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 Saccharomyce s cerevisiae with nonfermentable carbon sources, Expression of the CAT 8 gene is subject to glucose repression mainly caused by Mig1p. Unexpe ctedly, the deletion of the Mig1p-binding motif within the CAT8 promot er did not increase OiT8 transcription; moreover, it resulted in a los s of CAT8 promoter activation, Insertion experiments with a promoter t est plasmid confirmed that this regulatory 20-bp element influences gl ucose repression and derepression as well, This finding suggests an up stream activating function of this promoter region, which is Mig1p ind ependent, as Delta mig1 mutants are still able to derepress the CAT8 p romoter, No other putative binding sites such as a Hap2/3/4/5p site an d an Abf1p consensus site were functional with respect to glucose-regu lated CAT8 expression, Fusions of Cat8p with the Gal4p DNA-binding dom ain mediated transcriptional activation, This activation capacity was still carbon source regulated and depended on the Cat1p (Snf1p) protei n kinase, which indicated that Cat8p needs posttranslational modificat ion to reveal its gene-activating function, Indeed, Western blot analy sis on sodium dodecyl sulfate-gels revealed a single band (Cat8pI) wit h crude extracts from glucose-grown cells, whereas three bands (Cat8pI , -II, and -III) were identified in derepressed cells, Derepression-sp ecific Cat8pII and -III resulted from differential phosphorylation, as shown by phosphatase treatment, Only the most extensively phosphoryla ted modification (Cat8pIII) depended on the Cat1p (Snf1p) kinase, indi cating that another protein kinase is responsible for modification for m Cat8pII, The occurrence of Cat8pIII was strongly correlated with the derepression of gluconeogenic enzymes (phosphoenolpyruvate carboxykin ase and fructose-1,6-bisphosphatase) and gluconeogenic PCK1 mRNA, Furt hermore, 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 c urrent model that glucose derepression of gluconeogenic genes needs Ca t8p phosphorylation and additionally show that a still unknown transcr iptional activator is also involved.