Cat8 and Sip4 mediate regulated transcriptional activation of the yeast malate dehydrogenase gene MDH2 by three carbon source-responsive promoter elements

Malate dehydrogenase isoenzymes are localized in different cellular compart ments and fulfil important functions in intermediary metabolism. In the yea st Saccharomyces cerevisiae, three malate dehydrogenase genes, MDH1, MDH2 a nd MDH3, encoding mitochondrial, cytosolic and peroxisomal variants, have b een identified. We demonstrate the importance of transcriptional activators Hap4, Cats and Pip2 for the carbon source-dependent regulation of MDH1, MD H2 and MDH3, respectively. The control region of the MDH2 gene required for gluconeogenic growth with C-2 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 w eak UAS element showing a strong synergism when present in multiple copies. Cumulative mutagenesis of the natural MDH2 promoter confirmed the contribu tion of all three elements to transcriptional derepression under non-fermen tative growth conditions. The DNA-binding domains of zinc fluster proteins Cats and Sip4 synthesized in Escherichia coil could interact in vitro with CSRE motifs of MDH2, This result was confirmed by binding assays using prot ein extracts from yeast. Deregulated variants of Cats and Sip4 modified by heterologous transcriptional activation domains were able to alleviate gluc ose repression of MDH2 substantially. Although Sip4 turned out as the less effective activator, our findings demonstrate the general significance of b oth proteins for expression of gluconeogenic structural genes. Copyright (C ) 2000 John Wiley & Sons, Ltd.