerst2, Vibor Laketa2, Fatima Verissimo2, Cihan Cetin2, Holger Erfle2,6, Mariana G. Bexiga1, Vasanth R. Singan1, Jean-Karim Hériché3, Beate Neumann3, Alvaro Mateos2, Jonathon Blake4, Stephanie Bechtel5, Vladimir Benes4, Stefan Wiemann5, Jan Ellenberg2,3 and Rainer Pepperkok2,7 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. Within higher eukaryotic cells membrane traffic pathways connect the Extensive efforts over many years have revealed a significant number various membrane-bounded organelles, thereby ensuring that they of regulators associated with the secretory pathway. Early biochemical retain the correct complement of proteins and lipids to maintain approaches to identify individual machinery components have started cellular homeostasis. Typically, membrane traffic steps do not work to make way for more systematic strategies, including organellar in isolation, but rather are linked together in a carefully ordered proteomics and high-throughput imaging-based methods, in an sequence of events. The paradigm of this is the constitutive secretory effort to complete the cataloguing process5,6. More recently, RNA pathway, which ensures that newly synthesized material originating interference (RNAi)-mediated gene knockdown has been the method at the endoplasmic reticulum (ER) is correctly modified as it passes of choice, including two genome-wide secretion screens, in Drosophila through the Golgi complex and ultimately out to the cell surface. This cells7,8. Although these screens revealed several regulatory candidates, a pathway has the capacity to cope with a variety of cargo molecules, significant fraction (approximately 40%) do not seem to have a human and as such utilizes an extensive array of regulatory machinery1,2. At orthologue. Therefore, to discover how the secretion process is finely the heart of this machinery are cytoplasmic coat protein complexes tuned in human cells, we ideally require use of a human cell system. that identify cargo, reshape membranes into transport carriers and Largely as a result of the complex genetic architecture of mammalian provide links to other accessory proteins. In the early secretory systems, genome-wide RNAi screens in human cells are still relatively pathway it is the COPII and COPI coat complexes that perform these few in number. Indeed with regard to membrane traffic, the screens roles3,4, and therefore a greater understanding of their functional reported so far have targeted specific gene families9,10. We have previ- networks is a vital step towards understanding the regulation of the ously described a fully automated high content screening RNAi-based entire secretion process. approach to quantitatively monitor membrane transport in cultured 1School of Biology and Environmental Science & Conway Institute of Biomolecular and Biomedical Research, University College Dublin (UCD), Dublin 4, Ireland. 2Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany. 3MitoCheck Project Group, EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany. 4Gene Core, EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany. 5Division of Molecular Genome Analysis, German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. 6Present address: BioQuant Centre, University of Heidelberg, Im Neuenheimer Feld, 69120 Heidelberg, Germany. 7Correspondence should be addressed to J.C.S. or R.P. (e-mail: [email protected] or [email protected]) Received 13 January 2012; accepted 26 April 2012; published online 3 June 2012; DOI: 10.1038/ncb2510 764 NATURE CELL BIOLOGY VOLUME 14 j NUMBER 7 j JULY 2012 © 2012 Macmillan Publishers Limited. All rights reserved. RESOURCES a b No effect Mild inhibitorStrong inhibitor Secretion accelerator Preparation of siRNA-spotted chambers (22,627 genes/51,758 siRNAs/149 chambers) Plate HeLa Kyoto cells—reverse transfection (1.0×106 cells per chamber) 33 h at 37 °C Infect with recombinant adenoviruses (express CFP–tsO45G) 16 h at 39.5 °C Total CFP–tsO45G DAPI–nuclei Release tsO45G from ER 1 h at 32 °C Cell surface Fix cells, immunostain, image acquisition CFP–tsO45G (stain cell surface tsO45G, DAPI-stain nuclei) dev = +0.29 dev = +0.85 dev = +2.03 dev = –1.87 c Mild inhibitors Replicates QC siRNA mapping QC 4,551 siRNAs 3,005 siRNAs 2,469 siRNAs Primary secretion screen 4,176 genes 2,818 genes 2,308 genes Secretion inhibitors 700,000 images 4,120 siRNAs 4.1 replicates Strong inhibitors Replicates QC siRNA mapping QC 3,629 genes 3,587 siRNAs 2,050 siRNAs 1,651 siRNAs 3,273 genes 1,910 genes 1,531 genes e Strong inhibitors Validation screen Val. screen inhibitors d Strong inhibitors, dev >1.0 1,651 siRNAs 46,000 images 973 siRNAs 3,587 siRNAs 1,531 genes 4.6 replicates 757 genes Mild inhibitors, dev 0.75-1.0 4,551 siRNAs 3 ENSEMBL e!61 1,570 siRNAs 2 697 genes 1 Transcriptome sequencing 0 siRNA (1 – 51,758) Deviation –1 Expressed n.d. Not expressed 514 genes 40 genes 143 genes –2 –3 Further analysis 554 genes Figure 1 Primary and validation secretion screens in HeLa cells. (a) Overview inhibitors. (e) Results pipeline from validation secretion screens employing of experimental approach to monitor cell surface arrival of tsO45G in further siRNA sequences against each target gene identified in the primary downregulated cells. (b) Example images of cells showing either no effect, screen. Genes were considered as validated hits (697 genes) when secretion mild inhibition, strong inhibition or acceleration of secretion. Scale bar, could be inhibited by deviation values >1.0 with two or more independent 10 µm. (c) Results pipeline from primary secretion screens indicating quality siRNAs. Whole transcriptome sequencing established that 143 of these were control (QC) steps employed. From 22,627 genes, a total of 3,629 genes not expressed at detectable levels within the cells, and these candidates were found to inhibit secretion on knockdown. See also Supplementary were not included in further analyses. The remaining expressed genes (514) Table S1. (d) Graphical overview of effect on secretion of genome-wide and those that were not analysed in the transcriptome sequencing (40) were siRNA set used in this study (primary screen). Deviation values of 0.75–1.0 taken into secondary screens for further analysis. See also Supplementary were considered mild inhibitors; deviations >1.0 were considered strong Tables S4 and S5. human cells11, specifically assessing the arrival of a fluorescently labelled 22,000 human genes12 for interference with ER-to-plasma membrane transmembrane cargo protein at the cell surface. Here we apply and transport of the well-characterized secretory cargo membrane protein extend this approach, providing the first systematic survey of the human tsO45G (ref. 13). This approach allows single-cell quantification of genome with respect to the constitutive secretion process. the relative levels of fluorescently labelled tsO45G both inside the cell and at the plasma membrane (Fig. 1a,b). In total 700,000 images RESULTS (representing on average 4.1 replicates per siRNA) containing in total Genome-wide cell-based secretion screening 8.3 million cells were automatically acquired and transport of tsO45G We used our previously described RNAi-based high content screening to the plasma membrane was quantified11. We classified the secretion microscopy platform11 to screen a genome-wide library of over inhibitors as either mild (deviation 0.75–1.0) or strong (deviation >1.0) 51,000 small interfering RNAs (siRNAs) targeting approximately inhibitors (Fig. 1c,d). After taking account of replicate reproducibility NATURE CELL BIOLOGY VOLUME 14 j NUMBER 7 j JULY 2012 765 © 2012 Macmillan Publishers Limited. All rights reserved. RESOURCES and siRNA mapping (to ENSEMBL 61), the number of mild inhibitory inhibition of tsO45G transport (Fig. 2b and Supplementary Table S6). siRNAs was determined to be 2,469 siRNAs targeting 2,308 genes, and This increased detection of COPI subunits, compared with COPII the number of strong inhibitory siRNAs was 1,651 siRNAs targeting subunits, most likely reflects that isoforms exist for only two of the 1,531 genes. Compiling these data together revealed a final list of COPI subunits, and that all conventional secretory cargo must pass 3,629 genes, which on downregulation resulted in a marked inhibition through the Golgi