Snf1 Protein Kinase Regulates Phosphorylation of the Mig1 Repressor in Saccharomyces cerevisiae

In glucose-grown cells, the Mig1 DNA-binding protein recruits the Ssn6-Tup1 corepressor to glucose-repressed promoters in the yeast Saccharomyces cerevisiae. Previous work showed that Mig1 is differentially phosphorylated in response to glucose. Here we examine the role of Mig1 in regulating repression and the role of the Snf1 protein kinase in regulating Mig1 function. Immunoblot analysis of Mig1 protein from a snf1 mutant showed that Snf1 is required for the phosphorylation of Mig1; moreover, hxk2 and reg1 mutations, which relieve glucose inhibition of Snf1, correspondingly affect phosphorylation of Mig1. We show that Snf1 and Mig1 interact in the two-hybrid system and also coimmunoprecipitate from cell extracts, indicating that the two proteins interact in vivo. In immune complex assays of Snf1, coprecipitating Mig1 is phosphorylated in a Snf1-dependent reaction. Mutation of four putative Snf1 recognition sites in Mig1 eliminated most of the differential phosphorylation of Mig1 in response to glucose in vivo and improved the two-hybrid interaction with Snf1. These studies, together with previous genetic findings, indicate that the Snf1 protein kinase regulates phosphorylation of Mig1 in response to glucose.     

RPW8 promoter is involved in pathogen‐And stress‐inducible expression from Vitis pseudoreticulata

Based on previous cloning of VpRPW8‐e, we obtained a 1,126 bp VpRPW8‐e promoter sequence in this study. A large number of TATA‐boxes, CAAT‐boxes, and other cis‐acting elements were predicted including light‐responsive elements, hormone‐responsive elements, stress‐responsive elements, and growth‐ and development‐associated elements within the promoter sequence. To further investigate the function of this promoter, we examined its activity in response to biotic and abiotic stress. The VpRPW8‐e promoter was strongly activated by Plasmopara viticola infection, and activation also occurred when the orientation of the promoter was reversed, although to a lesser extent. Deletion analysis showed that the ?1,126 to ?475 bp region of VpRPW8‐e promoter had high activity. A promoter fragment 5′ deleted to ?475 bp (P_?475) was activated in response to heat and cold stress, and even more strongly in response to Phytophthora capsici and salicylic acid (SA). Furthermore, Transgenic Nicotiana benthamiana were generated, VpRPW8‐e driven by P_?475 enhanced resistance to Ph. capsici in N. benthamiana. Based on these results, the ?475 bp region was deduced to be an indispensable part of the VpRPW8‐e promoter. VpRPW8‐e promoter is involved in pathogen‐ and stress‐inducible expression.     

Regulation of Snf1 kinase. Activation requires phosphorylation of threonine 210 by an upstream kinase as well as a distinct step mediated by the Snf4 subunit

The yeast Snf1 kinase and its metazoan orthologues, the AMP-activated protein kinases, are activated in response to nutrient limitation. Activation requires the phosphorylation of a conserved threonine residue in the activation loop of the catalytic subunit. A phosphopeptide antibody was generated that specifically recognizes Snf1 protein that is phosphorylated in its activation loop on threonine 210. Using this reagent, we show that phosphorylation of threonine 210 correlates with Snf1 activity, since it is detected in cells subjected to glucose limitation but not in cells grown in abundant glucose. A Snf1 mutant completely lacking kinase activity was phosphorylated normally on threonine 210 in glucose-starved cells, eliminating the possibility that the threonine 210 modification is due to an autophosphorylation event. Cells lacking the Reg1 protein, a regulatory subunit for the Glc7 phosphatase, showed constitutive phosphorylation of Snf1 threonine 210. Exposure of cells to high concentrations of sodium chloride also induced phosphorylation of Snf1. Interestingly, Mig1, a downstream target of Snf1 kinase, is phosphorylated in glucose-stressed but not sodium-stressed cells. Finally, cells lacking the gamma subunit of the Snf1 kinase complex encoded by the SNF4 gene exhibited normal regulation of threonine 210 phosphorylation in response to glucose limitation but are unable to phosphorylate Mig1 efficiently. Our data indicate that activation of the Snf1 kinase complex involves two steps, one that requires a distinct upstream kinase and one that is mediated by the gamma subunit of the kinase itself.     

The β‐subunits of the Snf1 kinase in Saccharomyces cerevisiae,Gal83 and Sip2, but not Sip1, are redundant in glucose derepression and regulation of sterol biosynthesis

The conserved Snf1/AMP‐activated protein kinase family is one of the central components in the nutrient sensing and regulation of the carbon metabolism in eukaryotes. It is also involved in several other processes such as stress resistance, invasive growth and ageing. Snf1 kinase is composed of a catalytic α‐subunit Snf1, a regulatory γ‐subunit Snf4 and one of three possible β‐subunits, Sip1, Sip2 or Gal83. We used a systematic approach to study the role of the three β‐subunits by analysing all seven possible combinations of β‐subunit deletions together with the reference strain. Previous studies showed that the three β‐subunits are redundant for growth on alternative carbon sources. Here we report that the mutant strain with only SIP1 expressed (sip2Δgal83Δ) could utilize acetate, but neither ethanol nor glycerol, as alternative carbon source. We also showed that Gal83 is the most important isoform not only for the growth on non‐fermentable carbon sources, but also for regulation of ergosterol biosynthetic genes, under glucose‐limited condition. Furthermore, we found that Sip2, but not Sip1, can take over when Gal83 is deleted, but to a lesser extent. However, Sip1 may be sufficient for some other processes such as regulation of the nitrogen metabolism and meiosis.