Genetic analysis of pleiotropic effects of pho85 mutations in yeast Saccharomyces cerevisiae

The cyclin-dependent phosphoprotein kinase Pho85p is involved in the regulation of metabolism and cell cycle in the yeast Saccharomyces cerevisiae. It is known that mutations in the PHO85 gene lead to constitutive synthesis of Pho5p acidic phosphatase, a delay in cell growth on media containing nonfermentable carbon sources, sensitivity to high temperature, and other phenotypic effects. A lack of growth at 37 degrees C and on a medium with alcohol as the carbon source was shown to be associated with the rapid accumulation of nuclear ts and mitochondrial [rho-] mutations occurring in the background of gene PHO85 inactivation. Thus, Pho85p seems to play an important role in the maintenance of yeast genome stability.     

Calorie restriction extends the chronological lifespan of Saccharomyces cerevisiae independently of the Sirtuins

Calorie restriction (CR) extends the mean and maximum lifespan of a wide variety of organisms ranging from yeast to mammals, although the molecular mechanisms of action remain unclear. For the budding yeast Saccharomyces cerevisiae reducing glucose in the growth medium extends both the replicative and chronological lifespans (CLS). The conserved NAD(+)-dependent histone deacetylase, Sir2p, promotes replicative longevity in S. cerevisiae by suppressing recombination within the ribosomal DNA locus and has been proposed to mediate the effects of CR on aging. In this study, we investigated the functional relationships of the yeast Sirtuins (Sir2p, Hst1p, Hst2p, Hst3p and Hst4p) with CLS and CR. SIR2, HST2, and HST4 were not major regulators of CLS and were not required for the lifespan extension caused by shifting the glucose concentration from 2 to 0.5% (CR). Deleting HST1 or HST3 moderately shortened CLS, but did not prevent CR from extending lifespan. CR therefore works through a Sirtuin-independent mechanism in the chronological aging system. We also show that low temperature or high osmolarity additively extends CLS when combined with CR, suggesting that these stresses and CR act through separate pathways. The CR effect on CLS was not specific to glucose. Restricting other simple sugars such as galactose or fructose also extended lifespan. Importantly, growth on nonfermentable carbon sources that force yeast to exclusively utilize respiration extended lifespan at nonrestricted concentrations and provided no additional benefit when restricted, suggesting that elevated respiration capacity is an important determinant of chronological longevity.     

The wheat LEA protein Em functions as an osmoprotective molecule in Saccharomyces cerevisiae

The biased amino acid composition and aperiodic (random coil) configuration of Group 1 late embryogenesis-abundant (LEA) proteins imply that these proteins are capable of binding large amounts of water. While Group 1 LEAs have been predicted to contribute to osmotic stress protection in both embryonic and vegetative tissues, biochemical support has been lacking. We have used Saccharomyces cerevisiae as a model system to test the putative osmoprotective function of a wheat Group 1 LEA protein, Em. We demonstrate that expression of Em protein in yeast cells is not deleterious to growth in media of normal osmolarity and attenuates the growth inhibition normally observed in media of high osmolarity. Enhanced growth is observed in the presence of a variety of osmotically active compounds indicating that Em protein is capable of mitigating the detrimental effect of low water potential in a relatively non-specific manner. These results are the first biochemical demonstration of an osmoprotective function for a Group 1 LEA and suggest that the yeast expression system will be useful in dissecting the mechanism of protection through structure-function studies.     

Genetic Analysis of Pleiotropic Effects of pho85 Mutations in Yeast Saccharomyces cerevisiae

The cyclin-dependent phosphoprotein kinase Pho85p is involved in the regulation of metabolism and cell cycle in the yeast Saccharomyces cerevisiae. It is known that mutations in the PHO85gene lead to constitutive synthesis of Pho5p acidic phosphatase, a delay in cell growth on media containing nonfermentable carbon sources, sensitivity to high temperature, and other phenotypic effects. A lack of growth at 37°C and on a medium with alcohol as the carbon source was shown to be associated with the rapid accumulation of nuclear ts and mitochondrial [rho ^– ] mutations occurring in the background of gene PHO85 inactivation. Thus, Pho85p seems to play an important role in the maintenance of yeast genome stability.     

NatC Nalpha-terminal acetyltransferase of yeast contains three subunits, Mak3p, Mak10p, and Mak31p

The yeast Saccharomyces cerevisiae contains three types of N(alpha)-terminal acetyltransferases, NatA, NatB, and NatC, with each having a different catalytic subunit, Ard1p, Nat3p, and Mak3p, respectively, and each acetylating different sets of proteins with different N(alpha)-terminal regions. We show that the NatC N(alpha)-terminal acetyltransferases contains Mak10p and Mak31p subunits, in addition to Mak3p, and that all three subunits are associated with each other to form the active complex. Genetic deletion of any one of the three subunits results in identical abnormal phenotypes, including the lack of acetylation of a NatC substrate in vivo, diminished growth at 37 degrees C on media containing nonfermentable carbon sources, and the lack of maintenance or assembly of the L-A dsRNA viral particle.     

Direct screening of libraries of yeast clones for alpha-amylase activity on raw starch hydrolysis

High-throughput screening for high-activity barley alpha-amylase mutants expressed in Saccharomyces cerevisiae is hampered by the interference of reducing agents, particularly the glucose used in yeast growth media. The present investigation employed colorimetric and chemiluminescent detection systems that enable direct and rapid screening of activities on raw starch substrate. Active clones could be separated into two groups, based on high total activity or high specific activity.     

Antimicrobial and biological effects of ipemphos and amphos on bacterial and yeast strains

In this study, the antimicrobial effects of monophosphazenes such as SM ipemphos and amphos were examined on bacterial and yeast strains. In addition, the biological effects of these compounds were tested on the lipid level of Saccharomyces cerevisiae and Candida albicans cells. The SM has an antimicrobial effect on the bacterial and yeast strains within the range of 100 and 1500 microg. When the concentration was increased, the inhibition zone expanded on the growth media ( p < 0.01; p < 0.001). The ipemphos did not affect the bacterial and yeast cells in the 100 and 600 microg range. In addition, the amphos did not show an antimicrobial effect on the bacterial cells between 100 and 300 microg or on yeast cells at any of the administered concentrations. In vitro media, the biological effects of these molecules were compared with vitamin E, melatonin and fish oil on the yeast cells. We have found that monophosphazenes have growth effects on the cells in vitro media. The lipid level of S. cerevisiae cells was decreased by 300 microg doses of vitamin E, fish oil, and ipemphos (respectively; p < 0.05, p < 0.01, and p < 0. 001). In addition, the lipid levels of the same yeast cells were depressed by 1000-microg doses in all supplemented groups. However, it was observed that the highest decrease in lipid level of S. cerevisiae cells occurred in the amphos group ( p < 0.001). The lipid levels of the C. albicans cells were significantly reduced ( p < 0.01) by 300 microg of amphos and melatonin. In contrast, the vitamin E and fish oil significantly raised ( p < 0.01; p < 0.001) the lipid level of the same yeast cell, as compared with the control. In addition, the lipid level of these cells was increased by administration of 1000 microg vitamin E, and melatonin ( p < 0.01). In conclusion, while high concentrations of ipemphos and amphos have an antimicrobial effect on bacterial and yeast cells, amphos did not affect the yeast cells. While ipemphos and amphos increased cell growth in media, they reduced the lipid level of C. albicans and S. cerevisiae. In addition, the antioxidants such as vitamin E, melatonin, and fish oils affected the lipid level of yeast cells.     

Multicopy suppressors of phenotypes resulting from the absence of yeast VDAC encode a VDAC-like protein.

The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel). Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1. However, cells missing the POR1 gene (delta por1) were able to grow on yeast media containing a nonfermentable carbon source (glycerol) but not on such media at elevated temperature (37 degrees C). If VDAC normally provides the pathway for metabolites to pass through the outer membrane, some other protein(s) must be able to partially substitute for that function. To identify proteins that could functionally substitute for POR1, we have screened a yeast genomic library for genes which, when overexpressed, can correct the growth defect of delta por1 yeast grown on glycerol at 37 degrees C. This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amino acid sequence identity to the previously identified yeast VDAC protein (YVDAC1). YVDAC2 can functionally complement defects present in delta por1 strains only when it is overexpressed. Deletion of the POR2 gene alone had no detectable phenotype, while yeasts with deletions of both the POR1 and POR2 genes were viable and able to grow on glycerol at 30 degrees C, albeit more slowly than delta por1 single mutants. Like delta por1 single mutants, they could not grow on glycerol at 37 degrees C. Subcellular fractionation studies with antibodies which distinguish YVDAC1 and YVDAC2 indicate that YVDAC2 is normally present in the outer mitochondrial membrane. However, no YVDAC2 channels were detected electrophysiologically in reconstituted systems. Therefore, mitochondrial membranes made from wild-type cells, delta por1 cells, delta por1 delta por2 cells, and delta por1 cells overexpressing YVDAC2 were incorporated into liposomes and the permeability of resulting liposomes to nonelectrolytes of different sizes was determined. The results indicate that YVDAC2 does not confer any additional permeability to these liposomes, suggesting that it may not normally form a channel. In contrast, when the VDAC gene from Drosophila melanogaster was expressed in delta por1 yeast cells, VDAC-like channels could be detected in the mitochondria by both bilayer and liposome techniques, yet the cells failed to grow on glycerol at 37 degrees C. Thus, channel-forming activity does not seem to be either necessary or sufficient to restore growth on nonfermentable carbon sources, indicating that VDAC mediates cellular functions that do not depend on the ability to form channels.     

Molecular genetic analysis of volatile-anesthetic action.

The mechanism(s) and site(s) of action of volatile inhaled anesthetics are unknown in spite of the clinical use of these agents for more than 150 years. In the present study, the model eukaryote Saccharomyces cerevisiae was used to investigate the action of anesthetic agents because of its powerful molecular genetics. It was found that growth of yeast cells is inhibited by the five common volatile anesthetics tested (isoflurane, halothane, enflurane, sevoflurane, and methoxyflurane). Growth inhibition by the agents is relatively rapid and reversible. The potency of these compounds as yeast growth inhibitors directly correlates with their lipophilicity as is predicted by the Meyer-Overton relationship, which directly correlates anesthetic potency of agents and their lipophilicity. The effects of isoflurane on yeast cells were characterized in the most detail. Yeast cells survive at least 48 h in a concentration of isoflurane that inhibits colony formation. Mutants resistant to the growth-inhibitory effects of isoflurane are readily selected. The gene identified by one of these mutations, zzz4-1, has been cloned and characterized. The predicted ZZZ4 gene product has extensive homology to phospholipase A2-activating protein, a GO effector protein of mice. Both zzz4-1 and a deletion of ZZZ4 confer resistance to all five of the agents tested, suggesting that signal transduction may be involved in the response of these cells to volatile anesthetics.     

Evaluation of SCO1 deletion on Saccharomyces cerevisiae metabolism through a proteomic approach

The Saccharomyces cerevisiae gene SCO1 has been shown to play an essential role in copper delivery to cytochrome c oxidase. Biochemical studies demonstrated specific transfer of copper from Cox17p to Sco1p, and physical interactions between the Sco1p and Cox2p. Deletion of SCO1 yeast gene results in a respiratory deficient phenotype. This study aims to gain a more detailed insight on the effects of SCO1 deletion on S. cerevisiae metabolism. We compared, using a proteomic approach, the protein pattern of SCO1 null mutant strain and wild-type BY4741 strain grown on fermentable and on nonfermentable carbon sources. The analysis showed that on nonfermentable medium, the SCO1 mutant displayed a protein profile similar to that of actively fermenting yeast cells. Indeed, on 3% glycerol, this mutant displayed an increase of some glycolytic and fermentative enzymes such as glyceraldehyde-3-phosphate dehydrogenase 1, enolase 2, pyruvate decarboxylase 1, and alcohol dehydrogenase 1. These data were supported by immunoblotting and enzyme activity assay. Moreover, the ethanol assay and the oxygen consumption measurement demonstrated a fermentative activity in SCO1 mutant on respiratory medium. Our results suggest that on nonfermentable carbon source, the lack of Sco1p causes a metabolic shift from respiration to fermentation.     

Inhibition of yeast growth by molybdenum-hydroxylamido complexes correlates with their presence in media at differing pH values

The effects of Mo-hydroxylamido complexes on cell growth were determined in Saccharomyces cerevisiae to investigate the biological effects of four different Mo complexes as a function of pH. Studies with yeast, an eukaryotic cell, are particularly suited to examine growth at different pH values because this organism grows well from pH 3 to 6.5. Studies can therefore be performed both in the presence of intact complexes and when the complexes have hydrolyzed to ligand and free metal ion. One of the complexes we examined was structurally characterized by X-ray crystallography. Yeast growth was inhibited in media solutions containing added Mo-dialkylhydroxylamido complexes at pH 3-7. When combining the yeast growth studies with a systematic study of the Mo-hydroxylamido complexes' stability as a function of pH and an examination of their speciation in yeast media, the effects of intact complexes can be distinguished from that of ligand and metal. This is possible because different effects are observed with complex present than when ligand or metal alone is present. At pH 3, the growth inhibition is attributed to the forms of molybdate ion that exist in solution because most of the complexes have hydrolyzed to oxomolybdate and ligand. The monoalkylhydroxylamine ligand inhibited yeast growth at pH 5, 6 and 7, while the dialkylhydroxylamine ligands had little effect on yeast growth. Growth inhibition of the Mo-dialkylhydroxylamido complexes is observed when a complex exists in the media. A complex that is inert to ligand exchange is not effective even at pH 3 where other Mo-hydroxylamido complexes show growth inhibition as molybdate. These results show that the formation of some Mo complexes can protect yeast from the growth inhibition observed when either the ligand or Mo salt alone are present.     

Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism

Cancer cells are under intrinsic increased oxidative stress and vulnerable to free radical-induced apoptosis. Here, we report a strategy to hinder mitochondrial electron transport and increase superoxide O2. radical generation in human leukemia cells as a novel mechanism to enhance apoptosis induced by anticancer agents. This strategy was first tested in a proof-of-principle study using rotenone, a specific inhibitor of mitochondrial electron transport complex I. Partial inhibition of mitochondrial respiration enhances electron leakage from the transport chain, leading to an increase in O2. generation and sensitization of the leukemia cells to anticancer agents whose action involve free radical generation. Using leukemia cells with genetic alterations in mitochondrial DNA and biochemical approaches, we further demonstrated that As2O3, a clinically active anti-leukemia agent, inhibits mitochondrial respiratory function, increases free radical generation, and enhances the activity of another O2.-generating agent against cultured leukemia cells and primary leukemia cells isolated from patients. Our study shows that interfering mitochondrial respiration is a novel mechanism by which As2O3 increases generation of free radicals. This novel mechanism of action provides a biochemical basis for developing new drug combination strategies using As2O3 to enhance the activity of anticancer agents by promoting generation of free radicals.     

Characterization of two 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase isozymes from Saccharomyces cerevisiae

The Saccharomyces cerevisiae ADE16 and ADE17 genes encode 5-aminoimidazole-4-carboxamide ribonucleotide transformylase isozymes that catalyze the penultimate step of the de novo purine biosynthesis pathway. Disruption of these two chromosomal genes results in adenine auxotrophy, whereas expression of either gene alone is sufficient to support growth without adenine. In this work, we show that an ade16 ade17 double disruption also leads to histidine auxotrophy, similar to the adenine/histidine auxotrophy of ade3 mutant yeast strains. We also report the purification and characterization of the ADE16 and ADE17 gene products (Ade16p and Ade17p). Like their counterparts in other organisms, the yeast isozymes are bifunctional, containing both 5-aminoimidazole-4-carboxamide ribonucleotide transformylase and inosine monophosphate cyclohydrolase activities, and exist as homodimers based on cross-linking studies. Both isozymes are localized to the cytosol, as shown by subcellular fractionation experiments and immunofluorescent staining. Epitope-tagged constructs were used to study expression of the two isozymes. The expression of Ade17p is repressed by the addition of adenine to the media, whereas Ade16p expression is not affected by adenine. Ade16p was observed to be more abundant in cells grown on nonfermentable carbon sources than in glucose-grown cells, suggesting a role for this isozyme in respiration or sporulation.     

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.     

某些金属离子对酿酒酵母和粟酒裂殖酵母的细胞增殖具有不同的作用效应

由于配制培养液所用试剂污染的金属离子已能满足酵母细胞生长需要,本文采用金属离子蟹合剂EDTA去除自由离子,然后回加某些金属离子方法研究了这些金属离子对酵母细胞增殖的影响,结果发现某些金属离子对S．pombe和S．cereisiae的细胞增殖具有不同的作用效应。实验表明,完全抑制S．pombe和S．cerevisiae的细胞增殖所需的EDTA浓度分别为0．5mmol／L和5mmol／L。Fe3＋不能恢复EDTA对S．cervisiae增殖的抑制作用,却能恢复EDTA对S．pombe增殖的抑制作用。Mn2＋则恰好相反,基本恢复EDTA对S．cerevisiae的抑制作用,却对EDTA抑制S．pombe的抑制作用恢复得很差。Zn2＋和Cu2＋能够恢复EDTA对S．cerevisiae和S.pombe的抑制作用。Mg2＋却不能恢复EDTA的抑制作用。这表明酵母增殖需某些金属离子的参与。金属离子浓度的测定和分析表明,EDTA的加入几乎不影响培养液中的自由Mg2＋浓度,而主要是影响Cu2＋和Zn2＋,这与前人报道的结果不一样。     

A screen for nigericin-resistant yeast mutants revealed genes controlling mitochondrial volume and mitochondrial cation homeostasis

Little is known about the regulation of ion transport across the inner mitochondrial membrane in Saccharomyces cerevisiae. To approach this problem, we devised a screening procedure for facilitating the identification of proteins involved in mitochondrial ion homeostasis. Taking advantage of the growth inhibition of yeast cells by electroneutral K(+)/H(+) ionophore nigericin, we screened for genetic mutations that would render cells tolerant to this drug when grown on a nonfermentable carbon source and identified several candidate genes including MDM31, MDM32, NDI1, YMR088C (VBA1), CSR2, RSA1, YLR024C, and YNL136W (EAF7). Direct examination of intact cells by electron microscopy indicated that mutants lacking MDM31 and/or MDM32 genes contain dramatically enlarged, spherical mitochondria and that these morphological abnormalities can be alleviated by nigericin. Mitochondria isolated from the Deltamdm31 and Deltamdm32 mutants exhibited limited swelling in an isotonic solution of potassium acetate even in the presence of an exogenous K(+)/H(+) antiport. In addition, growth of the mutants was inhibited on ethanol-containing media in the presence of high concentrations of salts (KCl, NaCl, or MgSO(4)) and their mitochondria exhibited two- (Deltamdm31 and Deltamdm32) to threefold (Deltamdm31Deltamdm32) elevation in magnesium content. Taken together, these data indicate that Mdm31p and Mdm32p control mitochondrial morphology through regulation of mitochondrial cation homeostasis and the maintenance of proper matrix osmolarity.