A combination of three amino acids including 1.0 g/L isoleucine, 0.5 g/L methionine and 2.0 g/L phenylalanine was found to enhance ethanol tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae. When subjected to 20% (V/V) ethanol for 9 h at 30 degrees C, all cells died whereas 57% remained viable for the cells grown in the presence of the three amino acids. Based on the analysis of protein amino acid composition of plasma membranes and the determination of plasma membrane fluidity by measuring fluorescence anisotropy using diphenylhexatriene as a probe, it was found that the significantly increased ethanol tolerance of cells grown with the three amino acids was due to the incorporation of the supplementary amino acids into the plasma membranes, thus resulting in enhanced ability of the plasma membranes to efficiently counteract the fluidizing effect of ethanol when subjected to ethanol stress. This is the first time to report that plasma membrane fluidity can be influenced by protein amino acid composition of plasma membranes.
Amino Acids ; analysis ; physiology ; Cell Membrane ; chemistry ; Culture Media ; Drug Tolerance ; Ethanol ; pharmacology ; Flocculation ; Membrane Fluidity ; Saccharomyces cerevisiae ; chemistry ; drug effects ; growth & development ; Schizosaccharomyces ; chemistry ; drug effects ; growth & development

Although alterations in fatty acid composition of phospholipids in plasma membranes had no effect on activities of plasma membrane ATPases of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae cells grown in the absence of ethanol (basal enzymes), they significantly affected the susceptibilities of the enzymes to in vivo activation induced by ethanol: the maximal values for the activated enzymes in cells pregrown with 0.6 mmol/L palmitic, linoleic or linolenic acid respectively were 3.6, 1.5 and 1.2-fold higher than their respective basal levels (in cells grown without ethanol), whereas the corresponding value for cells pregrown in the absence of fatty acid was 2.3-fold, with the concentrations of ethanol for the above maximal in vivo activation of enzymes being 7%, 6%, 6% and 7% (V/V) respectively. The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the basal and the activated plasma membrane ATPases were essentially identical; however, the v(max) values of activated enzymes increased significantly. It was found that the characteristics of phospholipid fatty acid composition of plasma membrane leading to the enhanced ethanol tolerance of this strain, were also efficacious to increase the percentage of activation of plasma membrane ATPase per unit of ethanol. These data support a close correlation between the ethanol tolerance of this strain and the sensitivity of its plasma membrane ATPase to the in vivo ethanol-induced activation.
Adenosine Triphosphatases ; antagonists & inhibitors ; metabolism ; Cell Membrane ; chemistry ; enzymology ; Enzyme Activation ; Ethanol ; pharmacology ; Fatty Acids ; analysis ; Phospholipids ; analysis ; Saccharomyces cerevisiae ; drug effects ; enzymology ; growth & development ; Schizosaccharomyces ; drug effects ; enzymology ; growth & development

Ca2+ at 1.64 mmol/L markedly increased ethanol tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae. After 9 h of exposure to 20% (V/V) ethanol at 30 degrees C , no viability remained for the control whereas 50.0% remained for the cells both grown and incubated with ethanol in Ca2+ -added medium. Furthermore, when subjected to 15% (V/V) ethanol at 30 degrees C, the equilibrium nucleotide concentration and plasma membrane permeability coefficient (P' ) of the cells both grown and incubated with ethanol in Ca2+ -added medium accounted for only 50.0% and 29.3% those of the control respectively, indicating that adding Ca2+ can markedly reduce plasma membrane permeability of yeast cells under ethanol stress as compared with the control. Meanwhile, high viability levels acquired by the addition of Ca2+ exactly corresponded to the striking decreases in extracellular nucleotide concentration and P' achieved with identical approach. Therefore, the enhancing effect of Ca2+ on ethanol tolerance of this strain is closely related to its ability to decrease plasma membrane permeability of yeast cells subjected to ethanol stress.
Calcium ; pharmacology ; Cell Membrane ; drug effects ; metabolism ; Cell Membrane Permeability ; drug effects ; Ethanol ; pharmacology ; Saccharomyces cerevisiae ; drug effects ; growth & development ; metabolism ; Schizosaccharomyces ; drug effects ; growth & development ; metabolism ; Temperature

Investigation was undertaken for the purpose of examining any possible correlation between flocculence of a self-flocculating fusant of Schizosaccharomyces pombe mutant and Saccharomyces cerevisiae mutant (called fusant SPSC for short) and the tolerance of this strain to ethanol. When exposed to 18% (V/V) ethanol for 7 h at 30 degrees C, 52%, 37% and 9% of viability levels remained for the cells of fusant SPSC and its two parental strains, Sch. pombe mutant and S. cerevisiae mutant respectively. Analysis of phospholipid fatty acid composition of plasma membrane showed that the content of palmitic acid of each flocculating yeast (fusant SPSC or Sch. pombe mutant) was around 2-fold higher than that of free S. cerevisiae mutant, with remarkably lower contents of palmitoleic and oleic acids than the latter. When 0.1 mol/L sodium citrate was initially included in the medium in which cells of each flocculating yeast were grown, free cells rather than aggregates were finally obtained. Furthermore, the content of palmitic acid in the phospholipid fatty acid composition of the plasma membranes of the free cells of each flocculating yeast was found to decrease significantly, with a marked increase in the contents of palmitoleic and oleic acids. As a result, the characteristics of the phospholipid fatty acid composition of the plasma membranes of the free cells of each flocculating yeast were similar to those of S. cerevisiae mutant. Meanwhile, the disappearance of flocculence of each flocculating yeast caused by the action of sodium citrate brought about a steeply decreased tolerance of the free cells to ethanol, thus being equivalent to that of S. cerevisiae mutant. These data suggest that the stronger ethanol tolerance of each flocculating yeast is related to the higher content of palmitic acid in the phospholipid fatty acid composition of the plasma membranes. Thus, the enhancement by flocculence on the tolerance of yeast cells to ethanol as well as its mechanism are first reported in this work.
Bioreactors ; microbiology ; Carbohydrates ; Cell Membrane ; metabolism ; Drug Tolerance ; Ethanol ; metabolism ; pharmacology ; Fatty Acids ; metabolism ; Fermentation ; Flocculation ; Phospholipids ; metabolism ; Saccharomyces cerevisiae ; drug effects ; metabolism ; Schizosaccharomyces ; drug effects ; metabolism ; Zea mays ; metabolism