Glycerol 3-phosphate dehydrogenase regulates heat shock response in Saccharomyces cerevisiae

The aim of this work was to identify elements of adaptive regulatory mechanism for basal level of yeast histone deacetylase Sir2. Heat shock response (HSR) was altered in the absence of the NAD-dependent glycerol 3-phosphate dehydrogenase (Gpd1). Increase in HSR was lower in ΔGpd1 cells than wild-type cells. An inverse correlation existed between Gpd1 and Sir2; Sir2-deleted cells showed higher expression of Gpd1 while deletion of Gpd1 led to higher expression of Sir2. In the absence of Gpd1, basal activity of Sir2 promoter was higher and was increased further upon heat shock, suggesting higher Sir2 levels. No interaction between Gpd1 and Sir2 was detected without or with heat shock using immunoprecipitation. The results show that Gpd1 regulates HSR in yeast cells and likely blocks its uncontrolled activation. As uncontrolled stress adversely affects the cellular adaptive response, Gpd1 may be a component of the cell's catalogue to ensure a balanced response to unmitigated thermal stress.     

Heat shock proteins affect RNA processing during the heat shock response of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the splicing of mRNA precursors is disrupted by a severe heat shock. Mild heat treatments prior to severe heat shock protect splicing from disruption, as was previously reported for Drosophila melanogaster. In contrast to D. melanogaster, protein synthesis during the pretreatment is not required to protect splicing in yeast cells. However, protein synthesis is required for the rapid recovery of splicing once it has been disrupted by a sudden severe heat shock. Mutations in two classes of yeast hsp genes affect the pattern of RNA splicing during the heat shock response. First, certain hsp70 mutants, which overproduce other heat shock proteins at normal temperatures, show constitutive protection of splicing at high temperatures and do not require pretreatment. Second, in hsp104 mutants, the recovery of RNA splicing after a severe heat shock is delayed compared with wild-type cells. These results indicate a greater degree of specialization in the protective functions of hsps than has previously been suspected. Some of the proteins (e.g., members of the hsp70 and hsp82 gene families) help to maintain normal cellular processes at higher temperatures. The particular function of hsp104, at least in splicing, is to facilitate recovery of the process once it has been disrupted.     

Enzymatic synthesis of glutathione using engineered Saccharomyces cerevisiae

Two single gene cassettes, each containing one of the individual gene (γ-glutamylcysteine synthetase gene GSH1 or glutathione synthetase gene GSH2), were constructed under the control of alcohol dehydrogenase (ADH1) promoter and their respective native terminators. The recombinant plasmids constructed with Kan ^r or Hyg ^r as the selective markers and were transformed into Saccharomyces cerevisiae separately and jointly. Three engineered strains, GSH1-enhanced strain S.TS013/GSH1, GSH2-enhanced strain S.TS013/GSH2 and GSH1+GSH2 double-enhanced strain S.TS013/GSH1+GSH2, were constructed. Glutathione production using the recombinant strains to improve was then determined. By the cell dosage proportions of two engineered strains (S.TS013/GSH1, S.TS013/GSH2) and a two-stage reaction, GSH productivity increased by 84 and 59 % over that of the host strain and the S.TS013/GSH1+GSH2 strain, respectively.     

Trehalose accumulates in Saccharomyces cerevisiae during exposure to agents that induce heat shock response

The storage disaccharide, trehalose, is accumulated in yeast during a temperature shift from 30 to 45 degrees C. The response peaks at 90 min and is transient since levels of trehalose decline rapidly in cells returned to 30 degrees C. Storage of trehalose is inhibited when cells are incubated in the presence of acridine orange or ethidium bromide prior to and during temperature shift, suggesting a requirement for de novo RNA synthesis. Accumulation of trehalose occurs when cells are exposed to either ethanol, copper sulphate or hydrogen peroxide at 30 degrees C, indicating that the phenomenon may be a general response to physiological stress. Parallels are drawn between the trehalose accumulation response and the heat shock response in yeast.     

Ycf1p-dependent Hg(II) detoxification in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, disruption of the YCF1 gene increases the sensitivity of cell growth to mercury. Transformation of the resulting ycf1 null mutant with a plasmid harbouring YCF1 under the control of the GAL promoter largely restores the wild-type resistance to the metal ion. The protective effect of Ycf1p against the toxicity of mercury is especially pronounced when yeast cells are grown in rich medium or in minimal medium supplemented with glutathione. Secretory vesicles from S. cerevisiae cells overproducing Ycf1p are shown to exhibit ATP-dependent transport of bis(glutathionato)mercury. Moreover, using beta-galactosidase as a reporter protein, a relationship between mercury addition and the activity of the YCF1 promoter can be shown. Altogether, these observations indicate a defence mechanism involving an induction of the expression of Ycf1p and transport by this protein of mercury-glutathione adducts into the vacuole. Finally, possible coparticipation in mercury tolerance of other ABC proteins sharing close homology with Ycf1p was investigated. Gene disruption experiments enable us to conclude that neither Bpt1p, Yor1p, Ybt1p nor YHL035p plays a major role in the detoxification of mercury.     

The Skn7 response regulator of Saccharomyces cerevisiae interacts with Hsf1 in vivo and is required for the induction of heat shock genes by oxidative stress

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein-protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.     

The metabolic response of Saccharomyces cerevisiae to continuous heat stress

A study has been initiated to integrate molecular and physiological responses of Saccharomyces cerevisiae to heat stress conditions. We focus our research on a quantification of the energetics of the stress response. A series of continuous heat stresses was applied to exponentially growing cells of the strain X2180-1A at 28°C, by increasing the growth temperature to 37, 39, 40, 41, 42, or 43°C. Here, the results on cell growth and viability, as well as on anabolic and catabolic rates are presented. We observed a surprisingly thin line for the cells between growing, surviving, and dying, with regard to growth temperature. The heat stress showed a dual effect on catabolism: immediately after the temperature increase a strong peak was seen, after which a new, steady level was reached. In addition, the yield on glucose decreased with increasing temperature. Our results indicate that life at elevated temperatures is energetically unfavourable and a non-lethal heat stress invokes a redistribution of catabolic and anabolic fluxes.     

Induction of synthesis of Hsp104 of Saccharomyces cerevisiae in heat shock is controlled by mitochondria

Heat shock protein Hsp104 of Saccharomyces cerevisiae functions as a protector of cells against heat stress. When yeast are grown in media containing nonfermentable carbon sources, the constitutive level of this protein increases, which suggests an association between the expression of Hsp104 and yeast energy metabolism. In this work, it is shown that distortions in the function of mitochondria appearing as a result of mutation petite or after exposure of cells to the mitochondrial inhibitor sodium azide reduce the induction of Hsp104 synthesis during heat shock. Since the addition of sodium azide suppressed the formation of induced thermotolerance in the parent type and in mutant hsp104, the expression of gene HSP104 and other stress genes during heat shock is apparently regulated by mitochondria.     

Cryoprotection provided by heat shock treatment in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells exposed to 43 degrees C (normal being 30 degrees C) exhibit the synthesis of heat shock proteins (hsps). Time course studies indicated that the major hsps (97 kDa, 85 kDa and 70 kDa family) are induced within 10 min. of heat shock and attain maximum amount with two hours of treatment. The viability of cells decreased by 99% when directly frozen into liquid nitrogen. However, a prior heat shock (2 hours) increased the cell survival by 20-30 fold. Such an effect of prior heat shock treatment could be supported by light and electron microscopical studies. Differential scanning calorimetric analysis of whole cells revealed that heat shock treatment decreases the denaturation (delta H) of total cellular proteins. A direct correlation between the degree of hsp inducibility and protection against freezing and thawing injury was observed. Cycloheximide treatment curtailed the synthesis of hsps as well as protection against subsequent freezing. This suggests that prior heat shock treatment protects the cells from freezing injury and, furthermore, that hsps can act as biological cryoprotectants.