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Detection principle behind the competitive Gγ recruitment system.

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posted on 2014-09-22, 02:56 authored by Misato Kaishima, Nobuo Fukuda, Jun Ishii, Akihiko Kondo

(A) Schematic of the Gγcyto–X and Y1 genes. The Gγcyto–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 Gγcyto interacts with protein ‘Y1’, the Gβ and Gγcyto complex (Gβγcyto) migrates to the inner leaflet of the plasma membrane and restores the signaling function. If protein ‘X’ cannot interact with protein ‘Y1’, Gβγcyto 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 Gγcyto 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.