Fig. 4. Competition between LPP and zyxin for the binding site of -actinin as revealed by the three-hybrid system. Yeast reporter strain Y190 was cotransfected with the prey vector pACT2 encoding the -actinin rod (residues 264-725) and the bait vector pBridge. The latter vector contained the sequence for zyxin (residues 1-42) in multiple cloning site I and the sequences for zyxin (residues 1-42) or LPP (residues 1-61 or 1-40) in multiple cloning site II. Note that multiple cloning site II is under the control of the MET25 promoter, which is active in the absence of methionine, but inactive in its presence. Colonies that grew on leucine- and tryptophan-deficient plates were inoculated into liquid medium lacking leucine and tryptophan (black bars) or, alternatively, lacking leucine, tryptophan and methionine (grey bars). Expression of the reporter gene for ß-galactosidase was analyzed by a colorimetric assay. The results represent the means with standard deviation from at least three independent determinations.

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