During fermentation, increased ethanol concentration is a major stress for yeast cells. of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in and mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype of mutants resulted from severe cytosolic acidification. Interestingly, the mutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol. IMPORTANCE The yeast has been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains used in alcoholic fermentation. The results revealed that straight-chain alcohols induced cytosolic and vacuolar acidification through their membrane-permeabilizing effects. Contrary to expectations, a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress, but not in the maintenance of intracellular pH, seems to be important for protecting yeast cells against ethanol stress. These findings will expand our understanding of the mechanisms of ethanol tolerance and provide encouraging hints for the development of ethanol-tolerant yeast stresses. INTRODUCTION The budding yeast has been widely used in industrial fermentation, such as in the production of alcoholic beverages and ethanol gas. During fermentation, yeast cells encounter several environmental tensions, such as increased alcohol concentration, high osmolarity, and heat fluctuations. Among these, ethanol is usually a major stress factor that affects the vitality and viability of yeast cells, thereby leading to an arrest of the fermentation process (1). High ethanol concentrations are known to influence a number of metabolic processes, including the inhibition of glucose and amino acid transport, denaturation of the important glycolytic enzymes pyruvate kinase and hexokinase, and increased membrane permeability (1). Cellular membranes, especially the plasma membrane, have been suggested to be the main target of ethanol stress in yeast cells (2). Ethanol and other short-chain alcohols are believed to disturb the cellular membrane through the association of their aliphatic chains with the hydrophobic interior of membranes, thereby affecting membrane permeability and stability (3). The loss of membrane honesty decreases Belinostat (PXD101) manufacture the cell’s ability to maintain a concentration gradient across the plasma membrane, which is usually important for coupled transport of several metabolites, such as amino acids and ions (4, 5). Since the budding yeast normally lives in a slightly acidic environment with a high proton concentration, the increase in membrane permeability caused by alcohol may lead to an increased passive influx of protons across the membrane, thereby inducing intracellular acidification (6). Compounds with high lipophilicity, usually expressed in terms of the log octanol-water partition coefficient (log mutants lacking V-ATPase activity exhibit a more-alkaline vacuolar pH than the wild-type strain (16) and are unable to grow at pH values of >7 and <4 (17, 18). Moreover, a loss of V-ATPase activity causes ubiquitination and endocytosis of Pma1p, possibly to balance overall pH homeostasis (16, 19). These phenotypes of the mutants suggest that the V-ATPase and the plasma membrane H+-ATPases together play an interdependent role in the rules of intracellular pH homeostasis. In addition to the expected phenotypes of the mutants, Belinostat (PXD101) manufacture which are a direct result of impaired intracellular pH rules, these mutants also exhibited other pleiotropic phenotypes, including sensitivity to a variety of oxidants, such as H2O2 (20), sensitivity to transition metals, such as copper mineral and zinc (21, 22), poor growth under conditions of both high and low concentrations of calcium and iron (18, 23, 24), poor growth on Belinostat (PXD101) manufacture nonfermentable carbon sources (25), and defective vacuolar morphology and vacuolar protein sorting (26, 27). Some of these phenotypes can be very easily explained by an increased accumulation of reactive oxygen species (ROS) or defective vacuolar function, important for storage and sequestration of several metabolites and toxins in these mutants (20, 25). Based on these previous observations, it is Rabbit Polyclonal to IKK-gamma (phospho-Ser31) usually possible that the V-ATPase, possibly in collaboration with the plasma membrane H+-ATPase, is usually required for recovery from cytosolic acidification or other harmful.