Conformational changes play a role in regulating the activity of the proline utilization pathway-specific regulator in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the ability to use proline as a nitrogen sourc e requires the Put3p transcriptional regulator, which turns on the expressi on of the proline utilization genes, PUT1 and PUT2, in the presence of the inducer proline and in the absence of preferred nitrogen sources. Changes i n target gene expression occur through an alteration in activity of the DNA -bound Put3p, a member of the Zn(II)(2)Cys(6) binuclear cluster family of p roteins. Here, we report that the 'on' conformation can be mimicked in the absence of proline by the insertion of an epitope tag in several different places in the protein, as well as by specific amino acid changes that suppr ess a put3 mutation leading to non-inducibility of the pathway, In addition , the presence of proline causes a conformational change in the Put3 protei n detected by increased sensitivity to thrombin or vs protease. These findi ngs suggest that Put3p shifts from an inactive to an activate state via con formational changes.