Figure 3. Expression of non-codon-optimized EGFP and codon-optimized ymUkG1 as fusion-tagged proteins to report PPIs using Gγ recruitment systems.

(A) Flow cytometry analyses using the Gγ recruitment system for cytosolic target proteins. The Fc protein was used as the cytosolic target protein ‘X’ and was expressed as a fusion protein with Gγ_cyto (Gγ_cyto-Fc). Membrane-anchored Z variants (Z_WT, Z_K35A,, Z_I31A and Z₉₅₅) were expressed as ‘Y’ library candidate proteins. ‘Control’ indicates BFG2118 and UGFG2 yeast strains harboring the pGK413 mock plasmid (without the expression of ‘Y’). (B) Flow cytometry analyses using the Gγ recruitment system for membrane protein targets. The Fc protein was used as the membrane target protein ‘X’ and was expressed as a membrane-associated protein with the C-terminal lipid anchor (derived from Ste18p). Four Z variants (Z_WT, Z_K35A,, Z_I31A and Z₉₅₅) were used as cytosolic candidate ‘Y’ proteins and were expressed as fusion proteins with Gγ_cyto (Gγ_cyto-Z variants). ‘Control’ indicates the FC-G0 and UG2-FCG0 yeast strains (without the expression of ‘Gγ_cyto-Y’). The engineered strains were grown in media containing 5 μM α-factor, and mean fluorescence intensities (MFIs) were analyzed. The MFIs of 10,000 cells were measured by flow cytometry.