Regulation of glutamine synthetase from Saccharomyces cerevisiae by repression, inactivation and proteolysis

Glutamine synthetase activity is modulated by nitrogen repression and by two distinct inactivation processes. Addition of glutamine to exponentially grown yeast leads to enzyme inactivation. 50% of glutamine synthetase activity is lost after 30 min (a quarter of the generation time). Removing glutamine from the growth medium results in a rapid recovery of enzyme activity. A regulatory mutation (gdhCR mutation) suppresses this inactivation by glutamine in addition to its derepressing effect on enzymes involved in nitrogen catabolism. The gdhCR mutation also increases the level of proteinase B in exponentially grown yeast. Inactivation of glutamine synthetase is also observed during nitrogen starvation. This inactivation is irreversible and consists very probably of a proteolytic degradation. Indeed, strains bearing proteinase A, B and C mutations are no longer inactivated under nitrogen starvation.     

Slm9, a novel nuclear protein involved in mitotic control in fission yeast

In the fission yeast Schizosaccharomyces pombe, as in other eukaryotic cells, Cdc2/cyclin B complex is the key regulator of mitosis. Perhaps the most important regulation of Cdc2 is the inhibitory phosphorylation of tyrosine-15 that is catalyzed by Wee1 and Mik1. Cdc25 and Pyp3 phosphatases dephosphorylate tyrosine-15 and activate Cdc2. To isolate novel activators of Cdc2 kinase, we screened synthetic lethal mutants in a cdc25-22 background at the permissive temperature (25 degrees ). One of the genes, slm9, encodes a novel protein of 807 amino acids. Slm9 is most similar to Hir2, the histone gene regulator in budding yeast. Slm9 protein level is constant and Slm9 is localized to the nucleus throughout the cell cycle. The slm9 disruptant is delayed at the G(2)-M transition as indicated by cell elongation and analysis of DNA content. Inactivation of Wee1 fully suppressed the cell elongation phenotype caused by the slm9 mutation. The slm9 mutant is defective in recovery from G(1) arrest after nitrogen starvation. The slm9 mutant is also UV sensitive, showing a defect in recovery from the cell cycle arrest after UV irradiation.     

Long-term preservation of yeast cultures in liquid nitrogen

Nineteen strains of taxonomieally diverse yeast species tested survived freezing and subsequent five-year storage in liquid nitrogen at ™196 °C, using a medium M 2 composed of malt extract, yeast extract, peptone, calf serum and dimethyl sulfoxide. Viability of the yeast cultures after long-term storage ranged from 5 to 97 % (average 62 %) compared with the viability of the cultures prior to freezing. The use of liquid nitrogen refrigeration for preserving yeast cultures is strongly advocated.     

Split-dose and liquid-holding recovery after X-irradiation in diploid yeast Saccharomyces cerevisiae. II. Dependence of the recovery process on cellular protein metabolism.

In diploid yeast, split-dose recovery (SDR) after X-irradiation was affected, if incubation between split doses was performed in the presence of the protein-synthesis inhibitor, cycloheximide. In exponentially-growing cell-cultures, early SDR was undisturbed but complete recovery was not achieved. Concomitantly the cells show a decresing ability to perform subsequent liquid-holding recovery (LHR). In stationary-phase cell-cultures, SDR was completely suppressed. The cells show, however, recovery from potentially lethal damage in the presence of cycloheximide during incubation between the dose-fractions. The experimental results suggest that in diploid yeast SDR after X-irradiation is an enzymatic process dependent on a functioning protein metabolism.     

Effect of aeration on minimal medium recovery of heated Salmonella typhimurium.

The effect of presence or absence of air on minimal medium recovery of heated Salmonella typhimurium was investigated. It was determined that the expression of minimal medium recovery is not only dependent on heat and a nutritionally complex medium but also on air. Unlike in the presence of air, in the presence of nitrogen, cells were able to recover their ability to grow on Trypticase soy agar enriched with 0.5% yeast extract (TSY) when incubated in TSY broth. It was established that in the presence of nitrogen the number of heat-TSY- induced, single-straneded breaks in deoxyribonucleic acid (DNA) were less than in the presence of air. Furthermore, the DNA breaks in nitrogen were repaired, whereas DNA breaks in air were not. The ability of cells to grow on TSY agar corresponded well with their ability to repair damage to DNA.     

Recovery of yeast cells following the combined action of hyperthermia and ionizing radiation

Consecutive action of elevated temperature (50 degrees C) and gamma-irradiation on yeast cells Saccharomyces cerevisiae was studied. It was shown that yeast cells can recover from lethal thermal and radiation lesions after the combined action of the two factors. The efficiency of recovery does not depend upon the sequence of treatments. Heating (50 degrees C) before or after gamma-irradiation increases the radiation response of yeast when plating the cells on a nutrient agar containing 1.5 M KCl. The synergistic effect decreases with yeast cells kept in water at 28 degrees C before plating. The influence of one factor on the effectiveness of recovery from damages induced by the other was estimated.     

Autophagy in the fission yeast Schizosaccharomyces pombe

Autophagy is a non-selective degradation process in eukaryotic cells. The genome sequence of the fission yeast Schizosaccharomyces pombe has revealed that many of the genes required for autophagy are common between the fission yeast and budding yeast, suggesting that the basic machinery of autophagy is conserved between these species. Autophagy in fission yeast is specifically induced by nitrogen starvation based on monitoring a GFP-Atg8p marker. Upon nitrogen starvation, fission yeast cells exit the vegetative cell cycle and initiate sexual differentiation to produce spores. Most of the nitrogen used for de novo protein synthesis during sporulation derives from the autophagic protein degradation system. This review focuses on the recent advances in the role of autophagy in fission yeast.     

Some effects of radiation hormesis for bacterial and yeast cells

A comparative study of chronic and acute action of ionizing radiation on the processes of aging and dying off of bacterial and yeast cells was carried out. It was ascertained that chronic action of ionizing radiation, 2-10,000 times exceeded the natural background, resulted in slowing down of aging and dying off of both pro- and eukaryotic cells. A single acute irradiation of yeast also resulted in the retardation of dying off of the yeast cells surviving after irradiation. The data is presented demonstrating a great increase in the survival of yeast cells under their repeated irradiation after recovery from potentially lethal radiation.     

Survival and recovery of yeast cells after combined treatment with ionizing radiation and heat

Cell survival, recovery kinetics and inactivation forms after successive and simultaneous treatments with gamma rays (60Co) and high temperatures were studied in diploid yeast cells capable of recovery. Both the extent and the rate of the recovery were shown to be greatly decreased with increase in the duration of heat treatment (60 degrees C) followed by radiation and with increase in exposure temperature after simultaneous treatment with heat and radiation. A quantitative approach describing the recovery process was used to estimate the probability of recovery per unit time and the irreversible component of damage after the combined treatment with heat and radiation. It was shown that the probability of recovery was independent of the conditions of the treatment with heat and radiation, while the irreversible component gradually increased as a function of the duration of heat treatment (60 degrees C) after sequential treatment with heat and radiation and as a function of the exposure temperature after simultaneous treatment with heat and radiation. The increase in the irreversible component was accompanied by an increase in cell death without postirradiation division. It is concluded on this basis that the synergistic interaction of ionizing radiation and hyperthermia in yeast cells is not related to the impairment of the recovery capacity itself and that it may be attributed to an increased yield of irreversible damage.     

A medium designed for monitoring pitching yeast contamination in beer using a conductimetric technique

Nitrogen assimilation is the most readily utilized source of conductance changes when pitching yeast is grown in a glucose-based medium. A simple growth medium comprising yeast nitrogen base, in which nitrogen is supplied as ammonium sulphate, and glucose gave good growth but little change in conductance. Inclusion of a succinate buffer in the medium to remove protons liberated as a result of nitrogen uptake produced a large decrease in conductance and detection times that correlated well with enumeration of yeast by plate counting. The medium will allow more rapid and automated detection of pitching yeast survival in pasteurized beer although individual calibration for each beer type will be necessary.     

Nitrogen limitation and recovery in the cyanobacterium Aphanocapsa 6308

The effects of nitrogen limitation and recovery on nitrogen-containing macromolecules were followed in the cyanobacterium Aphanocapsa 6308. Removal of nitrogen from growth media triggers the degradation of the endogenous nitrogen reserves phycocyanin and cyanophycin granule polypeptide in the cyanobacterium Aphanocapsa 6308. Nitrogen recovery involves immediate synthesis of cyanophycin granule polypeptide with peak levels of 5–12% of cell dry weight found 8–12 h after a utilizable nitrogen source is added. A rapid decrease in cyanophycin granule polypeptide level then occurs and the level remains low even in light-limited stationary growth with all nitrogen sources tested except nitrate and ammonia. Protein and phycocyanin recoveries began 3 h after a utilizable nitrogen source was added. Data suggest continuous activity of the enzyme system synthesizing cyanophycin granule polypeptide in nitrogen-limited cells, but synthesis of a degrading system only after nitrogen recovery begins.Nonstandard Abbreviations CGP Cyanophycin granule polypeptide - CAP chloramphenicol - PC phycocyanin To whom offprint requests should be sent     

Effect of Sugar Transport Inactivation in Saccharomyces cerevisiae on Sluggish and Stuck Enological Fermentations

Sluggish and stuck (i.e., very delayed or incomplete) fermentations have been often observed in wine making. Some of them appeared to be associated with insufficient levels of yeast nutrients such as assimilable nitrogen. In these conditions, sugar transport catabolite inactivation, which is triggered by the protein synthesis arrest, may account in part for the inhibition of fermentation. Moreover, this mechanism of inhibition may explain the failure of added ammoniacal nitrogen to nitrogen-limited musts to restore or elevate rate of fermentation after the early yeast growth phase.     

Induction and repression of arginase and ornithine transaminase in baker's yeast

The arginase and the ornithine transaminase of baker's yeast are induced byl-arginine. Both enzymes have been shown to be repressed by nitrogen compounds. This is evidenced primarily by the decrease in specific enzyme activities caused by the addition of readily assimilable nitrogen compounds to a yeast culture with arginine, secondly by the derepression of both enzymes during nitrogen starvation of the yeast grown in various arginine-free media. This derepression equals both in rate and in amount the enzyme synthesis during the adaptation of the yeast to a medium withl-arginine as the sole nitrogen source. It is inhibited by various assimilable and non-assimilable amino acids. The derepression is the result of the nitrogen deficiency itself, since during the starvation of the yeast for sulphate, phosphate or magnesium, neither of the two enzymes is derepressed, and since it is independent of the nature of the carbon source in the nitrogen starvation medium, provided the latter is immediately assimilable.The enzymes are not subject to catabolite repression by glucose metabolites.It is concluded that the synthesis of arginase and ornithine transaminase in yeast is regulated by induction and repression. Arginine induces the enzymes; they are repressed by nitrogen compounds, probably in cooperation with one or more vitamins.Thanks are due to Professor E. G. Mulder for his frequent encouragement, to the Heineken's Brouwerij, Rotterdam and to the Landbouwhogeschoolfonds for research grants, and to Miss H. P. M. Klinkers, to Mr. P. J. Buysman and to Mr. G. J. K. Pesch for their skilful technical assistance.     

Relationship of low lysine and high arginine concentrations to efficient ethanolic fermentation of wheat mash.

Very high gravity wheat mashes containing 20 or more grams of carbohydrates per 100 mL were fermented completely by Saccharomyces cerevisiae, even though these mashes contained low amounts of assimilable nitrogen. Supplementation of wheat mashes with various amino acids or with yeast extract, urea, or ammonium sulfate reduced the fermentation time. However, lysine or glycine added as single supplements, inhibited yeast growth and fermentation. With lysine, yeast growth was severely inhibited, and a loss of cell viability as high as 80% was seen. Partial or complete reversal of lysine-induced inhibition was achieved by the addition of a number of nitrogen sources. All nitrogen sources that relieved lysine-induced inhibition of yeast growth also promoted uptake of lysine and restored cell viability to the level observed in the control. They also increased the rate of fermentation. Experiments with minimal media showed that for lysine to be inhibitory to yeast growth, assimilable nitrogen in the medium must be in growth-limiting concentrations or totally absent. In the presence of excess nitrogen, lysine stimulated yeast growth and fermentation. Results indicate that supplementing wheat mash with other nitrogen sources increases the rate of fermentation not only by providing extra nitrogen but also by reducing or eliminating the inhibitory effect of lysine on yeast growth.     

What is the function of nitrogen catabolite repression in Saccharomyces cerevisiae?

In contrast to the previously held notion that nitrogen catabolite repression is primarily responsible for the ability of yeast cells to use good nitrogen sources in preference to poor ones, we demonstrate that this ability is probably the result of other control mechanisms, such as metabolite compartmentation. We suggest that nitrogen repression is functionally a long-term adaptation to changes in the nutritional environment of yeast cells.     

Effect of nitrogen limitation and surplus upon trehalose metabolism in wine yeast

Trehalose metabolism in yeast has been related to stress and could be used as a stress indicator. Winemaking conditions are stressful for yeast and understanding trehalose metabolism under these conditions could be useful for controlling alcoholic fermentation. In this study, we analysed trehalose metabolism of a commercial wine yeast strain during alcoholic fermentation by varying the nitrogen levels from low (below adequate) to high (excess). We determined trehalose, nitrogen, sugar consumption and expression of NTH1, NTH2 and TPS1. Our results show that trehalose metabolism is slightly affected by nitrogen availability and that the main consumption of nitrogen occurs in the first 24 h. After this period, nitrogen is hardly taken up by the yeast cells. Although nitrogen and sugar are still available, no further growth is observed in high concentrations of nitrogen. Increased expression of genes involved in trehalose metabolism occurs mainly at the end of the growth period. This could be related to an adaptive mechanism for fine tuning of glycolysis during alcoholic tumultuous fermentation, as both anabolic and catabolic pathways are affected by such expression.     

Fission yeast homologue of Tip41-like proteins regulates type 2A phosphatases and responses to nitrogen sources

A fission yeast (Schizosaccharomyces pombe) gene encoding a member of the TIP41-like protein family was identified and characterized. Deletion of the fission yeast tip41 gene leads to slower growth when ammonium chloride is the nitrogen source, but the growth rate is not affected when adenine is the nitrogen source. The tip41 mutant cells also enter the G1 phase of the cell cycle earlier than wild-type cells in response to nitrogen starvation. Overexpression of tip41(+) causes cell death, spherical cell morphology and blocks the shift to G1 phase upon nitrogen starvation. Overexpression of tip41(+) increases the activity of type 2A phosphatase. In a ppa2 deletion strain with reduced PP2A activity, overexpression of tip41(+) no longer blocks the shift to G1 upon nitrogen starvation. These results suggest that fission yeast Tip41 plays a role in cellular responses to nitrogen nutrient conditions at least partly through regulation of type 2A phosphatase activity.