Abstract
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.
Highlights
Reactive oxygen species (ROS), such as superoxide anion (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (OH) are an unavoidable by-products of oxygen metabolism
To verify whether the heat-induced ROS production and membrane potential (MMP) rise were occurred in respiring yeast cells entering the stationary phase, cells grown in YEPD medium for 24 h were treated at 45 °C during 0, 10, 30 and 60 min and ROS production and the MMP value were determined by using 2′, 7′-dichlorofluorescein diacetate (H2DCF·DA) and MitoTracker Orange (MO), respectively
The heat-induced MMP increase was reduced in nde1Δ cells (Fig. 6b). These results suggest that, under heat shock conditions, external NADH dehydrogenase Nde1p is involved in ROS production in stationary phase yeast cells, probably via its ability to induce the hyperpolarization of inner mitochondrial membrane
Summary
Reactive oxygen species (ROS), such as superoxide anion (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (OH) are an unavoidable by-products of oxygen metabolism. External NADH dehydrogenases (Nde1p and Nde2p) are located on the exterior face of the inner mitochondria membrane and are involved in the oxidation of NADH produced in the cytosol[9,10,11] It was shown[12,13,14,15] that S. cerevisiae external and internal dehydrogenases are potential sites of ROS production. Diphenyleneiodonium, an inhibitor of flavin-containing enzymes, inhibited hydrogen peroxide formation in isolated mitochondria supplied by exogenous NADH suggesting that external NADH dehydrogenases produce ROS in S. cerevisiae mitochondria under resting conditions[12]. Deletion of external mitochondrial NADH dehydrogenase genes (NDE1 and NDE2) has been reported to reduce the heat-induced ROS production in S. cerevisiae cells and mitochondria[13]. Many of these are directly participating in redox reactions connected to the electron transport chain[18], suggesting their involvement in ROS production
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