Here we describe the protein interaction platform assay, a method for identifying interacting proteins in Saccharomyces cerevisiae. This assay relies on the reovirus scaffolding protein μNS, which forms large focal inclusions in living cells. When a query protein is fused to μNS and potential interaction partners are fused to a fluorescent reporter, interactors can be identified by screening for yeast that display fluorescent foci.A variety of methods have been developed to screen for interacting proteins in living cells, including yeast two-hybrid (Y2H) and protein-fragment complementation assays1 , 2. We recently demon-strated a complementary approach: the protein interaction plat-form (PIP) assay, in which we generate inclusions that serve as platforms for assembling interacting proteins in living mammalian cells. Here, we investigated whether this assay could be adapted to the yeast S. cerevisiae, in which (unlike in mammalian systems) plasmids for expressing proteins are readily maintained.PIP relies on the reovirus scaffolding protein μNS to form distinctively large focal cytoplasmic inclusions in mammalian cells3 ,4 . The C-terminal one-third of μNS (μNS residues 471-721) formed inclusions when expressed in S. cerevisiae (Fig. 1a). These inclusions did not perturb yeast growth, and mutations in the putative zinc-hook motif of μNS that disrupt the formation of platforms in mammalian cells 5 also disrupted their formation in yeast (Fig. 1b).The basic design of PIP as adapted to yeast involved fusing one protein (A) to μNS and a second protein (B) to GFP, so that if A and B interact, fluorescent green inclusions are visible in individual yeast cells (Fig. 1c,d). We created a series of recombination-based destination vectors using Gateway technology (Invitrogen) to facilitate the transfer of open reading frames such that they are fused to μNS at their N termini and expressed from either a constitutive (GPD) or an inducible (GAL1) yeast promoter. Many Gram-negative bacterial pathogens, including Shigella spp. and Salmonella spp., directly inject virulence proteins, called effectors, into host cells via specialized secretion systems. Some effectors require chaperones for delivery to the bacterial secretion apparatus. Shigella flexneri encodes four chaperones and at least 30
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