Cat8p, the activator of gluconeogenic genes in Saccharomyces cerevisiae, regulates carbon source-dependent expression of NADP-dependent cytosolic isocitrate dehydrogenase (Idp2p) and lactate permease (Jen1p).

The yeast transcriptional activator Cat8p has been identified as a factor that is essential for the derepression of genes involved in gluconeogenesis (like FBP1, PCK1, ACR1, ICL1 and MLS1) when only nonfermentable carbon sources are provided. Cat8p-dependent expression is mediated by cis-acting elements in the respective promoters, which are named UAS/CSREs (upstream activating sequence/carbon source responsive element). To establish whether the function of Cat8p is restricted to the activation of gluconeogenesis or is also involved in the regulation of a greater variety of genes, we investigated the transcriptional regulation of two genes, IDP2 and JEN1, which exhibit a similar expression pattern to gluconeogenic genes, although IDP2 at least is not linked directly to the gluconeogenic pathway. We identified functional UAS/CSRE elements in the promoters of both genes. Expression studies revealed that JEN1 is regulated negatively by the repressors Mig1p and Mig2p, and that Cat8p is needed for full derepression of the gene under non-fermentative growth conditions. Furthermore, we showed that Mig2p is also involved in the repression of CAT8 itself. The results presented in this study support a model in which Cat8p-dependent gene activation is not restricted to gluconeogenesis, but targets a wide variety of genes which are strongly derepressed under non-fermentative growth conditions.

The succinate/fumarate transporter Acr1p of Saccharomyces cerevisiae is part of the gluconeogenic pathway and its expression is regulated by Cat8p.

The product of the ACR1 gene is essential for growth of Saccharomyces cerevisiae on ethanol or acetate as sole carbon source, and its expression is subject to glucose repression. It was previously shown that Acr1p is a membrane protein which specifically transports succinate and fumarate. Its suggested function is to shuttle cytosolic succinate from the glyoxylate cycle into the mitochondria in exchange for fumarate, an activity that is essential during gluconeogenic growth on C2 compounds. In this study we show that ACR1 is coregulated with the genes coding for the key enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1 and PCK1, FBP1 respectively. We demonstrate that derepression of ACR1 is strictly dependent on the Zn2Cys6-type transcriptional activator Cat8p. A detailed deletion analysis of the ACR1 promoter revealed that 69% of the derepression of ACR1 is mediated by three cis-acting elements, located between positions -679 and -569 relative to the translational start, which show a high degree of similarity to the UAS/CSRE elements of PCK1, FBP1, ICL1 and MLS1. Our results, in conjunction with previous biochemical data, clearly identify Acr1p as an element which is directly involved in gluconeogenesis, functioning as the mitochondrial carrier which links the anaplerotic reactions of the glyoxylate cycle to the TCA cycle.

Glucose derepression of gluconeogenic enzymes in Saccharomyces cerevisiae correlates with phosphorylation of the gene activator Cat8p.

The Cat8p zinc cluster protein is essential for growth of Saccharomyces cerevisiae with nonfermentable carbon sources. Expression of the CAT8 gene is subject to glucose repression mainly caused by Mig1p. Unexpectedly, the deletion of the Mig1p-binding motif within the CAT8 promoter did not increase CAT8 transcription; moreover, it resulted in a loss of CAT8 promoter activation. Insertion experiments with a promoter test plasmid confirmed that this regulatory 20-bp element influences glucose repression and derepression as well. This finding suggests an upstream activating function of this promoter region, which is Mig1p independent, as delta mig1 mutants are still able to derepress the CAT8 promoter. No other putative binding sites such as a Hap2/3/4/5p site and an Abf1p consensus site were functional with respect to glucose-regulated CAT8 expression. Fusions of Cat8p with the Gal4p DNA-binding domain mediated transcriptional activation. This activation capacity was still carbon source regulated and depended on the Cat1p (Snf1p) protein kinase, which indicated that Cat8p needs posttranslational modification to reveal its gene-activating function. Indeed, Western blot analysis on sodium dodecyl sulfate-gels revealed a single band (Cat8pI) with crude extracts from glucose-grown cells, whereas three bands (Cat8pI, -II, and -III) were identified in derepressed cells. Derepression-specific Cat8pII and -III resulted from differential phosphorylation, as shown by phosphatase treatment. Only the most extensively phosphorylated modification (Cat8pIII) depended on the Cat1p (Snf1p) kinase, indicating that another protein kinase is responsible for modification form Cat8pII. The occurrence of Cat8pIII was strongly correlated with the derepression of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) and gluconeogenic PCK1 mRNA. Furthermore, glucose triggered the dephosphorylation of Cat8pIII, but this did not depend on the Glc7p (Cid1p) phosphatase previously described as being involved in invertase repression. These results confirm our current model that glucose derepression of gluconeogenic genes needs Cat8p phosphorylation and additionally show that a still unknown transcriptional activator is also involved.

CAT5, a new gene necessary for derepression of gluconeogenic enzymes in Saccharomyces cerevisiae.

PCK1 encoding phosphoenolpyruvate carboxykinase is transcriptionally regulated by two upstream activating elements. By screening for mutants that failed to derepress a UAS2PCK1-CYC1-lacZ reporter gene we isolated the new recessive derepression mutation cat5. The CAT5 gene encodes a protein of 272 amino acids showing a 42% identity to the ZC395.2 gene product of Caenorhabditis elegans whose function is unknown. Deletion of CAT5 caused a complete loss of glucose derepression affecting gluconeogenic key enzymes. Respiration, but not mitochondrial cytochrome c oxidase activity, was also affected. CAT5 expression is 5- to 6-fold repressed by glucose, and CAT5 transcriptional activation was dependent on CAT1 (SNF1), CAT8 and CAT5 itself. The CAT5 gene is necessary for UAS1PCK1 and UAS2PCK1 protein binding since a carbon source-specific interaction was no longer detectable in cat5 mutants. Glucose derepression of gluconeogenesis depends on the active Cat1 (Snf1) protein kinase and the Cat8 zinc cluster activator. Mig1p-independent overexpression of CAT8 did not stimulate activation of gluconeogenic promoters in cat1 and in cat5 mutants. Since Cat8p multicopy expression suppresses the ethanol growth deficiency in cat1 (snf1) mutants, these results indicate that activation of Cat8p by the Cat1p (Snf1p) kinase and the Cat5p protein might be necessary for release from glucose repression.