Evidence That Proteolysis of Gal4 Cannot Explain the Transcriptional Effects of Proteasome ATPase Mutations

• Published in: The Journal of biological chemistry 2001-03, Vol.276 (13), p.9825-9831
• Main Authors: Russell, Steven Jon, Johnston, Stephen Albert
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Alleles; Amino Acids - chemistry; Cycloheximide - pharmacology; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; DNA Repair; DNA-Binding Proteins; Fungal Proteins - genetics; Fungal Proteins - metabolism; GAL4 gene; Galactose - pharmacology; Genes, Recessive; Glucose - pharmacology; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; Mutation; Phenotype; Proteasome Endopeptidase Complex; proteasomes; Protein Structure, Tertiary; Protein Synthesis Inhibitors - pharmacology; Repressor Proteins - genetics; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; SUG1 gene; SUG2 gene; Temperature; Transcription Factors - metabolism; Transcription, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M010889200

The Gal system of Saccharomyces cerevisiae is a paradigm for eukaryotic gene regulation. Expression of genes required for growth on galactose is regulated by the transcriptional activator Gal4. The activation function of Gal4 has been localized to 34 amino acids near the C terminus of the protein. The gal4D allele of GAL4 encodes a truncated protein in which only 14 amino acids of the activation domain remain. Expression of GAL genes is dramatically reduced in gal4Dstrains and these strains are unable to grow on galactose as the sole carbon source. Overexpression of gal4D partially relieves the defect in GAL gene expression and allows growth on galactose. A search for extragenic suppressors of gal4D identified recessive mutations in the SUG1 and SUG2 genes, which encode ATPases of the 19S regulatory complex of the proteasome. The proteasome is responsible for the ATP-dependent degradation of proteins marked for destruction by the ubiquitin system. It has been commonly assumed that effects of SUG1 and SUG2mutations on transcription are explained by alterations in the proteolysis of gal4D protein. We have investigated this assumption. Surprisingly, we find that SUG1 and SUG2alleles that are unable to suppress gal4D cause a larger increase in gal4D protein levels than do suppressing alleles. In addition, mutations in genes encoding subunits of the proteolytic 20S sub-complex of the proteasome increase the levels of gal4D protein but do not rescue its transcriptional activity. Therefore, an alteration in the proteolysis of gal4D by the proteasome cannot explain the effects of mutations in SUG1 and SUG2 on expression of GAL genes. These findings suggest that the 19S regulatory complex may play a more direct role in transcription.