<i>GFP</i> transcription assays to test the selection of affinity-attenuated proteins.

<p>(A–C) Flow cytometry analyses. Fluorescence intensities of the engineered strains expressing membrane-anchored Z_WT (A), Z_K35A (B) and Z_I31A (C) as the parental ‘Y₁’ proteins. White bars indicate control yeast strain lacking expression of ‘Y₁’ and ‘Y₂’ (transformed with pGK415 mock vector to BFG2118). (D–F) Fluorescence microscope observations. Fluorescence micrographs of the engineered strains expressing membrane-anchored Z_WT (D), Z_K35A (E) and Z_I31A (F) as the parental ‘Y₂’ proteins. Five Z variants (ZZ, Z_WT, Z_K35A, Z_I31A and Z₉₅₅) were expressed as the cytosolic ‘Y₂’ candidate proteins in each strain. To investigate transmission of the signal, 5 µM of α-factor was used for each strain. Standard errors of three independent experiments are shown.