Figure 2

Schematic diagram of competitor-introduced Gγ recruitment systems to screen affinity-altered protein variants for cytosolic or membrane target proteins.

(A) Schematic outline of the previously established competitor-introduced Gγ recruitment system for cytosolic target proteins. Target protein ‘X’ should be expressed as a fusion with Gγ_cyto in the cytosol. Protein ‘Y₁’ should be anchored to the plasma membrane, whereas ‘Y₂’ should be expressed in the cytosol. By establishing ‘Y₁’ and ‘Y₂’ as the parental (known) proteins originally bound to target ‘X’ and the candidate variant proteins, respectively, ‘Y₁’ and ‘Y₂’ compete to bind to target ‘X.’ When ‘X’ has higher affinity for ‘Y₂,’ G-protein signaling is prevented due to the inability of Gγ_cyto to migrate to 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. (B) Schematic outline of competitor-introduced Gγ recruitment system for membrane protein targets. Target protein ‘X’ is a transmembrane or membrane-associated protein. Protein ‘Y₁’ should be fused to Gγ_cyto, whereas ‘Y₂’ should be expressed in the cytosol. By establishing ‘Y₁’ and ‘Y₂’ as the parental (known) proteins originally bound to the membrane target ‘X’ and the candidate variant proteins, respectively, ‘Y₁’ and ‘Y₂’ compete to bind to target ‘X.’ When ‘X’ has a higher affinity for ‘Y₂,’ G-protein signaling is prevented due to the inability of Gγ_cyto to migrate to the plasma membrane. When ‘X’ has higher affinity for ‘Y₁’ fused to Gγ_cyto, G-protein signaling is transmitted into the yeast cells and initiates the mating process.