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

Abstract

SummaryIn the yeast Saccharomyces cerevisiae, growth with a non-fermentable carbon source requires co-ordinate transcriptional activation of gluconeogenic structural genes by an upstream activation site (UAS) element, designated CSRE (carbon source-responsive element). The zinc cluster protein encoded by CAT8 is necessary for transcriptional derepression mediated by a CSRE. Expression of CAT8 as well as transcriptional activation by Cat8p is regulated by 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 (INO2 TAD ) and/ or a carbon source-independent promoter (MET25 ). Whereas a reporter gene driven by a CSRE-dependent synthetic minimal promoter showed a 40-fold derepression with wild-type CAT8, an almost constitutive expression was 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 at the level of ICL enzyme activity. Taking advantage of a Cat8p size variant, we demonstrate its binding to the CSRE. Our data show that carbon source-dependent transcriptional activation by Cat8p is the most important mechanism affecting the regulated expression of gluconeogenic structural genes.

Adr1 and Cat8 synergistically activate the glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast Saccharomyces cerevisiae

“…The desired mutation in plasmid pKW14 (ADH2-[CSRE Mut ]-lacZ URA3 2µ) was confirmed by DNA sequencing. Plasmids pAR33, pAR34 and pMH33, encoding deregulated variants of CAT8 and SIP4, have been described previously (carbon source-independent MET25 promoter, constitutive Ino2 or VP16 activation domains together with a FLAG epitope ; Rahner et al, 1999 ;Hiesinger et al, 2001).…”

“…Previous work demonstrated binding of Sip4 and Cat8 to CSRE sequence variants (Vincent & Carlson, 1998 ;Rahner et al, 1999 ;Roth & Schu$ ller, 2001 ;Hiesinger et al, 2001). To confirm the direct influence of Cat8 on UAS2, gel retardation experiments with protein extracts prepared from E. coli or yeast transformants were performed.…”

“…A functional Cat1\Snf1\Ccr1 protein kinase together with its auxiliary factors Cat3\Snf4 (Celenza & Carlson, 1986 ;Schu$ ller & Entian, 1987, 1988Celenza et al, 1989) and Sip1jSip2jGal83 (Yang et al, 1994 ;Schmidt & McCartney, 2000) is absolutely required for Cat8 function. Importantly, Cat8, as well as the related transcription factor Sip4, bind to CSRE ICL" , CSRE FBP" and CSRE MDH# motifs (Vincent & Carlson, 1998 ;Rahner et al, 1999 ;Roth & Schu$ ller, 2001). A CSRE-driven gene has been shown to be no longer regulated by carbon source with constitutively synthesized Cat8 and Sip4 variants containing heterologous activation domains (Vincent & Carlson, 1998 ;Rahner et al, 1999 ;Hiesinger et al, 2001).…”

“…Importantly, Cat8, as well as the related transcription factor Sip4, bind to CSRE ICL" , CSRE FBP" and CSRE MDH# motifs (Vincent & Carlson, 1998 ;Rahner et al, 1999 ;Roth & Schu$ ller, 2001). A CSRE-driven gene has been shown to be no longer regulated by carbon source with constitutively synthesized Cat8 and Sip4 variants containing heterologous activation domains (Vincent & Carlson, 1998 ;Rahner et al, 1999 ;Hiesinger et al, 2001). …”

The Transcriptional Activator Cat8p Provides a Major Contribution to the Reprogramming of Carbon Metabolism during the Diauxic Shift in Saccharomyces cerevisiae

“…At least one upstream activation sequence element, named carbon source-responsive element (CSRE) 1 was found in the promoter of all the genes identified as Cat8p targets. The binding of Cat8p on a CSRE motif has been recently shown (12).…”