Fig. 3. Mapping of the interaction domains of Six3 and Grg5 using a yeast two-hybrid assay. (A) Alignment of eh1-like motif identified in mouse Six3 and Six6 with the corresponding eh1 motifs present in the Drosophila, C. elegans and mouse engrailed protein. (B) Mapping of the interaction domains by using a yeast two-hybrid assay. The strength of the interaction between each pair of proteins was reflected by the growth rate of the transformants on both uracil-selective and histidine-selective plates. The N terminus and SD (Six31-183) of Six3 bound to Grg5 similarly to the full-length Six3. Removal of amino acids 1-183 (Six3184-333) abolished the interaction with Grg5. Binding to Grg5 was restored when the construct Six31-120 was used. Six373-120 also interacted strongly with Grg5; however, Six3121-183 did not interact with Grg5. Construct Six3F88E, including a point mutation at position 88 of the eh1-like motif of Six3 (phenylalanine was replaced by glutamic acid), abolished the interaction with Grg5. The fragment containing the Q domain and four amino acids of the SP domain of Grg5 (Grg51-134) interacted with Six3, whereas the fragment containing the C terminus of Grg5 (Grg5135-197) did not.
