Figure 1.

The reverse double two-hybrid strategy. (A) Two bait proteins (X and Tsg101) are expressed as Gal4 binding domain (GBD) and LexA fusion, respectively, whereas the prey protein (Y) is expressed as a triple fusion between the Gal4 activation domain (GAD) and a triple repeat of the PTAP motif. GBD-X and GAD-Y-PTAP are expressed from plasmids pGBKT7-X and pACT2-Y-PTAP, while the LexA-Tsg101 construct is integrated in the genome of the yeast strain. Left panel: The interaction between X and Y activates the transcription of the UAS_GAL-driven URA3 reporter, which converts 5-FoA into a toxic compound, while the PTAP-mediated interaction between the Y fusion protein and Tsg101 activate the lexA_op-driven HIS3 reporter. Therefore, these cells can grow in the absence of histidine but are sensitive to 5-FoA. Middle panel: Truncating mutations in Y that prevent binding to X confer resistance to 5-FoA but also disrupt the PTAP-mediated interaction with Tsg101 and thus prevent growth in the absence of histidine. Right panel: Missense mutations in Y that prevent binding to X do not disrupt the PTAP-mediated interaction with Tsg101 and thus allow growth both in the presence of 5-FoA and in the absence of histidine. (B) Selection of missense mutations in glucokinase (Y protein is GK) that prevent its interaction with the glucokinase regulatory protein (X protein is GKRP). GK mutations were generated by PCR random mutagenesis and in vivo gap repair. Colonies growing on histidine-free medium with 5-FoA were selected and plasmids expressing mutated GAD-GK-PTAP were purified and tested for two-hybrid interaction with GBD-GKRP or LexA-Tsg101 in strains carrying a UAS_GAL- (Y187) or lexA_op- (CTY10-5d) driven lacZ reporter, respectively. ß-Galactosidase lift filter assays are shown for nine representative mutant clones.