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. 2009 Sep 10;4(9):e6998. doi: 10.1371/journal.pone.0006998

Figure 8. SmDia/SmRho1-interaction in yeast.

Figure 8

Qualitative (A, B) and quantitative (C) Y2H analyses of SmDia-SmRho1 interaction. As bait, the RBD domain of SmDia was cloned into the bait vector pGBKT7 in fusion with the Gal4 DNA-binding domain (pGBKT7-DiaRBD). As prey, a mutated form of SmRho1 was generated lacking the CAAX-box at its C-terminal end and cloned into the pACT2 prey vector. Subsequently, this construct was employed to generate two variants containing point mutations at positions 15 and 64 (G15V or Q64L), respectively. A: Yeast cells were transformed with the pGBKT7-DiaRBD and the SmRho1 variants RhoWt (wildtype; 1), RhoG15V (2), or RhoQ64L (3) in pACT2. Transformed yeast cells were selected for the presence of the plasmids and for reporter gene expression (Trp−/Leu−/Ade−/His−). B: Control, the same transformed yeast cells grew on media selective for the presence of the plasmids (Trp−/Leu−). C: The β-Gal liquid assays (n = 2) demonstrated a reduced binding affinity for SmRho1 wildtype compared to the mutant forms RhoG15V or RhoQ64L, as indicated in A.