Phosphorylation of Ga14p at a single C-terminal residue is necessary for galactose-inducible transcription.
Gal4p regulates expression of genes necessary for galactose catabolism in Saccharomyces cerevisiae. We have previously shown that phosphorylation of Gal4p requires both its DNA binding and transcriptional-activation functions and have suggested that phosphorylation occurs as a consequence of interaction with general transcription factors. In this study, we show that phosphorylation occurs rapidly on a limited fraction of overexpressed Gal4p present in a sodium dodecyl sulfate-extractable subcellular fraction while a significant fraction remains stably unphosphorylated. Taken together with our previous observations, we conclude that Gal4p is phosphorylated only if it becomes localized to the nucleus and is capable of both DNA binding and transcriptional activation. We demonstrate that Gal4p is multiply phosphorylated at both the C and N termini, and we identify the precise locations of three sites of phosphorylation at serines 691, 696, and 699. Of these sites, only serine 699 must be phosphorylated for galactose-inducible transcription to occur. Mutation of S-699 to alanine significantly impairs GAL induction by galactose in GAL80+ cells but does not affect transcriptional activation by Gal4p in gal80- cells. In gal80- cells, Gal4p phosphorylation, including that of serine 699, is stimulated by the presence of both galactose and glucose, indicating that phosphorylation at this site is not specifically activated by galactose. Serine 699 phosphorylation requires Gal4p's DNA binding function and is influenced by the function of the RNA polymerase II holoenzyme component Gal11p. These results suggest that a phosphorylation on Gal4p, likely resulting from interaction with the holoenzyme, modulates the induction process by regulating interaction between Gal4p and Gal80p.
Sadowski
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I
Department of Biochemistry, University of British Columbia, Vancover, Canada.
Costa
C
C
Dhanawansa
R
R
D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000378:metabolism*; D005690:Galactose / Q000494:pharmacology*; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000187:drug effects*
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D010768:Phosphoserine; D005947:Glucose; D005690:Galactose