Fig. 4. Leu155 mutants of PDK1 do not interact with either PIF or the C–terminal fragment of PKA in the two-hybrid system. The Y190 yeast strain was transformed with vectors expressing the wild-type PDK1 or the indicated mutants of PDK1 fused to the Gal4 DNA-binding domain (GBD) together with vectors encoding the expression of either the 26 C–terminal residues of PRK2 (PIF) or the C–terminal fragment of PKA (PKA_CT residues 129–350) fused to a Gal4 activation domain (GAD). As a control, yeast were also co-transformed with vectors expressing the GAD and GBD domains only. The yeast were grown overnight at 30°C and β–galactosidase filter lift assays performed at 30°C for 4 h. An interaction between GBD–PDK1 and either GAD–PIF or GAD–PKA_CT induces the expression of β–galactosidase, which is detected as a blue colour (shown as black in the figure) in the filter lift assay.