过氧化氢诱导酿酒酵母细胞膜透性和组成的变化

以下简述了过氧化氢(H2O2)作为一种信号分子诱导并调节酿酒酵母(Saccharomyces cerevisiae)细胞膜的变化。H2O2是一种强氧化剂,可以跨膜扩散进入细胞中,形成跨膜梯度;当外源H2O2达到亚致死剂量时,酿酒酵母的细胞膜透性和流动性降低,产生跨膜梯度,从而限制H2O2向细胞内的扩散速率,保护细胞免受氧化胁迫的伤害。研究表明,由H2O2引起的膜透性和流动性的变化与膜的组成有关:当酵母细胞对H2O2产生适应时,与膜组成和微区域变化有关的几个基因的表达发生了改变。膜组成的变化和微区域的调整还可能与H2O2依赖的信号途径有关,即以H2O2为信号分子,调节膜的变化并赋予细胞对氧化压力更高的适应性,但这种信号分子的具体传递途径及机制还需要进一步研究。     

Initiation of meiosis and sporulation in Saccharomyces cerevisiae does not require a decrease in cyclic AMP.

Meiosis and sporulation of Saccharomyces cerevisiae are initiated in a guanine auxotroph by guanine deprivation (E. Bautz Freese, Z. Olempska-Beer, A. Hartig, and E. Freese, Dev. Biol. 102:438-451, 1984). We used this condition to examine a hypothesis (K. Matsumoto, I. Uno, and T. Ishikawa, Cell 32:417-423, 1983) that initiation of meiosis requires a low level of cAMP. We found that, after guanine deprivation, the intracellular concentration of cAMP transiently decreased not more than 20% and not at all if the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was added to the medium. Under these conditions, at least 76% of the cells sporulated in the absence of IBMX, and almost 100% sporulated in its presence. The sporulating cells continually excreted cAMP and utilized the gluconeogenic carbon source. The cells failed to sporulate efficiently and to form four-spored asci if simultaneously deprived of guanine and carbon. After guanine deprivation in glucose medium, sporulation remained suppressed and intracellular cAMP was unchanged. We conclude that, under conditions of guanine starvation, cAMP deficiency is not required for initiation of meiosis and sporulation, cAMP is produced in excess and excreted to the medium, the cells sporulate better if the cAMP concentration is increased by addition of IBMX, the cells require a gluconeogenic carbon source for complete and efficient sporulation, and suppression of sporulation by glucose is not mediated by cAMP.     

TRK2 is not a low-affinity potassium transporter in Saccharomyces cerevisiae.

TRK1 and TRK2 encode proteins involved in K+ uptake in Saccharomyces cerevisiae. A kinetic study of Rb+ influx in trk1 TRK2, trk1 TRK2D, and trk1 trk2 mutants reveals that TRK2 shows moderate affinity for Rb+. K(+)-starved trk1 delta TRK2 cells show a low-affinity component accounting for almost the total Vmax of the influx and a moderate-affinity component exhibiting a very low Vmax. Overexpression of TRK2 in trk1 delta TRK2D cells increases the Vmax of the moderate-affinity component, and this component disappears in trk1 delta trk2 delta cells. In contrast, the low-affinity component of Rb+ influx in trk1 delta TRK2 cells is not affected by mutations in TRK2. Consistent with the different levels of activity of the moderate-affinity Rb+ influx, trk1 delta TRK2 cells grow slowly in micromolar K+, trk1 delta TRK2D cells grow rapidly, and trk1 delta trk2 delta cells fail to grow. The existence of a unique K+ uptake system composed of several proteins is also discussed.     

Either of the major H2A genes but not an evolutionarily conserved H2A.F/Z variant of Tetrahymena thermophila can function as the sole H2A gene in the yeast Saccharomyces cerevisiae.

H2A.F/Z histones are conserved variants that diverged from major H2A proteins early in evolution, suggesting they perform an important function distinct from major H2A proteins. Antisera specific for hv1, the H2A.F/Z variant of the ciliated protozoan Tetrahymena thermophila, cross-react with proteins from Saccharomyces cerevisiae. However, no H2A.F/Z variant has been reported in this budding yeast species. We sought to distinguish among three explanations for these observations: (i) that S. cerevisiae has an undiscovered H2A.F/Z variant, (ii) that the major S. cerevisiae H2A proteins are functionally equivalent to H2A.F/Z variants, or (iii) that the conserved epitope is found on a non-H2A molecule. Repeated attempts to clone an S. cerevisiae hv1 homolog only resulted in the cloning of the known H2A genes yHTA1 and yHTA2. To test for functional relatedness, we attempted to rescue strains lacking the yeast H2A genes with either the Tetrahymena major H2A genes (tHTA1 or tHTA2) or the gene (tHTA3) encoding hv1. Although they differ considerably in sequence from the yeast H2A genes, the major Tetrahymena H2A genes can provide the essential functions of H2A in yeast cells, the first such case of trans-species complementation of histone function. The Tetrahymena H2A genes confer a cold-sensitive phenotype. Although expressed at high levels and transported to the nucleus, hv1 cannot replace yeast H2A proteins. Proteins from S. cerevisiae strains lacking yeast H2A genes fail to cross-react with anti-hv1 antibodies. These studies make it likely that S. cerevisiae differs from most other eukaryotes in that it does not have an H2A.F/Z homolog. A hypothesis is presented relating the absence of H2A.F/Z in S. cerevisiae to its function in other organisms.     

Mutations in XRS2 and RAD50 delay but do not prevent mating-type switching in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a large number of genes in the RAD52 epistasis group has been implicated in the repair of chromosomal double-strand breaks and in both mitotic and meiotic homologous recombination. While most of these genes are essential for yeast mating-type (MAT) gene switching, neither RAD50 nor XRS2 is required to complete this specialized mitotic gene conversion process. Using a galactose-inducible HO endonuclease gene to initiate MAT switching, we have examined the effect of null mutations of RAD50 and of XRS2 on intermediate steps of this recombination event. Both rad50 and xrs2 mutants exhibit a marked delay in the completion of switching. Both mutations reduce the extent of 5'-to-3' degradation from the end of the HO-created double-strand break. The steps of initial strand invasion and new DNA synthesis are delayed by approximately 30 min in mutant cells. However, later events are still further delayed, suggesting that XRS2 and RAD50 affect more than one step in the process. In the rad50 xrs2 double mutant, the completion of MAT switching is delayed more than in either single mutant, without reducing the overall efficiency of the process. The XRS2 gene encodes an 854-amino-acid protein with no obvious similarity to the Rad50 protein or to any other protein in the database. Overexpression of RAD50 does not complement the defects in xrs2 or vice versa.     

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H2O2) through the plasma membrane decreases during adaptation to H2O2 by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H2O2 the anisotropy of the plasma membrane increases. Adaptation to H2O2 was studied at several times (15min up to 90min) by applying the steady-state H2O2 delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H2O2 was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H2O2 adaptation. H2O2 diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H2O2 permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H2O2 is mediated by modulation of the biophysical properties of the plasma membrane.     

Modulation of plasma membrane lipid profile and microdomains by H2O2 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the rate of hydrogen peroxide (H₂O₂) diffusion through the plasma membrane decreases during adaptation to H₂O₂ by a still unknown mechanism. Here, adaptation to H₂O₂ was observed to modulate rapidly the expression of genes coding for enzymes involved in ergosterol and lipid metabolism. Adaptation to H₂O₂ also alters plasma membrane lipid composition. The main changes were the following: (a) there was a decrease in oleic acid (30%) and in the ratio between unsaturated and saturated long-chain fatty acids; (b) the phosphatidylcholine:phosphatidylethanolamine ratio increased threefold; (c) sterol levels were unaltered but there was an increased heterogeneity of sterol-rich microdomains and increased ordered domains; (d) the levels of the sterol precursor squalene increased twofold, in agreement with ERG1 gene down-regulation; and (e) C26:0 became the major very long chain fatty acid owing to an 80% decrease in 2-hydroxy-C26:0 levels and a 50% decrease in C20:0 levels, probably related to the down-regulation of fatty acid elongation (FAS1, FEN1, SUR4) and ceramide synthase (LIP1, LAC1) genes. Therefore, H₂O₂ leads to a reorganization of the plasma membrane microdomains, which may explain the lower permeability to H₂O₂, and emerges as an important regulator of lipid metabolism and plasma membrane lipid composition.     

Exploring the evolution of multicellularity in Saccharomyces cerevisiae under bacteria environment: An experimental phylogenetics approach

There have been over 25 independent unicellular to multicellular evolutionary transitions, which have been transformational in the complexity of life. All of these transitions likely occurred in communities numerically dominated by unicellular organisms, mostly bacteria. Hence, it is reasonable to expect that bacteria were involved in generating the ecological conditions that promoted the stability and proliferation of the first multicellular forms as protective units. In this study, we addressed this problem by analyzing the occurrence of multicellularity in an experimental phylogeny of yeasts (Sacharomyces cerevisiae) a model organism that is unicellular but can generate multicellular clusters under some conditions. We exposed a single ancestral population to periodic divergences, coevolving with a cocktail of environmental bacteria that were inoculated to the environment of the ancestor, and compared to a control (no bacteria). We quantified culturable microorganisms to the level of genera, finding up to 20 taxa (all bacteria) that competed with the yeasts during diversification. After 600 generations of coevolution, the yeasts produced two types of multicellular clusters: clonal and aggregative. Whereas clonal clusters were present in both treatments, aggregative clusters were only present under the bacteria treatment and showed significant phylogenetic signal. However, clonal clusters showed different properties if bacteria were present as follows: They were more abundant and significantly smaller than in the control. These results indicate that bacteria are important modulators of the occurrence of multicellularity, providing support to the idea that they generated the ecological conditions‐promoting multicellularity.     

The Saccharomyces cerevisiae chromosome III left telomere has a type X, but not a type Y'', ARS region.

A yeast Saccharomyces cerevisiae telomeric region was isolated by chromosome walking from HML alpha, the most distal known gene on the chromosome III left (IIIL) end. The terminal heterodisperse 3.3-kilobase (kb) SalI fragment on chromosome IIIL, 8.6 kb distal to HML alpha, was cloned in a circular vector to generate a telomeric probe. Southern hybridization and DNA sequencing analyses indicated that 0.6 kb (+/- 200 base pairs) of 5'-C1-3A-3' simple tandem repeat sequence, adjacent to a 1.2-kb type X ARS region, constitutes the telomere on the chromosome IIIL end, and no type Y' ARS region homologies exist between HML alpha and the IIIL terminus.     

Optimal cofactor swapping can increase the theoretical yield for chemical production in Escherichia coli and Saccharomyces cerevisiae

Maintaining cofactor balance is a critical function in microorganisms, but often the native cofactor balance does not match the needs of an engineered metabolic flux state. Here, an optimization procedure is utilized to identify optimal cofactor-specificity “swaps” for oxidoreductase enzymes utilizing NAD(H) or NADP(H) in the genome-scale metabolic models of Escherichia coli and Saccharomyces cerevisiae. The theoretical yields of all native carbon-containing molecules are considered, as well as theoretical yields of twelve heterologous production pathways in E. coli. Swapping the cofactor specificity of central metabolic enzymes (especially GAPD and ALCD2x) is shown to increase NADPH production and increase theoretical yields for native products in E. coli and yeast—including l-aspartate, l-lysine, l-isoleucine, l-proline, l-serine, and putrescine—and non-native products in E. coli—including 1,3-propanediol, 3-hydroxybutyrate, 3-hydroxypropanoate, 3-hydroxyvalerate, and styrene.     

Deletion of the ATP20 gene in Ustilago maydis produces an unstable dimer of F1FO-ATP synthase associated with a decrease in mitochondrial ATP synthesis and a high H2O2 production

The F₁F_O-ATP synthase uses the energy stored in the electrochemical proton gradient to synthesize ATP. This complex is found in the inner mitochondrial membrane as a monomer and dimer. The dimer shows higher ATPase activity than the monomer and is essential for cristae folding. The monomer-monomer interface is constituted by subunits a, i/j, e, g, and k. The role of the subunit g in a strict respiratory organism is unknown. A gene knockout was generated in Ustilago maydis to study the role of subunit g on mitochondrial metabolism and cristae architecture. Deletion of the ATP20 gene, encoding the g subunit, did not affect cell growth or glucose consumption, but biomass production was lower in the mutant strain (gΔ strain). Ultrastructure observations showed that mitochondrial size and cristae shape were similar in wild-type and gΔ strains. The mitochondrial membrane potential in both strains had a similar magnitude, but oxygen consumption was higher in the WT strain. ATP synthesis was 20 % lower in the gΔ strain. Additionally, the mutant strain expressed the alternative oxidase in the early stages of growth (exponential phase), probably as a response to ROS stress. Dimer from mutant strain was unstable to digitonin solubilization, avoiding its isolation and kinetic characterization. The isolated monomeric state activated by n-dodecyl-β-D-maltopyranoside showed similar kinetic constants to the monomer from the WT strain. A decrease in mitochondrial ATP synthesis and the presence of the AOX during the exponential growth phase suggests that deletion of the g gene induces ROS stress.     

Structural and functional analysis of a truncated form of Saccharomyces cerevisiae ATP sulfurylase: C-terminal domain essential for oligomer formation but not for activity

ATP sulfurylase catalyzes the first step in the activation of sulfate by transferring the adenylyl-moiety (AMP approximately ) of ATP to sulfate to form adenosine 5'-phosphosulfate (APS) and pyrophosphate (PP(i)). Subsequently, APS kinase mediates transfer of the gamma-phosphoryl group of ATP to APS to form 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and ADP. The recently determined crystal structure of yeast ATP sulfurylase suggests that its C-terminal domain is structurally quite independent from the other domains, and not essential for catalytic activity. It seems, however, to dictate the oligomerization state of the protein. Here we show that truncation of this domain results in a monomeric enzyme with slightly enhanced catalytic efficiency. Structural alignment of the C-terminal domain indicated that it is extremely similar in its fold to APS kinase although not catalytically competent. While carrying out these structural and functional studies a surface groove was noted. Careful inspection and modeling revealed that the groove is sufficiently deep and wide, as well as properly positioned, to act as a substrate channel between the ATP sulfurylase and APS kinase-like domains of the enzyme.     

APT1, but Not APT2, Codes for a Functional Adenine Phosphoribosyltransferase in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has two separate genes (APT1 and APT2) that encode two potentially different forms of adenine phosphoribosyltransferase (APRT). However, genetic analysis indicated that only APT1 could code for a complementing activity. Cloning and expression of both the APT1 and APT2 genes in Escherichia coli showed that although discrete proteins (APRT1 and APRT2) were made by these genes, only APRT1 had detectable APRT activity. Northern and Western blot analyses demonstrated that only APT1 was transcribed and translated under normal physiological conditions in yeast. Phylogenetic analysis revealed that APRT1 and APRT2 are evolutionary closely related and that they arise from a gene duplication event. We conclude that APT1 is the functional gene in S. cerevisiae and that APT2 is a pseudogene.