Fig. 6. Protein interactions with GL3 versus gl3-sst. Comparison of protein interactions using a yeast two-hybrid assay between GL3 and gl3-sst with (A) GL1, (B) TTG1, (C) TRY. D shows the ability of TRY to compete for the GL1 binding sites of GL3. Using the pBridge vector (Clontech), a third protein (TRY) under the control of a methionine-repressible promoter was expressed in a yeast interaction assay comparing the interactions of GL3 or gl3-sst with GL1. The assay was performed at varying methionine concentrations (0 µM, 15 µM, 30 µM, and 125 µM). The GL3 and gl3-sst proteins were compared as GAL4 activation domain (AD) fusions whereas GL1, TTG1 and TRY were expressed as GAL4 binding domain fusions (BD) except in D where TRY was expressed as a free protein (no AD or BD domain). The samples were normalized by using OD₅₅₀ readings. The strength of the interaction was determined by using ß-galactosidase activity as measured using an ONPG assay.