The Gal3, Gal80, and Gal4 proteins of Saccharomyces cerevisiae comprise a signal transducer that governs the galactose-inducible Gal4p-mediated transcription activation of GAL regulon genes. In the absence of galactose, Gal80p binds to Gal4p and prohibits Gal4p from activating transcription, whereas in the presence of galactose, Gal3p binds to Gal80p and relieves its inhibition of Gal4p. We have found that immunoprecipitation of full-length Gal4p from yeast extracts coprecipitates less Gal80p in the presence than in the absence of Gal3p, galactose, and ATP. We have also found that retention of Gal80p by GSTG4AD (amino acids [aa] 768 to 881) is markedly reduced in the presence compared to the absence of Gal3p, galactose, and ATP. Consistent with these in vitro results, an in vivo two-hybrid genetic interaction between Gal80p and Gal4p (aa 768 to 881) was shown to be weaker in the presence than in the absence of Gal3p and galactose. These compiled results indicate that the binding of Gal3p to Gal80p results in destabilization of a Gal80p-Gal4p complex. The destabilization was markedly higher for complexes consisting of G4AD (aa 768 to 881) than for full-length Gal4p, suggesting that Gal80p relocated to a second site on full-length Gal4p. Congruent with the idea of a second site, we discovered a two-hybrid genetic interaction involving Gal80p and the region of Gal4p encompassing aa 225 to 797, a region of Gal4p linearly remote from the previously recognized Gal80p binding peptide within Gal4p aa 768 to 881.Signaling systems controlling the activation and repression states of genes often include interactions among multiple proteins. One well-studied example is the galactose-responsive system that governs the expression state of the galactose pathway genes (GAL genes) of the yeast Saccharomyces cerevisiae and the closely related milk yeast Kluyveromyces lactis (16,52,57,62,63,74,78,79,81). Galactose signaling operates through a three-component switch consisting of the Gal3 (or Gal1), Gal80, and Gal4 proteins. In the presence of galactose, the GAL genetic switch operates to elicit activation of the GAL genes, and in the absence of galactose, the switch operates to inhibit activation of the GAL genes (32,40,56).The Gal4 protein component of the GAL genetic switch exhibits both site-specific DNA binding and transcription activation, and these activities reside in separable protein domains (11,36,43). Amino acids 1 to 65 of S. cerevisiae Gal4p specify binding to a 17-bp DNA site (UAS GAL