Deregulation of gluconeogenic structural genes by variants of the transcriptional activator Cat8p of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, growth with a non-fermentable carbon source requires co-ordinate transcriptional activation of gluconeogenic st ructural genes by an upstream activation site (UAS) element, designated CSR E (carbon source-responsive element). The zinc cluster protein encoded by C AT8 is necessary for transcriptional derepression mediated by a CSRE. Expre ssion of CAT8 as well as transcriptional activation by Cat8p is regulated b y the carbon source, requiring a functional Cat1p (= Snf1p) protein kinase. The importance of both regulatory levels was investigated by construction of CAT8 variants with a constitutive transcriptional activation domain (INO 2(TAD)) and/or a carbon source-independent promoter (MET25). Whereas a repo rter gene driven by a CSRE-dependent synthetic minimal promoter showed a 40 -fold derepression with wild-type CAT8, an almost constitutive expression w as found with a MET25-CAT8-INO2(TAD) fusion construct due to a dramatically increased gene activation under conditions of glucose repression. Similar results were obtained with the mRNA of the isocitrate lyase gene ICL1 and a t the level of ICL enzyme activity. Taking advantage of a Cat8p size varian t, we demonstrate its binding to the CSRE. Our data show that carbon source -dependent transcriptional activation by Cat8p is the most important mechan ism affecting the regulated expression of gluconeogenic structural genes.