Detection principle behind the competitive Gγ recruitment system.

(A) Schematic of the Gγ_cyto–X and Y₁ genes. The Gγ_cyto–X fusion gene is designed to exclude the lipidation motif of the yeast endogenous Gγ (Ste18p). The lipidation motif is artificially attached to the Y₁ protein to be anchored on the plasma membrane. (B) Schematic outline of previously established Gγ recruitment system to detect PPIs using yeast G-protein signaling. When protein ‘X’ fused to Gγ_cyto interacts with protein ‘Y₁’, the Gβ and Gγ_cyto complex (Gβγ_cyto) migrates to the inner leaflet of the plasma membrane and restores the signaling function. If protein ‘X’ cannot interact with protein ‘Y₁’, Gβγ_cyto is released into the cytosol and signaling is blocked. (C) Schematic outline of the competitive method for creating affinity-altered proteins. Protein “Y₁” should be anchored on the plasma membrane, whereas “Y₂” should be expressed in the cytosol. By putting “Y₁” and “Y₂” as the parental (known) proteins originally bound to the target ‘X’ or to the candidate variant proteins, respectively, ‘Y₁’ and ‘Y₂’ compete to bind against target ‘X’. When “X” has higher affinity for “Y₂”, G-protein signaling is prevented by sequestration of Gγ_cyto from the plasma membrane. When “X” has higher affinity for “Y₁”, G-protein signaling is transmitted into the yeast cells and invokes the mating process. Thus, affinity-enhanced proteins or affinity-attenuated proteins can be screened in a specific manner. In our system, a transcription assay using the GFP reporter gene fused to the signal-responsive FIG1 gene allows detection of the signaling. Mating growth selection to isolate the methionine- and lysine-prototrophic diploids can also detect the signaling and permits the selective screening of signal-promoted cells.