Multiple Positive and Negative Elements Involved in the Regulation of Expression of GSY1 in Saccharomyces cerevisiae

Abstract

GSY1 is one of the two genes encoding glycogen synthase in Saccharomyces cerevisiae. Both the GSY1 message and the protein levels increased as cells approached stationary phase. A combination of deletion analysis and site-directed mutagenesis revealed a complex promoter containing multiple positive and negative regulatory elements. Expression of GSY1 was dependent upon the presence of a TATA box and two stress response elements (STREs). Expression was repressed by Mig1, which mediates responses to glucose, and Rox1, which mediates responses to oxygen. Characterization of the GSY1 promoter also revealed a novel negative element. This element, N1, can repress expression driven by either an STRE or a heterologous element, the UAS of CYC1. Repression by N1 is dependent on the number of these elements that are present, but is independent of their orientation. N1 repressed expression when placed either upstream or downstream of the UAS, although the latter position is more effective. Gel shift analysis detected a factor that appears to bind to the N1 element. The complexity of the GSY1 promoter, which includes two STREs and three distinct negative elements, was surprising. This complexity may allow GSY1 to respond to a wide range of environmental stresses.The yeast Saccharomyces cerevisiae is exposed to a wide variety of environmental stressors, such as growth into stationary phase, heat shock, and osmotic shock. Cells respond to these stressors by modifying many metabolic processes, presumably making cells more resistant to these stressors. Among these changes are increased activities of the glycogen metabolic enzymes and the deposition of glycogen up to 23% of the dry weight of the cell (see below).Glycogen metabolism is highly conserved from yeast to mammals. The regulation of glycogen metabolism in S. cerevisiae closely parallels the more extensively studied counterparts in mammals (1, 2). Regulation is mediated primarily by effects on the activities of glycogen synthase and glycogen phosphorylase. These enzymes are regulated, in part, by protein phosphorylation. Cyclic AMP appears to play a central role in the regulation of these enzymes in S. cerevisiae, as it does in mammals, although the precise mechanisms remain to be identified. The parallels between glycogen metabolism in S. cerevisiae and mammals extends to the level of protein sequence. Glycogen synthase and glycogen phosphorylase from this yeast and mammals are 50 and 49% identical, respectively (3-6).Glycogen metabolism is also regulated at the level of gene expression in S. cerevisiae. The protein levels of the enzymes involved in glycogen metabolism increase in parallel with glycogen accumulation as cells approach stationary phase or when nutrients are depleted (7,8). This increase in the level of glycogen metabolic enzyme activity appears to result, in part, from the regulation at the level of transcription. Northern blot analysis has shown that the levels of mRNA expressed from GPH1 (encoding glycogen phosphorylase), GLC3 (glycogen branching enzyme)...

Combinatorial control by the protein kinases PKA, PHO85 and SNF1 of transcriptional induction of the Saccharomyces cerevisiae GSY2 gene at the diauxic shift

“…With respect to the Msn2/4p-dependent branch of the pathway, the control of GSY2 is more complicated than previously anticipated, because this gene is still responsive to Msn2/4p when the STREs have been deleted from its promoter. Similar conclusions have been drawn with respect to the transcriptional control of GSY1 by Msn2/4p (Unnikrishnan et al 2003), and from a genome-wide analysis of msn2msn4 mutant, which identified more than 200 genes that are subject to control by this transcription factor, although only 47 of them harbour STREs (Causton et al 2001). Therefore, one might consider that Msn2/4p could bind to a degenerate STRE, or that the effect of the msn2msn4 deletion on a GSY2 gene that lacks STREs is indirect, being mediated by a STRE-regulated factor.…”

“…Using a mutant allele of GSY2 that lacks STREs in its promoter, we demonstrated that STREs are dispensable for GSY2 induction and glycogen accumulation during diauxic growth on glucose, while they are essential for the response to stress. These data also indicated that, under these growth conditions, the glycogen synthase isoform 1 encoded by GSY1 cannot substitute for GSY2, despite the fact that the two genes are under similar transcriptional control (Unnikrishnan et al 2003).…”

Carbohydrate utilization and the lager yeast transcriptome during brewery fermentation

“…During fermentation, the upregulation of many of these genes may likewise be controlled by glucose derepression. Why GSY1 transcription is relatively high at the beginning of the fermentation is unclear, particularly since this gene is negatively regulated by the Rox1 protein, which accumulates in the presence of oxygen (Unnikrishnan et al, 2003). The absence of any upregulation in the genes GLG1, GSY1, GDB1 and GPH1 emphasizes the complexity of the brewery fermentation process compared with controlled laboratory experiments and emphasizes the difficulty in extrapolating from small-to full-scale fermentations.…”

Characterization and gene expression profiles of thermotolerant Saccharomyces cerevisiae isolates from Thai fruits

“…Trehalose protects proteins from thermal denaturation and aggregation through stabilizing protein structures and activities (15,16). In addition, accumulation of glycogen, possibly as a reserve carbohydrate, is correlated with thermotolerance (14,17).…”