Contribution of Cat8 and Sip4 to the transcriptional activation of yeast gluconeogenic genes by carbon source-responsive elements

Abstract

The carbon source-responsive element (CSRE) functions as an activating promoter motif of gluconeogenic genes in Saccharomyces cerevisiae. The positively acting regulatory genes CAT8 and SIP4 encode CSRE-binding proteins which contribute unequally to the regulated expression of a CSRE-dependent reporter gene (85% and 15%, respectively, under conditions of glucose derepression). Deregulated variants of Cat8 and Sip4 are able to bind to the CSRE and allow glucose-insensitive gene activation, even in the absence of the other protein, arguing against the physiological significance of heterodimer formation. Gel retardation assays provide evidence for a different binding affinity of Cat8 and Sip4 to at least some CSRE sequence variants. Both efficient biosynthesis of and transcriptional activation by Sip4 require a functional CAT8 gene, while Cat8 was not dependent on SIP4. Thus, our data suggest that the apparent minor importance of Sip4 may be the result of autoregulatory cross-talk among the isofunctional activators Cat8 and Sip4. The derepression deficiency of a CSRE-dependent reporter gene in a strain lacking the Cat1 (Snf1) protein kinase can be suppressed by Sip4 fused to a strong heterologous activation domain. This finding agrees with the idea that phosphorylation by Cat1 may convert Sip4 into a functional activator.

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

“…Strains of S. cerevisiae used in this work were derived from the regulatory wild-type JS91.15-23 (MATα ura3 leu2 his3 trp1 can1). Regulatory mutants EMY1 (∆cat8 ::HIS3 ; Rahner et al, 1996), MHY14 (∆sip4 ::kanMX4), SRH24 (∆cat8 ::HIS3 ∆sip4 ::kanMX4), JS96.20-1 (∆cat1 ::LEU2 ; Hiesinger et al, 2001), JS95.14-1 (∆adr1 ::LEU2) and JS95.16-1 (∆adr1 ::LEU2 ∆cat8 ::HIS3 ; Kratzer & Schu$ ller, 1997) were obtained by introduction of the null alleles indicated. Synthetic complete medium used for selective growth of transformants has been described previously (Schu$ ller & Entian, 1987).…”

“…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).…”

“…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). …”

“…A DNAbinding factor which is specifically required for UAS2-dependent gene activation and directly interacts with UAS2 has not yet been identified. We have previously analysed the genetic control of structural genes involved in the glyoxylate cycle and gluconeogenesis [ICL1, FBP1 (Scho$ ler & Schu$ ller, 1993, 1994, MLS1 (Caspary et al, 1997) and MDH2 (Roth & Schu$ ller, 2001)]. This led to the identification of a common cis-acting element, designated CSRE (carbon source-responsive element ; consensus CCRTYSRNCCG ; reviewed by Entian & Schu$ ller, 1997 ;Gancedo, 1998), which could confer glucose-sensitive gene regulation to a synthetic minimal promoter.…”