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Figure 6


Fig. 6. Rho proteins and GAP interactions. (A) Two-hybrid analysis of the AgRho1 proteins and potential GTPase-activating proteins. The C-terminal lipid-modification motif of the small GTP-binding proteins was truncated from the coding sequence to allow two-hybrid testing. The glutamate-to-histidine exchange in switch II mimics the GTP-bound state of the proteins. The histidine-to-tyrosine exchange is indicated by H39Y, the reverse mutation in AgRho1b by Y40H. The left panel shows a growth control on medium selective for prey and bait plasmids. The medium in the right panel is also selective for interaction. (B) Pull-down experiment in the presence of aluminium fluoride. Glutathion-S-Transferase (GST) or AgRho1a proteins fused to GST were bound to glutathion-sepharose and washed. The eluted proteins were subjected to western blot analysis. The blot was probed with {alpha}-GST and {alpha}-6HIS. (C) The H39Y mutation increases interaction strength of AgRho1a with AgLrg1. The indicated prey/bait combinations from A were additionally tested on medium with different concentrations of 3-aminotriazole to visualize the increase in interaction strength of the AgRho1aH39Y mutant. The first image on the left side is the growth control. (D) Two-hybrid analysis of Rho1 proteins from S. cerevisiae and Kluyveromyces lactis. The procedure used was identical to the one described for A.





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