Stress tolerance in a yeast sterol auxotroph: role of ergosterol, heat shock proteins and trehalose

The role of ergosterol in yeast stress tolerance, together with heat shock proteins (hsps) and trehalose, was examined in a sterol auxotrophic mutant of Saccharomyces cerevisiae. Ergosterol levels paralleled viability data, with cells containing higher levels of the sterol exhibiting greater tolerances to heat and ethanol. Although the mutant synthesised hsps and accumulated trehalose upon heat shock to the same levels as the wild-type cells, these parameters did not relate to stress tolerance. These results indicate that the role of ergosterol in stress tolerance is independent of hsps or trehalose.     

Thermal resistance in fungi: the role of heat shock proteins and trehalose

The parallel synthesis of heat shock proteins and trehalose in response to heat shock did not allow the role of these compounds in the acquisition of thermotolerance by fungal cells to be established for a long time. This review analyses experimental data obtained with the use of mutant fungal strains and shows differences in the thermoprotective functions of trehalose and heat shock proteins in relation to cell membranes and macromolecules. The main emphasis has been placed on data demonstrating the thermoprotective role of trehalose in fungi, the present-day understanding of its biological functions, and mechanisms of trehalose interaction with subcellular structures and cell macromolecules.     

Molecular genetics of heat tolerance and heat shock proteins in cereals

Heat stress is common in most cereal-growing areas of the world. In this paper, we summarize the current knowledge on the molecular and genetic basis of thermotolerance in vegetative and reproductive tissues of cereals. Significance of heat stress response and expression of heat shock proteins (HSPs) in thermotolerance of cereal yield and quality is discussed. Major avenues for increasing thermotolerance in cereals via conventional breeding or genetic modification are outlined.     

Thermotolerance and trehalose accumulation induced by heat shock in yeast cells of Candida albicans

Candida albicans yeast cells growing exponentially on glucose are extremely sensitive to severe heat shock treatments (52.5°C for 5 min). When these cultures were subjected to a mild temperature preincubation (42°C), they became thermotolerant and displayed higher resistance to further heat stress. The intracellular content of trehalose was very low in exponential cells, but underwent a marked increase upon non-lethal heat exposure. The accumulation of trehalose is likely due to heat-induced activation of the trehalose-6-phosphate synthase complex, whereas the external trehalase remained practically unmodified. After a temperature reversion shift (from 42°C to 28°C), the pool of trehalose was rapidly mobilized without any concomitant change in trehalase activity. These results support an important role of trehalose in the mechanism of acquired thermotolerance in C. albicans and seem to exclude the external trehalase as a key enzyme in this process.     

Alterations in fatty acid composition and trehalose concentration ofSaccharomyces brewing strains in response to heat and ethanol shock

Summary The effects of heat and ethanol shock on fatty acid composition and intracellular trehalose concentration of lager and ale brewing yeasts were examined. Exposure of cells to heat shock at 37°C or 10% (v/v) ethanol for 60 min resulted in a significant increase in the ratio of the total unsaturated to saturated fatty acyl residues and the intracellular trehalose concentration of cells. A similar increase in the amount of unsaturated fatty acids was observed in cells after 24 h of fermentation of 16°P (degree Plato) or 25°P wort, at which time more than 2% (v/v) ethanol was present in the growth medium. These results suggest that unsaturated fatty acids and high concentrations of intracellular trehalose may protect the cells from the inhibitory effects of heat and ethanol shock.     

Expression of heat shock proteins in turtle and mammal hearts: relationship to anoxia tolerance

Heat shock proteins (HSPs) may play a cardioprotective role during hypoxia or ischemia. We hypothesized that cardiac tissue from hypoxia-tolerant animals might have high levels of specific HSPs. We measured myocardial HSP60 and HSP72/73 in painted and softshell turtles during normoxia and anoxia (12 h) and after recovery (12 or 24 h). We also measured myocardial HSPs in normoxic rats and rabbits. During normoxia, hearts from the most highly anoxia-tolerant species, the painted turtle, expressed the highest levels of HSP60 (22.6+/-2.0 mg/g total protein) followed by softshells (11.5+/-0.8 mg/g), rabbits (6.8+/-0.9 mg/g), and rats (4.5+/-0.5 mg/g). HSP72/73 levels, however, were not significantly different. HSP60 levels in hearts from both painted and softshell turtles did not deviate significantly from control values after either 12 h of anoxia or 12 or 24 h of recovery. The pattern of changes observed in HSP72/73 was quite different in the two turtle species. In painted turtles anoxia induced a significant increase in myocardial HSP72/73 (from 2.8+/-0.1 mg/g normoxic to 3.9+/-0.2 mg/g anoxic, P<0.05). By 12 h of recovery, HSP72/73 had returned to control levels (2.7+/-0.1 mg/g) and remained there through 24 h (2.6+/-0.2 mg/g). In softshell turtles, HSP72/73 decreased significantly after 12 h of anoxia (from 2.4+/-0.4 mg/g normoxic to 1.3+/-0.2 mg/g anoxic, P<0.05). HSP72/73 levels were still slightly below control after 12 h of recovery (2.1+/-0.1 mg/g) and then rose to significantly above control after 24 h of recovery (4.1+/-0.7 mg/g, P<0.05). We also conclude that anoxia-tolerant and anoxia-sensitive turtles exhibit different patterns of myocardial HSP changes during anoxia and recovery. Whether these changes correlate with their relative degrees of anoxia tolerance remains to be determined.     

Role of unsaturated lipid and ergosterol in ethanol tolerance of model yeast biomembranes

We present a combined atomic force microscopy and fluorescence microscopy study of the behavior of a ternary supported lipid bilayer system containing a saturated lipid (DPPC), an unsaturated lipid (DOPC), and ergosterol in the presence of high ethanol (20 vol %). We find that the fluorescent probe Texas Red DHPE preferentially partitions into the ethanol-induced interdigitated phase, which allows the use of fluorescence imaging to investigate the phase behavior of the system. Atomic force microscopy and fluorescence images of samples with the same lipid mixture show good agreement in sample morphology and area fractions of the observed phases. Using area fractions obtained from fluorescence images over a broad range of compositions, we constructed a phase diagram of the DPPC/DOPC/ergosterol system at 20 vol % ethanol. The phase diagram clearly shows that increasing unsaturated lipid and/or ergosterol protects the membrane by preventing the formation of the interdigitated phase. This result supports the hypothesis that yeast cells increase ergosterol and unsaturated lipid content to prevent interdigitation and maintain an optimal membrane thickness as ethanol concentration increases during anaerobic fermentations. Changes in plasma membrane composition provide an important survival factor for yeast cells to deter ethanol toxicity.     

集胞藻PCC6803中S2P同源蛋白基因sll0862缺失突变株对热胁迫与氧化胁迫的响应

原核生物中S2P参与应答外界环境刺激,然而行光合作用的蓝细菌-集胞藻PCC6803的S2P同源蛋白功能未知。【目的】考察集胞藻PCC6803中S2P同源蛋白sll0862是否参与外界环境刺激的应答。【方法】监测在高温和氧化胁迫的条件下sll0862基因缺失突变株与野生株在生长速率或存活率上的差异,利用水样调制叶绿素荧光仪(water-PAM,脉冲-振幅-调制叶绿素荧光仪)测量在高温和氧化胁迫的条件下突变株与野生株叶绿素荧光参数的差异,来考察其光合作用差异。【结果】sll0862突变株与野生株在正常的培养环境中生长速率并无差异,但是将sll0862突变株与野生株在48℃加热处理半小时后,sll0862突变株的存活率明显低于野生株。当初始OD730值为0.1的藻液中添加终浓度为1 mmol/L双氧水的时候,sll0862突变株的生长速率比野生株明显低,而且氧化胁迫条件下突变株与野生株的调制叶绿素荧光有差异。【结论】集胞藻PCC6803中sll0862基因的缺失导致突变体对高温与氧化胁迫响应出现缺陷,提示有功能的sll0862参与响应热和氧化胁迫。研究结果为进一步阐述S2P同源蛋白sll0862在集胞藻PCC6803中的功能奠定基础。     

Membrane lipids and ethanol tolerance in the mouse. The influence of dietary fatty acid composition

Mice of the TO Swiss strain received diets containing different amounts of saturated or unsaturated fat throughout life. These diets produced characteristic changes in cardiac phospholipid fatty acid composition, but produced no significant differences in fatty acid composition of phospholipids from a crude membrane fraction of brain. When littermates of these animals were exposed to ethanol vapour in an inhalation chamber it was observed that mice which had received a diet high in saturated fat lost the righting reflex at an estimated concentration of ethanol in blood higher than that required for mice receiving a control diet, or a diet rich in polyunsaturated fat. Analysis of the brain membrane fraction from those animals which had received ethanol revealed that mice receiving the highly saturated fat diet now had a significantly greater proportion of saturated fatty acids in brain membrane phospholipids. These results are discussed in relation to the hypothesis that brain membrane lipid composition may influence the behavioural response to ethanol.     

Induction of heat shock proteins and thermotolerance by ethanol in Saccharomyces cerevisiae

The temperature sensitivity of $\text{Saccharomyces}\text{cerevisiae}$ and the conditions of moderate heat pretreatment required to induce thermotolerance are established. Ethanol is identified as an inducer of heat shock proteins and an inducer of thermotolerance.     

Stress proteins in oligodendrocytes: differential effects of heat shock and oxidative stress

Heat shock proteins (HSP) or stress proteins serve as biomarkers to identify the contribution of stress situations underlying the pathogenesis of degenerative diseases of the CNS. We have analyzed by immunoblot technique the constitutive and inducible occurrence of stress proteins in cultured rat brain oligodendrocytes subjected to heat shock or oxidative stress exerted by hydrogen peroxide, or a combination of both. The data demonstrate that oligodendrocytes constitutively express HSP32, HSP60 and the cognate form of the HSP70 family of proteins, HSC70. After heat shock, HSP25, alpha B-crystallin and HSP70 were up-regulated, while after oxidative stress the specific induction of HSP32 and alpha B-crystallin was observed. HSP32 represents heme oxygenase 1 (HO-1), a small stress protein with enzymatic activity involved in the oxidative degradation of heme which participates in iron metabolism. The presence of the iron chelators phenanthroline or deferoxamine (DFO), which previously has been shown to protect oligodendrocytes from oxidative stress-induced onset of apoptosis, caused a marked stimulation of HSP32 without affecting HSP70. This indicates that DFO possibly exerts its protective role by directly influencing the antioxidant capacity of HO-1. In summary, HSP in oligodendrocytes are differentially stimulated by heat stress and oxidative stress. Heme oxygenase-1 has been linked to inflammatory processes and oxidative stress, its specific up-regulation after oxidative stress in oligodendrocytes suggests that it is an ideal candidate to investigate the involvement of oxidative stress in demyelinating diseases.     

Stress proteins and cross-protection by heat shock and salt stress in Bacillus subtilis.

Bacillus subtilis induced a set of general stress proteins in response to a salt or heat stress. Cells subjected to a mild heat stress showed a protective response which enabled them to survive otherwise lethal temperatures (e.g. 52 degrees C). In a similar way bacteria were enabled to survive toxic concentrations of NaCl by pretreatment with lower salt concentrations. A mild heat shock induced a cross-protection against lethal salt stress. The pretreatment of cells with low salt, however, was less effective in the induction of thermotolerance than a preceding mild heat stress. Three stress proteins were identified on the basis of their N-terminal amino acid sequences as homologues of GroEL, DnaK and ClpP of Escherichia coli. The role of general and specific stress proteins in the induction of thermotolerance/salt tolerance and cross-protection is discussed.     

Synthesis and subcellular location of peroxisomal membrane proteins in a peroxisome-deficient mutant of the yeast Hansenula polymorpha.

We have studied the synthesis and subcellular location of peroxisomal membrane proteins (PMPs) in cells of a peroxisome-deficient (per) mutant of the methylotrophic yeast Hansenula polymorpha. Western blot analysis of methanol-induced cells of the per mutant, which had been growing in a continuous culture on a glucose/methanol mixture, indicated that various PMPs were normally synthesized. As in wild type (WT) cells, the levels of PMP synthesis appeared to be dependent on specific cultivation conditions, e.g. the carbon source used for growth. In contrast to WT controls, PMPs in methanol-induced per mutants were not subject to proteolytic degradation. Biochemical and immuno(cyto)chemical studies suggested that the PMPs in methanol-induced per cells were located in small proteinaceous aggregates, separated from peroxisomal matrix proteins that were also present in the cytosol. Vesicular membranous structures, resembling the morphology of intact peroxisomes, were never detected irrespective of the growth conditions employed.     

Stress tolerance in fungi -- to kill a spoilage yeast

The fungal spoilage of ingredients of food manufacture is an economic problem, often causes product loss and may constitute a health hazard. To effectively combat fungal food spoilage, a mechanistic understanding of tolerance for, and adaptation to, the preservation method used is crucial. Both are dependent on the genetic make-up and growth history of the organism. In the post-genomic era we are arriving at a situation in which, in the model organism Saccharomyces cerevisiae, physiological data, classical molecular biology and whole-genome responses can be combined to obtain explanatory and predictive models for growth. For food spoilage fungi we have not yet reached such a level of understanding, but we may use the knowledge gained for S. cerevisiae for the prevention of spoilage.     

Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast

The plasma membrane potential is mainly considered as the driving force for ion and nutrient translocation. Using the yeast Saccharomyces cerevisiae as a model organism, we have discovered a novel role of the membrane potential in the organization of the plasma membrane. Within the yeast plasma membrane, two non-overlapping sub-compartments can be visualized. The first one, represented by a network-like structure, is occupied by the proton ATPase, Pma1, and the second one, forming 300-nm patches, houses a number of proton symporters (Can1, Fur4, Tat2 and HUP1) and Sur7, a component of the recently described eisosomes. Evidence is presented that sterols, the main lipid constituent of the plasma membrane, also accumulate within the patchy compartment. It is documented that this compartmentation is highly dependent on the energization of the membrane. Plasma membrane depolarization causes reversible dispersion of the H(+)-symporters, not however of the Sur7 protein. Mitochondrial mutants, affected in plasma membrane energization, show a significantly lower degree of membrane protein segregation. In accordance with these observations, depolarized membranes also considerably change their physical properties (detergent sensitivity).     

A soybean 101-kD heat shock protein complements a yeast HSP104 deletion mutant in acquiring thermotolerance.

A cDNA clone encoding a 101-kD heat shock protein (HSP101) of soybean was isolated and sequenced. Genomic DNA gel blot analysis indicated that the corresponding gene is a member of a multigene family. The mRNA for HSP101 was not detected in 2-day-old etiolated soybean seedlings grown at 28 degrees C but was induced by elevated temperatures. DNA sequence comparison has shown that the corresponding gene belongs to the Clp (caseinolytic protease) (or Hsp100) gene family, which is evolutionarily conserved and found in both prokaryotes and eukaryotes. On the basis of the spacer length between the two conserved ATP binding regions, this gene has been identified as a member of the ClpB subfamily. Unlike other Clp genes previously isolated from higher plants, the expression of this soybean Hsp101 gene is heat inducible, and it does not have an N-terminal signal peptide for targeting to chloroplasts. Transformation of the soybean Hsp101 gene into a yeast HSP104 deletion mutant complemented restoration of acquired thermotolerance, a process in which cells survive an otherwise lethal heat stress after they are given a permissive heat treatment.