Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1? mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.     

Free intracellular amino acid pools during autonomous oscillations in Saccharomyces cerevisiae

In the present work dynamic changes of free intracellular amino acid pools during autonomous oscillations of Saccharomyces cerevisiae were quantified in glucose-limited continuous cultivations. At a dilution rate of D = 0.22 h(-1) cyclic changes with a period of 120 min were found for many variables such as carbon dioxide production rate, dissolved oxygen, pH, biomass content, and various metabolite concentrations. On the basis of the observed dynamic patterns, free intracellular amino acids were classified to show oscillatory, stationary, or chaotic behavior. Amino acid pools such as serine, alanine, valine, leucine, or lysine were subjected to clear oscillations with a frequency of 120 min, identical to that of other described cultivation variables, indicating that there is a direct correlation between the periodic changes of amino acid concentrations and the metabolic oscillations on the cellular level. The oscillations of these amino acids were unequally phase-delayed and had different amplitudes of oscillation. Accordingly, they exhibited different patterns in phase plane plots vs. intracellular trehalose. Despite the complex and marked metabolic changes during oscillation, selected intracellular amino acids such as histidine, threonine, isoleucine, or arginine remained about constant. Concentrations of glutamate and glutamine showed a chaotic behavior. However, the ratio of glutamate to glutamine concentration was found to be oscillatory, with a period of 60 min and a corresponding figure eight-shaped pattern in a plot vs. trehalose concentration. Considering the described diversity, it can be concluded that the observed periodic changes are neither just the consequence of low or high rates of protein biosynthesis/degradation nor correlated to changing cell volumes during oscillation. The ratio between doubling time (189 min) and period of oscillation of intracellular amino acids (120 min) was 1:6. The fact that there is a close relationship between doubling time and period of oscillation underlines that the described autonomous oscillations are cell-cycle-associated.     

Changes in the amino acid pool in a nickel-resistant strain of Saccharomyces cerevisiae

In a Ni-resistant strain of S. cerevisiae , the histidine content of the amino acid pool was increased by culture in a Ni-supplemented medium, while the content of other amino acids was decreased. The toxicity of nickel to yeast was reduced by addition of histidine to the medium. It is concluded that histidine content plays an important role in Ni-resistance in yeast.     

酿酒酵母细胞衰老机理的研究进展

酿酒酵母的细胞衰老研究作为生命科学领域的前沿课题,对解析高等真核生物衰老的分子机制具有重要意义。迄今为止,在酵母中已经确立的衰老模式有两种,即复制型衰老和时序型衰老。细胞衰老的影响因子较多,涉及到很多过程,所以研究起来非常复杂。综述了两种细胞衰老机制的研究进展。     

SKO1 deficiency extends chronological lifespan in Saccharomyces cerevisiae

ABSTRACT

Sko1 plays a key role in the control of gene expression by osmotic and oxidative stress in yeast. We demonstrate that the decrease in chronological lifespan (CLS) of hog1Δ cells was suppressed by SKO1 deletion. sko1Δ single mutant cells were shown to have a longer CLS, thus implicating Sko1 in the regulation of their CLS.     

Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

Isolation and characterization of mutants resistant to amino acid analogues obtained from Saccharomyces cerevisiae strains

Mutants resistant to the amino acid analogues dl-thiaisoleucine, dl-4-azaleucine, 5,5,5-trifluoro-dl-leucine and l-O-methylthreonine, were isolated from Saccharomyces cerevisiae wine yeast strains. The fermentative production of secondary metabolites by the mutants was tested in grape must. Higher alcohols, acetaldehyde and acetic acid concentration varied depending on strain and analogue. Most of the mutants produced increased amounts of amyl alcohol. A remarkable variability in the level of n-propanol, isobutanol, acetaldehyde and acetic acid was observed. In practical application, the use of mutants resistant to amino acid analogues can improve the quality of wines by reducing or increasing the presence of some secondary compounds.     

Anthranilic acid release in adenosine-inhibited cultures of Saccharomyces cerevisiae and its inhibition by thiamin

Adenosine, at 1 mM concentrations or above, was found to have a fungistatic effect on Saccharomyces cerevisiae. A substance with amethyst fluorescence was detected in the medium of adenosine-inhibited cultures of S. cerevisiae. This compound was isolated and physicochemically identified as anthranilic acid. Both the inhibition of growth and release of anthranilic acid induced by adenosine were abrogated by thiamin or by the pyrimidine portion of thiamin, 2-methyl-4-amino-5-hdroxymethyl-pyrimidine (hydroxymethyl-pyrimidine); the latter was found to restore intracellular thiamin content that had been reduced by adenosine. It was demonstrated that effects of thiamin and hydroxymethylpyrimidine on S. cerevisiae cultured with adenosine resulted from their inhibition of adenosine uptake by growing yeast cells.     

三角褐指藻D5-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的表达

D5-脂肪酸脱氢酶是合成花生四烯酸的关键酶。根据已报道的D 5-脂肪酸脱氢酶基因设计引物, 分别从三角褐指藻基因组DNA和总cDNA中扩增得到1520 bp和1410 bp的特异片段, 序列分析结果显示, 结构基因中含有一个大小为110 bp的内含子, 这是国内外首次报道。将D5-脂肪酸脱氢酶基因亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0中, 在大肠杆菌中筛选到含有目的片段的重组质粒pYPTD5, 用电击转化的方法将重组质粒pYPTD5转化到营养缺陷型酿酒酵母菌株INVSc1中, 在缺省培养基中筛选得到酿酒酵母转化菌株YPTD5。在合适的培养条件下, 添加外源底物双高g-亚麻酸和诱导物半乳糖, 培养并收集菌体。通过脂肪酸甲酯气相色谱分析, 表明三角褐指藻D5-脂肪酸脱氢酶基因在酿酒酵母中获得了高效的表达, 将双高g-亚麻酸转化为花生四烯酸, 其底物转化率达到了45.9%。     

代谢工程改造酿酒酵母合成葡萄糖二酸

葡萄糖二酸是一种高附加值的有机酸,广泛用于食品、医药和化工领域。为获得生产葡萄糖二酸的微生物细胞工厂,通过共表达小鼠来源的肌醇加氧酶(MIOX)及恶臭假单胞菌来源的醛酸脱氢酶(Udh),在酿酒酵母Saccharomyces cerevisiae CEN.PK2-1C中构建了葡萄糖二酸合成途径,产量为(28.28±3.15)mg/L。在此基础上,通过调控前体肌醇的合成途径,发现肌醇-1-磷酸合成酶(INO1)是葡萄糖二酸合成途径的限速酶,过量表达INO1,葡萄糖二酸产量达到(107.51±10.87)mg/L,提高了2.8倍。进一步弱化竞争支路中磷酸果糖激酶(PFK1)的表达,最终葡萄糖二酸的产量达到(230.22±10.75)mg/L,为进一步获得高产葡萄糖二酸细胞工厂提供基础。     

Autophagy contributes to sulfonylurea herbicide tolerance via GCN2-independent regulation of amino acid homeostasis

Sulfonylurea (SU) herbicides inhibit branched-chain amino acid (BCAA) biosynthesis by targeting acetolactate synthase. Plants have evolved target-site resistance and metabolic tolerance to SU herbicides; the GCN2 (general control non-repressible 2) pathway is also involved in SU tolerance. Here, we report a novel SU tolerance mechanism, autophagy, which we call ‘homeostatic tolerance,’ is involved in amino acid signaling in Arabidopsis. The activation and reversion of autophagy and GCN2 by the SU herbicide tribenuron-methyl (TM) and exogenous BCAA, respectively, confirmed that TM-induced BCAA starvation is responsible for the activation of autophagy and GCN2. Genetic and biochemical analyses revealed a lower proportion of free BCAA and more sensitive phenotypes in atg5, atg7, and gcn2 single mutants than in wild-type seedlings after TM treatment; the lowest proportion of free BCAA and the most sensitive phenotypes were found in atg5 gcn2 and atg7 gcn2 double mutants. Immunoblotting and microscopy revealed that TM-induced activation of autophagy and GCN2 signaling do not depend on the presence of each other, and these 2 pathways may serve as mutually compensatory mechanisms against TM. TM inhibited the TOR (target of rapamycin), and activated autophagy in an estradiol-induced TOR RNAi line, suggesting that TM-induced BCAA starvation activates autophagy, probably via TOR inactivation. Autophagy and GCN2 were also activated, and independently contributed to TM tolerance in plants conferring metabolic tolerance. Together, these data suggest that autophagy is a proteolytic process for amino acid recycling and contributes to GCN2-independent SU tolerance, probably by its ability to replenish fresh BCAA.     

Phospholipase D1 is required for efficient mating projection formation in Saccharomyces cerevisiae

Phospholipase D1 (PLD1) is an important enzyme involved in lipid signal transduction in eukaryotes. A role for PLD1 in signaling in Saccharomyces cerevisiae was examined. Pheromone response in yeast is controlled by a well-characterized protein kinase cascade. Loss of PLD1 activity was found to impair pheromone-induced changes in cellular morphology that result in formation of mating projections. The rate at which projections appeared following pheromone treatment was delayed, suggesting that PLD1 facilitates the execution of a rate-limiting step in morphogenesis. Mutants were found to be less sensitive to pheromone, again arguing that PLD1 is acting at a rate-limiting step. The fact that morphogenesis is most dramatically affected indicates that PLD1 functions primarily in the morphogenic branch of the pheromone response pathway.     

酿酒酵母衰老机制研究进展

酿酒酵母衰老机制的研究对解析高等真核生物衰老的分子机制具有重要意义。酿酒酵母有两种衰老形式：时序衰老（chronologicalaging）和复制衰老（replicative aging）。酿酒酵母衰老研究中通常使用的寿命定义有两种：世代寿命和时序寿命。前者是指单个酿酒酵母细胞在死亡之前的分裂次数;后者是指一定数量的酵母细胞在后二次生长和稳定期的存活时间。本文分别综述了这两种衰老形式的分子机制及两者的相同点和不同点。     

Biosynthesis of L-ascorbic acid (vitamin C) by Saccharomyces cerevisiae

Saccharomyces cerevisiae cells incubated with D-glucose (D-Glc), D-galactose or D-mannose (D-Man) synthesised D-erythroascorbic acid (D-EAA) but not L-ascorbic acid (L-AA). Accumulation of D-EAA was observed in cells incubated with D-arabinose (D-Ara) whilst accumulation of L-AA occurred in cells incubated with L-galactose (L-Gal), L-galactono-1,4-lactone and L-gulono-1,4-lactone. When S. cerevisiae cells were incubated with D-[U-(14)C]Glc, D-[U-(14)C]Man or L-[1-(14)C]Gal, incorporation of radioactivity into L-AA was observed only with L-[1-(14)C]Gal. Pre-incubation of yeast cells with D-Ara substantially reduced the incorporation of L-[1-(14)C]Gal into L-AA. Our results indicate that, under appropriate conditions, yeast cells can synthesise L-AA via the pathway naturally used for D-EAA biosynthesis.