Effect of growth rate on ethanol tolerance ofSaccharomyces cerevisiae

Δ^5,7 Saccharomyces cerevisiae cells growing in chemostat at a specific growth rate of 0.075/h exhibited higher ethanol tolerance measured as ethanol-induced death and anaerobic growth inhibition than the cells growing at 0.2/h, the difference being dependent on the carbon-to-nitrogen molar proportion in the medium. The observed difference in sensitivity to ethanol of anaerobic growth between the slowly and rapidly-growing cells was completely reversed as a result of a block in sterol synthesis causing a negligible synthesis of Δ^5,7. Two physiological parameters, budding frequency and membrane composition, evidently affected ethanol tolerance. Differences between the Δ^5,7 and deficient strains documented a profound effect of the quality of the sterol present on the physiological state of the cell.     

Ethanol-induced death in yeast exhibits features of apoptosis mediated by mitochondrial fission pathway

Cell death in yeast (Saccharomyces cerevisiae) involves several apoptotic processes. Here, we report the first evidence of the following processes, which are also characteristic of apoptosis, in ethanol-induced cell death in yeast: chromatin condensation and fragmentation, DNA cleavage, and a requirement for de novo protein synthesis. Mitochondrial fission protein, Fis1, appears to mediate ethanol-induced apoptosis and ethanol-induced mitochondrial fragmentation. However, mitochondrial fragmentation in response to elevated ethanol levels was not correlated with cell death. Further, in the presence of ethanol, generation of reactive oxygen species was elevated in mutant fis1Delta cells. Our characterization of ethanol-induced cell death in yeast as being Fis1-mediated apoptosis is likely to pave the way to overcoming limitations in large-scale fermentation processes, such as those employed in the production of alcoholic beverages and ethanol-based biofuels.     

Metabolism of delta24-sterols by yeast mutants blocked in removal of the C-14 methyl group.

We have investigated the metabolism of exogenously provided delta24-sterols by whole cell cultures of a polyene-resistant mutant (D10) of Candida albicans blocked at removal of the C-14 methyl group. Comparison of the relative efficiencies of transmethylation at C-24 of selected sterol substrates revealed the following substrate preferences of the Candida delta24-sterol methyltransferase (EC 2.1.1.41): zymosterol greater than 4alpha-methylzymosterol greater than 14alpha-methylzymosterol. Exogenous 4,4-dimethylzymosterol was not transmethylated by mutant D10. Incorporation of the 14C-labelled methyl group of S-adenosyl-L-[methyl-14C]methionine into the sterols of a D10 culture preloaded with zymosterol indicated that zymosterol was a better (40 X) substrate than endogenous lanosterolmfeeding zymosterol to D10 and a polyene-resistant strain of Saccharomyces cerevisiae (Nys-P100) that was also blocked at removal of the C-14 methyl group gave 24-methyl sterols possessing delta22 and ring B unsaturation. Mutant D10 was able to produce ergosterol from zymosterol whereas Nys-P100 produced ergosta-7,22-dienol. When grown in the presence of 3 micrometer 25-aza-24,25-dihydrozymosterol, a known inhibitor of the delta24-sterol methyltransferase, Nys-P100 accumulated 14alpha-methylzymosterol, a minor metabolite in this mutant under normal growth conditions and hitherto unidentified as a yeast sterol.     

A novel screening for inhibitors of a pleiotropic drug resistant pump, Pdr5, in Saccharomyces cerevisiae

Yeast is an excellent model system of eukaryotes for the study of molecular mechanisms of ATP-binding cassette transporters. Pdr5 protein is a yeast Saccharomyces cerevisiae ATP-binding cassette transporter conferring resistance to several unrelated drugs. Here, we described a novel drug screening system designated to detect compounds that inhibit the function of Pdr5. An indicator strain with increased drug sensitivity was constructed with an ergosterol-deficient background (delta syr1/erg3 null mutation). The sensitivity of the indicator strain (delta syr1/erg3 delta pdr5 delta snq2) to the Pdr5 substrates, cycloheximide and cerulenin, was increased 16-fold and 4-fold against wild type, respectively. The screening system is mainly based on the growth inhibition of the PDR5-overexpressed indicator strain with the combination of a sample and cycloheximide or cerulenin. The effect of an mdr inhibitor, FK506 on the screening system was clearly detected even at a low concentration (approximately 0.5 microg/ml). In addition, accumulation of rhodamine 6G in the cells was detected as a result of Pdr5 inhibition by FK506. These results indicated that the screening system is useful for a sensitive screening of Pdr5-specific inhibitors with low toxicity.     

Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae.

A sterol C-14 reductase (erg24-1) mutant of Saccharomyces cerevisiae was selected in a fen1, fen2, suppressor background on the basis of nystatin resistance and ignosterol (ergosta-8,14-dienol) production. The erg24-1 allele segregated genetically as a single, recessive gene. The wild-type ERG24 gene was cloned by complementation onto a 12-kb fragment from a yeast genomic library, and subsequently subcloned onto a 2.4-kb fragment. This was sequenced and found to contain an open reading frame of 1,314 bp, predicting a polypeptide of 438 amino acids (M(r) 50,612). A 1,088-bp internal region of the ERG24 gene was excised, replaced with a LEU2 gene, and integrated into the chromosome of the parental strain, FP13D (fen1, fen2) by gene replacement. The ERG24 null mutant produced ergosta-8,14-dienol as the major sterol, indicating that the delta 8-7 isomerase, delta 5-desaturase and the delta 22-desaturase were inactive on sterols with the C14 = 15 double bond.     

Improvements in ethanol tolerance of Kluyveromyces fragilis in jerusalem artichoke juice

Alcoholic fermentation of Jerusalem artichoke juice, a natural complex medium, allowed the production of 13% (v/v) ethanol utilizing an inulin-fermenting strain of Kluyveromyces fragilis, strongly sensitive to ethanol. However, the fermentation of a simple medium with a similar concentration of fermentable sugars (235 g/L) as saccharose stopped prematurely when only 7% (v/v) ethanol had been produced. Differences in the two fermentation profiles were attributed to the significantly lower ethanol tolerance of K. fragilis IGC 2671 in the simple medium with 2% saccharose as compared with diluted J.a. juice with a similar sugar concentration, in fact, (1) in diluted J. a. juice, growth was possible up to 8% (v/v) added ethanol compared with 6% (v/v) in simple medium and (2) ethanol-induced inhibition of the specific growth and fermentation rate as well as ethanol-induced stimulation of the specific death rate were much more drastic in simple medium. Present results show that (1) the complex composition of the medium used for alcoholic fermentation plays a marked role in the ability of the yeast to tolerate and produce ethanol; (2) J. a. juice proved a very appropriate medium for a productive alcoholic fermentation, namely, in processes based on strains with a low ethanol resistance; and (3) to characterize and compare the ethanol tolerance of fermenting yeasts, the standardization of the medium composition must be taken in consideration.     

In vivo interaction of trivalent chromium with yeast plasma membrane, as revealed by EPR spectroscopy

The in vivo effects of CrCl(3) on an ergosterol-producing 33 erg(+) strain of the eukaryotic yeast Candida albicans, and on its ergosterol-deficient erg(-)2 mutant, were studied by using electron paramagnetic resonance spectroscopy. A 5-doxylstearic acid spin probe was applied to label the membranes. The absence of ergosterol, an increased accumulation of Delta(8) sterols, a decreased fatty acid chainlength and a lower proportion of unsaturated fatty acids of the erg(-)2 mutant resulted in a higher membrane rigidity and an increased sensitivity to Cr(III) than those of the parental 33 erg(+) strain. Cr(III) significantly increased the fluidity of the spin labelled membranes, this being more pronounced for the erg(-)2 mutant. The break in the slopes measured for the erg(-)2 mutant was decreased (DeltaAT approximately 4 degrees C) from 17 to 13 degrees C. Cr(III) treatment for 10 h caused a loss of metabolites adsorbing at 260 nm: this loss was 40% for 33 erg(+) and 60% for erg(-)2. This decriptification process might be the main cause of growth inhibition and cell killing by the impermeable Cr(III) ions.     

Delta14-sterol reductase in Saccharomyces cerevisiae.

An in vitro assay for delta14-sterol reductase from yeast was developed, using ergosta-8,14-dien-3beta-ol as the substrate. The kinetics and localization of the enzyme were examined. The inhibition of the enzyme by the antimycotic agent, 15-azasterol, was verified.     

Internal trehalose protects endocytosis from inhibition by ethanol in Saccharomyces cerevisiae

Endocytosis in Saccharomyces cerevisiae is inhibited by concentrations of ethanol of 2 to 6% (vol/vol), which are lower than concentrations commonly present in its natural habitats. In spite of this inhibition, endocytosis takes place under enological conditions when high concentrations of ethanol are present. Therefore, it seems that yeast has developed some means to circumvent the inhibition. In this work we have investigated this possibility. We identified two stress conditions under which endocytosis was resistant to inhibition by ethanol: fermentation during nitrogen starvation and growth on nonfermentable substrates. Under these conditions, yeast accumulates stress protectors, primarily trehalose and Hsp104, a protein required for yeast to survive ethanol stress. We found the following. (i) The appearance of ethanol resistance was accompanied by trehalose accumulation. (ii) Mutant cells unable to synthesize trehalose also were unable to develop resistance. (iii) Mutant cells that accumulated trehalose during growth on sugars were resistant to ethanol even under this nonstressing condition. (iv) Mutant cells unable to synthesize Hsp104 were able to develop resistance. We conclude that trehalose is the major factor in the protection of endocytosis from ethanol. Our results suggest another important physiological role for trehalose in yeast.     

Internal Trehalose Protects Endocytosis from Inhibition by Ethanol in Saccharomyces cerevisiae

Endocytosis in Saccharomyces cerevisiae is inhibited by concentrations of ethanol of 2 to 6% (vol/vol), which are lower than concentrations commonly present in its natural habitats. In spite of this inhibition, endocytosis takes place under enological conditions when high concentrations of ethanol are present. Therefore, it seems that yeast has developed some means to circumvent the inhibition. In this work we have investigated this possibility. We identified two stress conditions under which endocytosis was resistant to inhibition by ethanol: fermentation during nitrogen starvation and growth on nonfermentable substrates. Under these conditions, yeast accumulates stress protectors, primarily trehalose and Hsp104, a protein required for yeast to survive ethanol stress. We found the following. (i) The appearance of ethanol resistance was accompanied by trehalose accumulation. (ii) Mutant cells unable to synthesize trehalose also were unable to develop resistance. (iii) Mutant cells that accumulated trehalose during growth on sugars were resistant to ethanol even under this nonstressing condition. (iv) Mutant cells unable to synthesize Hsp104 were able to develop resistance. We conclude that trehalose is the major factor in the protection of endocytosis from ethanol. Our results suggest another important physiological role for trehalose in yeast.     

ESR determination of membrane order parameter in yeast sterol mutants.

ESR investigations designed to determine membrane order parameter in sterol mutants of Saccharomyces cerevisiae were conducted using the membrane probe, 5-doxyl stearic acid. These mutants are blocked in the ergosterol biosynthetic pathway and thus do not synthesize ergosterol, the end product sterol. They do not require exogenous ergosterol for growth and, therefore, incorporate ergosterol biosynthetic intermediates in their membrane. Increasing order parameter is reflective of an increase in membrane rigidity. Single mutants involving B-ring delta 8 leads to delta 7 isomerization (erg 2) and C-24 methylation (erg 6) showed greater membrane rigidity than wild-type during exponential growth. A double mutant containing both lesions (erg 6/2) showed an even greater degree of membrane rigidity. During stationary phase the order of decreasing membrane rigidity was erg 6 greater than erg 6/2 greater than erg 2 = wild-type. The increased membrane order parameter was attributed to the presence of substituted sterols rather than increased sterol content or altered fatty acid synthesis.     

Alternative pathways of sterol synthesis in yeast. Use of C(27) sterol tracers to study aberrant double-bond migrations and evaluate their relative importance

Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the delta(8)-delta(7) isomerase (Erg2p) or delta(5) desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5 alpha-cholest-8-en-3beta-ol, 8-dehydrocholesterol (delta(5,8) sterol), or isodehydrocholesterol (delta(6,8) sterol), together with the corresponding 3 alpha-3H isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven delta(22E) sterols formed by action of the yeast C-22 desaturase (Erg5p). These products were separated by silver-ion high performance liquid chromatography (Ag(+)-HPLC) and identified by gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag(+)-HPLC. When delta(8)-delta(7) isomerization was blocked, exogenous delta(8) sterol underwent desaturation to delta(5,8), delta(6,8), and delta(8,14) sterols. Formation of delta(5,8) sterol was strongly favored over delta(6,8) sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis. The delta(5,8) sterol was metabolically almost inert except for delta(22) desaturation, whereas the delta(6,8) sterol was readily converted to delta(5,7), delta(5,7,9(11)), and delta(7,9(11)) sterols. The combined results indicate aberrant metabolic pathways similar to those in mammalian systems. However, delta(5,7) sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of delta(5,8) and delta(5,7,9(11)) sterols in wild-type yeast compared to Smith-Lemli-Opitz individuals.     

The immunosuppressant SR 31747 blocks cell proliferation by inhibiting a steroid isomerase in Saccharomyces cerevisiae.

SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion.     

Identification of essential amino acid residues in a sterol 8,7-isomerase from Zea mays reveals functional homology and diversity with the isomerases of animal and fungal origin

A putative 8,7SI (sterol 8,7-isomerase) from Zea mays, termed Zm8,7SI, has been isolated from an EST (expressed sequence tag) library and subcloned into the yeast erg2 mutant lacking 8,7SI activity. Zm8,7SI restored endogenous ergosterol synthesis. An in vitro enzymatic assay in the corresponding yeast microsomal extract indicated that the preferred Delta(8)-sterol substrate possesses a single C4alpha methyl group, in contrast with 8,7SIs from animals and fungi, thus reflecting the diversity in the structure of their active site in relation to the distinct sterol biosynthetic pathways. In accordance with the proposed catalytic mechanism, a series of lipophilic ammonium-ion-containing derivatives possessing a variety of structures and biological properties, potently inhibited the Zm8,7SI in vitro. To evaluate the importance of a series of conserved acidic and tryptophan residues which could be involved in the Zm8,7SI catalytic mechanism, 20 mutants of Zm8,7SI were constructed as well as a number of corresponding mutants of the Saccharomyces cerevisiae 8,7SI. The mutated isomerases were assayed in vivo by sterol analysis and quantification of Delta(5,7)-sterols and directly in vitro by examination of the activities of the recombinant Zm8,7SI mutants. These studies have identified His(74), Glu(78), Asp(107), Glu(121), Trp(66) and Trp(193) that are required for Zm8,7SI activity and show that binding of the enzyme-substrate complex is impaired in the mutant T124I. They underline the functional homology between the plant and animal 8,7SIs on one hand, in contrast with the yeast 8,7SI on the other hand, in accordance with their molecular diversity and distinct mechanisms.     

Energy metabolism in Saccharomyces cerevisiae. Effect of progressive starvation on respiration and pyridine nucleotide reduction linked to ethanol oxidation in intact cells

The progressive effects of aerobic starvation on endogenous and ethanol-linked respiration and pyridine nucleotide reduction have been studied in the yeast Saccharomyces cerevisiae. Three distinct phases of pyridine nucleotide reduction were observed when ethanol was added to unstarved yeast: an initial phase of rapid reduction and accelerating respiration (A); a steady-state phase of reduction with maximal respiration (B); a final phase of rapid reduction at anaerobiosis (C).During the first 5 hr of aeration, the steady-state Phase B was replaced by a phase of slow pyridine nucleotide reduction, while Phases A and C were unaffected. During this period, both endogenous pyridine nucleotide reduction and endogenous respiration decreased sharply.Between 5 and 22 hr of aeration, the endogenous level of reduced pyridine nucleotide declined further, while endogenous respiration remained unchanged. Concurrently, the extent of the Phase A reduction doubled.The addition of ethanol to aerobic, unstarved yeast stimulated a rapid pyridine nucleotide reduction, with further reduction occurring at anaerobiosis. Under anaerobic conditions, the addition of ethanol to unstarved yeast caused little further reduction of pyridine nucleotide. Two hours of starvation decreased the extent of the endogenously supported anaerobic reduction and correspondingly increased the ethanol-induced reduction. These results suggest that, in unstarved yeast, reducing equivalents derived from ethanol under aerobic conditions and those derived from endogenous carbohydrate under anaerobic conditions have access to the same pool of pyridine nucleotide. With starvation, this pool becomes accessible to ethanol-derived (or ethanol-mobilized) reducing equivalents under anaerobic conditions.     

Effect of ammonium ions on †5,7-sterol synthesis inSaccharomyces cerevisiae

The effect of ammonium concentration in the medium on †^5,7-sterol synthesis was examined. Higher concentrations of this nitrogen source in the medium decreased sterol synthesis and accumulation during growth. An intermittent supply with ammonium resulted in a proportional synthesis of †^5,7-sterols and biomass. The carbon to nitrogen molar ratio of ≧ 40 allowed the maximum accumulation of †^5,7-sterols with our strain of baker’s yeast.     

Sterol methylation in Saccharomyces cerevisiae.

Various nystatin-resistant mutants defective in S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) were shown to possess alleles of the same gene, erg6. The genetic map location of erg6 was shown to be close to trp1 on chromosome 4. Despite the single locus for erg6, S-adenosylmethionine: delta 24-sterol-C-methyltransferase enzyme activity was found in three separate fractions: mitochondria, microsomes, and the "floating lipid layer." The amount of activity in each fraction could be manipulated by assay conditions. The lipids and lipid synthesis of mutants of Saccharomyces cerevisiae defective in the delta 24-sterol-C-methyltransferase were compared with a C5(6) desaturase mutant and parental wild types. No ergosterol (C28 sterol) could be detected in whole-cell sterol extracts of the erg6 mutants, the limits of detection being less than 10(-11) mol of ergosterol per 10(8) cells. The distribution of accumulated sterols by these mutants varied with growth phase and between free and esterified fractions. The steryl ester concentrations of the mutants were eight times higher than those of the wild type from exponential growth samples. However, the concentration of the ester accumulated by the mutants was not as great in stationary-phase cells. Whereas the head group phospholipid composition was the same between parental and mutant strains, strain-dependent changes in fatty acids were observed, most notably a 40% increase in the oleic acid content of phosphatidylethanolamine of one erg6 mutant, JR5.     

Ergosterol is required for targeting of tryptophan permease to the yeast plasma membrane

It was known that the uptake of tryptophan is reduced in the yeast erg6 mutant, which is defective in a late step of ergosterol biosynthesis. Here, we show that this is because the high affinity tryptophan permease Tat2p is not targeted to the plasma membrane. In wild-type cells, the plasma membrane localization of Tat2p is regulated by the external tryptophan concentration. Tat2p is transported from the Golgi apparatus to the vacuole at high tryptophan, and to the plasma membrane at low tryptophan. However, in the erg6 mutant, Tat2p is missorted to the vacuole at low tryptophan. The plasma membrane targeting of Tat2p is dependent on detergent-insoluble membrane domains, suggesting that sterol affects the sorting through the organization of lipid rafts. The erg6 mutation also caused missorting to the multivesicular body pathway in late endosomes. Thus, sterol composition is crucial for protein sorting late in the secretory pathway. Tat2p is subject to polyubiquitination, which acts as a vacuolar-targeting signal, and the inhibition of this process suppresses the Tat2p sorting defects of the erg6 mutant. The sorting mechanisms of Tat2p that depend on both sterol and ubiquitin will be discussed.     

Interaction between RAX and PKR modulates the effect of ethanol on protein synthesis and survival of neurons

Ethanol exposure inhibits protein synthesis and causes cell death in the developing central nervous system. The double-stranded RNA (dsRNA)-activated protein kinase (PKR), a serine/threonine protein kinase, plays an important role in translational regulation and cell survival. PKR has been well known for its anti-viral response. Upon activation by viral infection or dsRNA, PKR phosphorylates its substrate, the alpha-subunit of eukaryotic translation initiation factor-2 (eIF2alpha) leading to inhibition of translation initiation. It has recently been shown that, in the absence of a virus or dsRNA, PKR can be activated by direct interactions with its protein activators, PACT, or its mouse homologue, RAX. We have demonstrated that exposure to ethanol increased the phosphorylation of PKR and eIF2alpha in the developing cerebellum. The effect of ethanol on PKR/eIF2alpha phosphorylation positively correlated to the expression of PACT/RAX in cultured neuronal cells. Using PKR inhibitors and PKR null mouse fibroblasts, we verified that ethanol-induced eIF2alpha phosphorylation was mediated by PKR. Overexpression of a wild-type RAX dramatically enhanced sensitivity to ethanol-induced PKR/eIF2alpha phosphorylation, as well as translational inhibition and cell death. In contrast, overexpression of a mutant (S18A) RAX inhibited ethanol-mediated PKR/eIF2alpha activation. Ethanol promoted PKR and RAX association in cells expressing wild-type RAX but not in cells expressing S18A RAX. S18A RAX functioned as a dominant negative protein and blocked ethanol-induced inhibition of protein synthesis and cell death. Our results suggest that the interactions between PKR and PACT/RAX modulate the effect of ethanol on protein synthesis and cell survival in the central nervous system.