Negative control of the Mig1p repressor by Snf1p‐dependent phosphorylation in the absence of glucose

Abstract

Mig1p, a zinc-finger protein that is related to the Krox/Egr, Wilms' tumor and Sp1 proteins, mediates glucose repression in the yeast Saccharomyces cerevisiae. Mig1p is inactive in the absence of glucose, and this inhibition is dependent on the Snf1p (Cat1p) protein kinase. The regulation is mediated by an internal part of Mig1p, and it can be transferred to a Mig1-viral protein 16 (VP16) fusion protein that functions as an activator [Östling, J., Carlberg, M. & Ronne, H. (1996) Mol. Cell. Biol. 16, 753Ϫ761]. We have used Mig1-VP16 to identify three target sites for phosphorylation that mediate Snf1p-dependent inhibition of its activity in the absence of glucose. Two of the sites, Ser278 and Ser311, fit the consensus sequence for phosphorylation by the kinase Snf1p, as determined in vitro. However, a third phosphorylated site, Ser108, does not resemble a Snf1p site. We tested the effect of deleting residues 181Ϫ245, which contain two conserved alanine-leucine-serine motifs. We found that the deletion produces a partially constitutive activator, indicating that this region plays a general negative role in regulating Mig1p.Keywords : MIG1 ; SNF1 ; Saccharomyces cerevisiae; glucose repression; yeast. Glucose repression is a metabolic regulatory response inWe have described previously the R1 domain, an internal region in the Mig1 protein, which plays a key role in its negative yeast and other fungi, by which the organism adapts for optimal control by Snf1p [21]. The R1 domain can confer Snf1p-depengrowth on glucose by repressing genes that encode enzymes that dent carbon-source control to a Mig1-VP16 (viral protein 16) are required for use of other carbon sources [1Ϫ4]. This includes hybrid protein, which functions as an activator in yeast [21], and genes that are involved in metabolism of alternative sugars, such it contains two sites that match the known consensus target site as the GAL and SUC genes, and genes involved in gluconeogenfor Snf1p [17]. It was shown recently that nuclear translocation esis, the Krebs' cycle and respiration. A key effector in glucose of a Mig1-GFP (green fluorescent protein) fusion is regulated repression is the zinc-finger protein Mig1p [5, 6]. Mig1p binds by the carbon source and that this regulation requires a region to the sequence SYGGRG (where Y is C or T, and R is G or that largely overlaps with the R1 domain [22]. Moreover, the A), which is present in glucose-repressed promoters [7], and it protein is constitutively found in the nucleus in a snf1 ssn6 is thought that it recruits the general corepressor complex to double mutant, suggesting that Snf1p-dependent control of the target promoter [8]. The corepressor complex contains two Mig1p activity may involve the same mechanism [22]. subunits, Tup1 and Cyc8p (or Ssn6p), and it is the Tup1p subunitThe two putative Snf1p sites in the R1 domain are conserved that inhibits transcription [9, 10]. A second Mig1p-related yeast in Mig1p from the yeasts Kluyveromyces lactis and Kluyveroprotein, known as Mig2p or Mlz1p, contributes to repr...

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