FIG. 3.

Effect of PP1-binding site mutations on the two-hybrid interaction between Reg1 and Glc7. (A) Two-hybrid interaction as measured by lexA_op-lacZ reporter gene expression. Strain KDY107 was transformed with combinations of plasmids that allowed the simultaneous expression of wild-type (WT) or mutant versions of LexA-Reg1 and a GAD fusion protein. Transformants were grown in SM lacking leucine and uracil and containing 5% glucose. β-Galactosidase activity was assayed as a measure of lexA_op-lacZ reporter gene expression. Stimulation of reporter gene expression by GAD-Glc7 reflects an interaction with LexA-Reg1. Each measurement is the average of four to nine independent transformants. Each error bar represents the standard deviation of the measurement. (B) Western blot analysis of wild-type and mutant LexA-Reg1 proteins. Cell extracts were prepared from transformants expressing GAD-Glc7 and the various LexA-Reg1 proteins. Twofold serial dilutions, as represented by the dark triangles, starting at 50 μg of protein, were loaded in reverse order onto an SDS–5.5% acrylamide gel. Proteins were transferred to nitrocellulose, and LexA-Reg1 fusion proteins were detected with monoclonal antibodies directed against LexA.