Galactose induction of the GAL1 gene requires conditional degradation of the Mig2 repressor

Abstract

Skp1 an essential component of the SCF (Skp1/cullin/F-box) E3 ubiquitin ligases, which target proteins for degradation by the 26S proteasome. We generated a skp1dM mutant strain that is defective for galactose induction of the GAL1 gene and we have found that galactose-induced protein degradation of the repressor Mig2 is defective in this strain. Mig2 degradation was also abolished in cells lacking the protein kinase Snf1 and the F-box protein Das1, suggesting that Snf1 triggers galactose-induced protein degradation of Mig2 by SCFDas1. Chromatin immunoprecipitation showed that Mig2 associates with the GAL1 promoter upon the galactose-induced exit of Mig1 in skp1dM cells, but not in wild-type cells, suggesting that the conditional degradation of Mig2 is required to prevent it from binding to the GAL1 promoter under inducing conditions. A galactose-stable deletion derivative of Mig2 caused a strong Mig (multi-copy inhibition of GAL gene expression) phenotype, confirming that galactose induction of the GAL1 gene requires the degradation of the repressor Mig2. Our results shed new light on the conflicting reports about the functional role of the degradation of transcriptional activators and indicate that gene expression studies interfering with proteasome degradation should take the stabilization of potential repressors into account.

Coregulated Expression of the Na+/Phosphate Pho89 Transporter and Ena1 Na+-ATPase Allows Their Functional Coupling under High-pH Stress

“…This is remarkable because for many years, it has been generally accepted that, in contrast to Mig1, Mig2 is not a substrate for the Snf1 kinase (75, 76). Recent work proposed that galactose induces fast degradation of Mig2 by a mechanism that involves Snf1-dependent Mig2 phosphorylation (77), although it is worth noting that those authors observed an almost complete depletion of Mig2 after only 20 min upon shifting of cells to galactose, whereas in our hands, the amount of Mig2 did not significantly change after 30 min of alkaline stress. In any case, our data support a role for Mig2 in repression of PHO89 expression, and it is reasonable to assume that activation of Snf1 results in a relief of such repression.…”

The Filamentous Growth MAPK Pathway Responds to Glucose Starvation Through the Mig1/2 Transcriptional Repressors in Saccharomyces cerevisiae

“…Snf1 phosphorylates Mig1 (Ostling and Ronne 1998;Treitel et al 1998;Smith et al 1999), which relieves its transcriptional repression function and promotes its export from the nucleus. Mig2 is a functional homolog of Mig1 (Lutfiyya and Johnston 1996), but Mig2 is not regulated by Snf1 and has a localization (Lutfiyya et al 1998;Fernandez-Cid et al 2012) and turnover pattern (Lim et al 2011) that is distinct from Mig1. In high-glucose conditions, Mig1 and Mig2 repress the expression of genes involved in the metabolism of poor carbon sources, in part through Mig1-dependent recruitment of the co-repressor Tup1/Ssn6 (Treitel and Carlson 1995).…”

Mediator Acts Upstream of the Transcriptional Activator Gal4

“…Phosphorylated Mig2 is ubiquitinated by the E3 ubiquitin ligases SCF Das1/Ufo1 [37]. SNF1 also signals Mediator the absence of glucose via the Snf1-Srb11 interaction.…”

“…Das1 and Ufo1 (but not Mdm30) also target the repressor Mig2 for galactose-induced protein degradation [37]. However, the additional gene deletion of MIG2 did not increase galactose induction of GAL1 mRNA in the ΔUFO1 strain and had only a very small effect on the galactose induction of the GAL1 mRNA in the ΔDAS1 strain [37].…”

“…Das1 and Ufo1 (but not Mdm30) also target the repressor Mig2 for galactose-induced protein degradation [37]. However, the additional gene deletion of MIG2 did not increase galactose induction of GAL1 mRNA in the ΔUFO1 strain and had only a very small effect on the galactose induction of the GAL1 mRNA in the ΔDAS1 strain [37]. Therefore, an additional target for Das1 and Ufo1 had been proposed, and we have now shown here that Gal80 is this functionally relevant target, as Gal80—like Mig2 [37]—became stable in galactose-induced cells lacking Das1 and Ufo1 (Figure S7A and S7B), and the additional gene deletion of GAL80 suppressed the gal − phenotype of both the ΔDAS1 and the ΔUFO1 strains (Figure S7C).…”