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 microNS, which forms large focal inclusions in living cells. When a query protein is fused to microNS and potential interaction partners are fused to a fluorescent reporter, interactors can be identified by screening for yeast that display fluorescent foci.

Figure 1

Development of PIP assay in yeast. (a,b) Yeast cells expressing wild-type (a) or mutant (b) alleles of GFP-μNS at 3–4 h post-induction of fusion protein expression. The left panel in a is a fluorescence image; the right panel shows simultaneous imaging of phase and fluorescence illumination. (c,d) Schematic and representative images of PIP. When fused to μNS, protein A is recruited to large platforms. If protein B does not interact with protein A (c), then the localization pattern of protein B is unaltered in the presence of μNS–protein A. Alternatively, if protein B interacts with protein A (d), then a protein B– GFP fusion appears as fluorescent foci. Scale bars are approximately 2 μm.

Figure 2

Detection of previously characterized chaperone-effector interactions via PIP. Each image is representative of yeast expressing designated effectors (e) and chaperones (c). The effectors and chaperones are fused to either GFP or μNS as designated. (−) indicates an absence of chaperones or effectors in these cells. (a) Yeast expressing nontoxic effectors IpaB and IpaC with or without their cognate chaperone IpgC. The image of GFP-IpgC (alone) is duplicated. (b) Yeast expressing toxic effectors and their respective chaperones, as indicated. IpgB2* refers to the mutant protein IpgB2 W62A. Images in the first three rows in b represent 3-s exposures normalized to display pixels visible over a defined linear range, thus demonstrating relative levels of expression of these effectors. Scale bars are approximately 2 μm.

Figure 3

S. flexneri effector-chaperone interactions by PIP. Yeast co-expressing the designated effectors and chaperones were visualized at 3–4 h post-induction of fusion protein expression. The first column shows yeast expressing each effector fused to GFP. The second through fifth columns show yeast co-expressing each effector fused to GFP along with each of the four chaperones fused to μNS. The last column shows yeast co-expressing the effectors fused to μNS and Spa15 fused to GFP. Scale bars are approximately 2 μm.