Protein interaction platforms: visualization of interacting proteins in yeast

Abstract

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

Identification of ZapD as a Cell Division Factor That Promotes the Assembly of FtsZ in Escherichia coli

“…The strains that conditionally express the fluorophore fusion plasmid were mated with those that conditionally express the NS fusion plasmid, and diploids were selected. Visualizations of live yeast expressing both the flurophore and NS fusions were conducted using 96-well glass-bottomed plates as described previously (48).…”

Systematic Analysis of Bacterial Effector-Postsynaptic Density 95/Disc Large/Zonula Occludens-1 (PDZ) Domain Interactions Demonstrates Shigella OspE Protein Promotes Protein Kinase C Activation via PDLIM Proteins

“…Derivatives for mammalian expression tagged at the C terminus with Myc were generated by cloning into pcDNA3 (Invitrogen) and pBABE-puro fragments containing the coding sequence, amplified by PCR with primers that include the sequence encoding Myc. For expression of genes in yeast for analysis by the protein interaction platform assay, appropriate sequences were PCR-amplified and cloned into pDNR221, pAG416, and pBYO11 plasmids, as described previously (12).…”

“…Protein Interaction Platform Assay and Analysis-Protein interaction assays in yeast assays were performed as described previously (12). Briefly, yeast cells were co-transformed with plasmids that encode NS or NS-PDLIM7 and GFP or GFPOspE1.…”

“…OspE expressed exogenously in mammalian cells was insoluble in nonionic detergent, likely due to its interactions with the cytoskeleton. Thus, to test whether OspE and PDLIM7 interact when co-expressed in vivo, a protein interaction assay was performed in yeast (12). PDLIM7 was fused to the reovirus scaffolding protein NS, which forms large focal inclusions in living cells.…”

Expanded Roles for Multicargo and Class 1B Effector Chaperones in Type III Secretion

“…This was the first evidence that T3SC could interact with multiple effectors and hence introduced the concept of "promiscuous" chaperones, i.e., chaperones that interact with multiple binding partners (35). In fact, a protein interaction platform assay has since revealed that Spa15 interacts with additional Shigella effectors, including IpgB2, OspB, OspC1, OspC2, OspD1, and OspD2 (for a total of nine) (67). Spa15 is involved in the efficient secretion of all nine of these effectors.…”