The role of flavin-containing enzymes in mitochondrial membrane hyperpolarization and ROS production in respiring Saccharomyces cerevisiae cells under heat-shock conditions.

Heat shock is known to accelerate mitochondrial ROS production in Saccharomyces cerevisiae cells. But how yeast mitochondria produce ROS under heat-shock condition is not completely clear. Previously, it was shown that ROS production in heat-stressed fermenting yeast cells was accompanied by mitochondrial membrane potential (MMP) increase. In the current investigation the relationship between ROS production and MMP was studied in respiring yeast cells in stationary phase, using diphenyleneiodonium chloride (DPI), an inhibitor of flavin-containing proteins, as well as the mutants deleted for NDE1, NDE2 and NDI1 genes, encoding flavin-containing external and internal NADH dehydrogenases. It was shown that heat shock induced a transient burst in mitochondrial ROS production, which was paralleled by MMP rise. ROS production and MMP was significantly suppressed by DPI addition and deletion of NDE1. The effect of DPI on ROS production and MMP rise was specific for respiring cells. The results obtained suggest that the functioning of mitochondrial flavin-binding enzymes, Nde1p for instance, is required for the hyperpolarization of inner mitochondrial membrane and ROS production in respiring S. cerevisiae cells under heat-shock conditions.

Relation between cell death progression, reactive oxygen species production and mitochondrial membrane potential in fermenting Saccharomyces cerevisiae cells under heat-shock conditions.

Moderate heat shock increased reactive oxygen species (ROS) production that led to cell death in glucose-grown Saccharomyces cerevisiae cells. Conditions that disturb mitochondrial functions such as treatment by uncouplers and petite mutation were shown to inhibit ROS production and protects cell from thermal death. Hence, mitochondria are responsible for ROS production and play an active role in cell death. An increase in ROS production was accompanied by hyperpolarization of inner mitochondrial membrane. All agents suppressing hyperpolarization also suppressed heat-induced ROS production. It was supposed that generation of ROS under moderate heat shock in glucose-grown S. cerevisiae cells is driven by the mitochondrial membrane potential.

Chaperone effects on prion and nonprion aggregates.

Exposure to high temperature or other stresses induces a synthesis of heat shock proteins. Many of these proteins are molecular chaperones, and some of them help cells to cope with heat-induced denaturation and aggregation of other proteins. In the last decade, chaperones have received increased attention in connection with their role in maintenance and propagation of the Saccharomyces cerevisiae prions, infectious or heritable agents transmitted at the protein level. Recent data suggest that functioning of the chaperones in reactivation of heat-damaged proteins and in propagation of prions is based on the same molecular mechanisms but may lead to different consequences depending on the type of aggregate. In both cases the concerted and balanced action of "chaperones' team," including Hsp104, Hsp70, Hsp40 and possibly other proteins, determines whether a misfolded protein is to be incorporated into an aggregate, rescued to the native state or targeted for degradation.

Nuclear-mitochondrial cross-talk during heat shock in Arabidopsis cell culture.

Apart from energy generation, mitochondria perform a signalling function determining the life and death of a cell under stress exposure. In the present study we have explored patterns of heat-induced synthesis of Hsp101, Hsp70, Hsp17.6 (class I), Hsp17.6 (class II) and Hsp60, and the development of induced thermotolerance in Arabidopsis thaliana cell culture under conditions of mitochondrial dysfunction. It was shown that treatment by mitochondrial inhibitors and uncouplers at the time of mild heat shock downregulates HSP synthesis, which is important for induced thermotolerance in plants. The exposure to elevated temperature induced an increase in cell oxygen consumption and hyperpolarization of the inner mitochondrial membrane. Taken together, these facts suggest that mitochondrial functions are essential for heat-induced HSP synthesis and development of induced thermotolerance in A. thaliana cell culture, suggesting that mitochondrial-nuclear cross-talk is activated under stress conditions. Treatment of Arabidopsis cell culture at 50 degrees C initiates a programmed cell death determined by the time course of viability decrease, DNA fragmentation and cytochrome c release from mitochondria. As treatment at 37 degrees C protected Arabidopsis cells from heat-induced cell death, it may be suggested that Hsp101, Hsp70 and small heat-shock proteins, the synthesis of which is induced under these conditions, are playing an anti-apoptotic role in the plant cell. On the other hand, drastic heat shock upregulated mitochondrial Hsp60 synthesis and induced its release from mitochondria to the cytosol, indicating a pro-apoptotic role of plant Hsp60.

Do mitochondria regulate the heat-shock response in Saccharomyces cerevisiae?

A mild heat shock induces the synthesis of heat-shock proteins (hsps), which protect cells from damage during more extreme heat exposure. The nature of the signals that induce transcription of heat shock-regulated genes remains conjectural. In this work we studied the role of mitochondria in regulating hsps synthesis in Saccharomyces cerevisiae. The results obtained clearly indicate that a mild heat shock elicits a hyperpolarization of the inner mitochondrial membrane and such an event is one of several signals triggering the chain of reactions that activates the expression of the HSP104 gene and probably the expression of other heat shock-regulated genes in S. cerevisiae. The uncouplers or mitochondrial inhibitors which are capable of dissipating the potential on the inner mitochondrial membrane under particular experimental conditions prevent the synthesis of Hsp104 induced by mild heat shock and thus inhibit the development of induced thermotolerance. It is suggested that cAMP-dependent protein kinase A is participating in the mitochondrial regulation of nuclear genes.

Sodium azide reduces the thermotolerance of respiratively grown yeasts.

The effect of sodium azide in heat shock-induced cell death was studied in Debaryomyces vanrijiae, Candida albicans, and Saccharomyces cerevisiae yeasts. The results presented demonstrate that the azide addition induced a drastic decrease in the thermotolerance of glucose-grown D. vanrijiae. In contrast, glucose-grown S. cerevisiae and C. albicans cells treated with NaN(3) became more resistant to heat shock than control cells. Nevertheless, in galactose medium the decrease of thermotolerance of S. cerevisiae and C. albicans cells was observed in the presence of sodium azide. It was suggested that the decreasing effect of sodium azide on thermotolerance takes place only when the yeast cell is incapable of using fermentation for ATP synthesis and obtains energy via oxidative phosphorylation.