Replacement of a conserved tyrosine by tryptophan in Gal3p of Saccharomyces cerevisiae reduces constitutive activity: implications for signal transduction in the GAL regulon.
The ability of Saccharomyces cerevisiae to utilize galactose is regulated by the nucleo-cytoplasmic shuttling of a transcriptional repressor, the Gal80 protein. Gal80 interacts with the transcriptional activator Gal4 in the nucleus and inhibits its function, preventing induction of the GAL genes. In response to galactose, the relative amounts of Gal80 in the cytoplasm and the nucleus are modulated by the action of a signal transducer, Gal3. Although it has been speculated that Gal3 binds galactose, this has not been experimentally demonstrated. In this study, we show that replacement of a conserved tyrosine in Gal3 by tryptophan leads to a reduction of its constitutive activity in the absence of galactose. In addition, this mutant protein was fully functional in vivo only when high concentrations of galactose were present in the medium. When overexpressed, the mutant was found to activate the genes GAL1 and GAL7/10 differentially. The implications of these findings for the fine regulation of GAL genes, and its physiological significance, are discussed.
Lakshminarasimhan
Anirudha
A
Laboratory of Molecular Genetics, School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, 400 076, India. jayadeva@btc.iitb.ac.in
Bhat
Paike Jayadeva
PJ
D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins; D005690:Galactose / Q000737:chemistry / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D018085:Regulon; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; C055002:GAL80 protein, S cerevisiae; C099289:Gal3 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014443:Tyrosine; D014364:Tryptophan; D005690:Galactose