Dissecting regulatory networks by means of two‐dimensional gel electrophoresis: Application to the study of the diauxic shift in the yeast Saccharomyces cerevisiae

Abstract

Using a proteomic approach based on the two-dimensional (2-D) gel analysis of synthesized proteins, we investigated the involvement of the Snf1 kinase pathway in the regulation of gene expression during the diauxic shift in Saccharomyces cerevisiae. For this purpose, we used a mutant strain deleted for SNF4, the gene coding for the activator subunit of Snf1p. The levels of synthesis of 82 spots were found to be affected by the absence of Snf4p at the diauxic shift. Half of the proteins which exhibit a reduced synthesis in the mutant strain are proteins whose genes are controlled by the transcriptional activator Cat8p, a target of Snf1p. Proteins with an increased level of synthesis in the mutant strain were also observed. Among them are glycolytic enzymes whose synthesis is strongly reduced when wild-type cells enter the diauxic shift. This observation suggests that Snf1p exerts a negative control on the expression of glycolytic genes during the diauxic transition. The results obtained in this study were compiled with those previously obtained by similar proteomic approach with other regulatory factors involved in the diauxic shift. This compilation illustrates how 2-D gel electrophoresis can be used to elucidate the network of regulators participating to complex biological process.

Glucose Signaling in Saccharomyces cerevisiae

“…Thus, in addition to its well-established role in derepressing glucose-repressed genes, Snf1 also somehow represses transcription of glucose-induced genes (including HXT1) (372). Snf1 repression of glycolytic gene expression has been further substantiated by proteomic analysis of cells lacking Snf4, the ␥ subunit of the Snf1 kinase complex (140).…”

Proteomic Analysis of a Nutritional Shift-up in Saccharomyces cerevisiae Identifies Gvp36 as a BAR-containing Protein Involved in Vesicular Traffic and Nutritional Adaptation

“…Of great interest is the finding that the shift-up transition induces the modulation of a substantial number of proteins involved in the response to stress, mainly oxidative stress. Various dynamics of response are observed: a transient peak of expression for cytochrome c peroxidase 1 and Tsa1, a protein that protects cells from free radical damage, a linear decrease for superoxide dismutase and for chaperone Hsp78, whereas the glycerol phosphatase Rhr2, which is involved in glycerol biosynthesis and in the cellular response to various kinds of stress increased linearly during the shift-up (1,21,54).…”

“…Genome-wide transcriptional analysis also identified distinguishable functional clusters of genes that are co-regulated when glucose becomes limiting (1). Proteomic analysis has also been later extended to mutants in genes within the Snf1 kinase pathway, a major regulatory pathway involved in sensing of intracellular glucose and/or energy charge in yeast (21).…”

The AMP-activated Protein Kinase Snf1 Regulates Transcription Factor Binding, RNA Polymerase II Activity, and mRNA Stability of Glucose-repressed Genes in Saccharomyces cerevisiae

“…Snf1 activates Cat8 by direct phosphorylation but activates Adr1 by promoting its dephosphorylation (6,18). Together they activate over 300 target genes involved in metabolic pathways that allow growth in the absence of a fermentable carbon source (25)(26)(27). Many of these genes are regulated directly by binding of Adr1 and/or Cat8 to their promoters (28).…”