Cat8 and Sip4 mediate regulated transcriptional activation of the yeast malate dehydrogenase geneMDH2 by three carbon source-responsive promoter elements

Abstract

Malate dehydrogenase isoenzymes are localized in different cellular compartments and fulfil important functions in intermediary metabolism. In the yeast Saccharomyces cerevisiae, three malate dehydrogenase genes, MDH1, MDH2 and MDH3, encoding mitochondrial, cytosolic and peroxisomal variants, have been identified. We demonstrate the importance of transcriptional activators Hap4, Cat8 and Pip2 for the carbon source‐dependent regulation of MDH1, MDH2 and MDH3, respectively. The control region of the MDH2 gene required for gluconeogenic growth with C2 substrates contains three sequence elements similar to the previously identified carbon source‐responsive element (CSRE). In a synthetic test system, each of these sequences turned out to be a weak UAS element showing a strong synergism when present in multiple copies. Cumulative mutagenesis of the natural MDH2 promoter confirmed the contribution of all three elements to transcriptional derepression under non‐fermentative growth conditions. The DNA‐binding domains of zinc cluster proteins Cat8 and Sip4 synthesized in Escherichia coli could interact in vitro with CSRE motifs of MDH2. This result was confirmed by binding assays using protein extracts from yeast. Deregulated variants of Cat8 and Sip4 modified by heterologous transcriptional activation domains were able to alleviate glucose repression of MDH2 substantially. Although Sip4 turned out as the less effective activator, our findings demonstrate the general significance of both proteins for expression of gluconeogenic structural genes. Copyright © 2000 John Wiley & Sons, Ltd.

Accelerated Alcoholic Fermentation Caused by Defective Gene Expression Related to Glucose Derepression inSaccharomyces cerevisiae

“…As shown in Fig. 1 22,23,40) and gluconeogenic enzymes (MDH2 and FBP1), 18,22,23,41) reached similar levels in the two strains upon diauxic shift (about 1 d). With increasing cultivation time, however, the expression levels of these genes in K701 gradually decreased, whereas those in X2180 remained relatively high, leading to significantly different values at 7 d. The expression levels in K701 at 7 d ranged from 8.0% (CIT3) to 59.9% (DLD1) of those in X2180.…”

Combined Global Localization Analysis and Transcriptome Data Identify Genes That Are Directly Coregulated by Adr1 and Cat8

“…We cannot detect Cat8 binding in vivo to the HAP4 CSRE-like region, so the observed Cat8-dependent binding might be due to another Cat8-dependent factor, as suggested by Brons et al (6). A strong candidate is Sip4, which binds to some CSREs and requires CAT8 for full expression (23,25,39). Our failure to detect Cat8 binding to the SIP4 promoter in vivo, despite evidence of binding in vitro (25), might reflect different sensitivities of the assays.…”

“…For Cat8, in vitro binding has been demonstrated by electrophoretic mobility shift assays to the CSRE (carbon source-responsive element) sequences from ACS1, ADH2, ICL2, ICL1, FBP1, MDH2, MLS1, and PCK1 (9,28,35,36,39,47,51). All of these genes are known to be regulated by Cat8 under derepressing conditions.…”

“…Coregulated promoters are cooccupied by Adr1 and Cat8. To determine which genes are regulated directly by both Adr1 and Cat8, we compared the data sets containing the direct targets of each factor and then made individual evaluations of candidate genes using gene-specific expression or binding data from the literature (4,5,9,24,28,36,39,46,47,50,51), from Fig. 1, and from our unpublished data.…”

Interaction of the Srb10 Kinase with Sip4, a Transcriptional Activator of Gluconeogenic Genes in Saccharomyces cerevisiae

“…Members of the zinc cluster transcriptional activator family have been shown to bind to DNA as dimers (22). Thus, it seemed likely that Sip4 forms homodimers and possible that Sip4 forms heterodimers with Cat8, another zinc cluster activator that binds to the CSRE and has a major role in the activation of gluconeogenic genes (9,21,25). We addressed this issue because of the further possibility that Srb10 has a role in dimerization.…”