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Genome-wide RNAi screening identies 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 Hrich3, 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 inuencing secretion. Cloning, uorescent-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 various membrane-bounded organelles, thereby ensuring that they retain the correct complement of proteins and lipids to maintain cellular homeostasis. Typically, membrane traffic steps do not work in isolation, but rather are linked together in a carefully ordered sequence of events. The paradigm of this is the constitutive secretory pathway, which ensures that newly synthesized material originating at the endoplasmic reticulum (ER) is correctly modified as it passes through the Golgi complex and ultimately out to the cell surface. This pathway has the capacity to cope with a variety of cargo molecules, and as such utilizes an extensive array of regulatory machinery1,2. At
the heart of this machinery are cytoplasmic coat protein complexes that identify cargo, reshape membranes into transport carriers and provide links to other accessory proteins. In the early secretory pathway it is the COPII and COPI coat complexes that perform these roles3,4, and therefore a greater understanding of their functional networks is a vital step...