酿酒酵母孢子壁结构及其合成相关基因研究进展

细胞壁是酵母细胞区别于哺乳动物细胞的重要特征结构。酵母细胞壁的结构组成、合成、再生等与酵母自身繁殖及环境胁迫压力密切相关。目前,酵母孢子壁的形成机理、调控过程机制及孢子壁合成相关基因的功能尚未研究清楚。本文以酿酒酵母为例,简要描述酵母孢子壁的形成过程,重点阐述孢子壁甘露糖层、葡聚糖层、壳聚糖层和二酪氨酸层的结构组成及其合成相关基因的国内外研究进展,以期为抗真菌药物的新靶点研究提供参考。     

The fission yeast spore is coated by a proteinaceous surface layer comprising mainly Isp3

The spore is a dormant cell that is resistant to various environmental stresses. As compared with the vegetative cell wall, the spore wall has a more extensive structure that confers resistance on spores. In the fission yeast Schizosaccharomyces pombe, the polysaccharides glucan and chitosan are major components of the spore wall; however, the structure of the spore surface remains unknown. We identify the spore coat protein Isp3/Meu4. The isp3 disruptant is viable and executes meiotic nuclear divisions as efficiently as the wild type, but isp3∆ spores show decreased tolerance to heat, digestive enzymes, and ethanol. Electron microscopy shows that an electron-dense layer is formed at the outermost region of the wild-type spore wall. This layer is not observed in isp3∆ spores. Furthermore, Isp3 is abundantly detected in this layer by immunoelectron microscopy. Thus Isp3 constitutes the spore coat, thereby conferring resistance to various environmental stresses.     

Electron microscopy of spore germination and cell wall formation in Mucor rouxii

Summary The fine structure of ungerminated and aerobically germinated sporangiospores of Mucor rouxii was compared. The germination process may be divided into two stages: I, spherical growth; II, emergence of a germ tube. In both stages, germination is growth in its strictest sense with overall increases in cell organelles; e.g., the increase in mitochondria is commensurate with the overall increase in protoplasmic mass. Noticeable changes occurring during germination are the disappearance of electron-dense lipoid bodies, formation of a large central vacuole and, most strikingly, formation of a new cell wall. Unlike many other fungi, M. rouxii does not germinate by converting the spore wall into a vegetative wall. Instead, as in other Mucorales, a vegetative wall is formed de novo under the spore wall during germination stage I. This new wall grows out, rupturing the spore wall, to become the germ tube wall. Associated with the apical wall of the germ tube is an apical corpuscle previously described. The vegetative wall exhibits a nonlayered, uniformly microfibrillar appearance in marked distinction to the spore wall which is triple-layered, with two thin electron dense outer layers, and a thick transparent inner stratum. The lack of continuity between the spore and vegetative walls is correlated with marked differences in wall chemistry previously reported. A separate new wall is also formed under the spore wall during anaerobic germination leading to yeast cell formation. On the other hand, in the development of one vegetative cell from another, such as in the formation of hyphae from yeast cells, the cell wall is structurally continuous. This continuity is correlated with a similarity in chemical composition of the cell wall reported earlier.     

Multiple roles of Spo11 in meiotic chromosome behavior

Spo11, a type II topoisomerase, is likely to be required universally for initiation of meiotic recombination. However, a dichotomy exists between budding yeast and the animals Caenorhabditis elegans and Drosophila melanogaster with respect to additional roles of Spo11 in meiosis. In Saccharomyces cerevisiae, Spo11 is required for homolog pairing, as well as axial element (AE) and synaptonemal complex (SC) formation. All of these functions are Spo11 independent in C.elegans and D.melanogaster. We examined Spo11 function in a multicellular fungus, Coprinus cinereus. The C.cinereus spo11-1 mutant shows high levels of homolog pairing and occasionally forms full-length AEs, but no SC. In C.cinereus, Spo11 is also required for maintenance of meiotic chromosome condensation and proper spindle formation. Meiotic progression in spo11-1 is aberrant; late in meiosis basidia undergo programmed cell death (PCD). To our knowledge, this is the first example of meiotic PCD outside the animal kingdom. Ionizing radiation can partially rescue spo11-1 for both AE and SC formation and viable spore production, suggesting that the double-strand break function of Spo11 is conserved and is required for these functions.     

Synthesis of Drosophila melanogaster alcohol dehydrogenase in yeast

Expression systems for the heterologous expression of Drosophila melanogaster alcohol dehydrogenase (ADH) in Saccharomyces cerevisiae have been designed, analyzed and compared. Four different yeast/Escherichia coli shuttle vectors were constructed and used to transform four different yeast strains. Expression was detectable in ADH- yeast strains, from either a constitutive promoter, yeast ADH1 promoter (ADCp), or a regulated promoter, yeast GALp. The highest amount of D. melanogaster ADH was obtained from a multicopy plasmid with the D. melanogaster Adh gene expressed constitutively under the control of yeast ADCp promoter. The D. melanogaster enzyme was produced in cell extracts, as assessed by Coomassie blue staining and Western blotting after polyacrylamide-gel electrophoresis and it was fully active and able to complement the yeast ADH deficiency. Results show that D. melanogaster ADH subunits synthesized in yeast are able to assemble into functional dimeric forms. The synthesized D. melanogaster ADH represents up to 3.5% of the total extracted yeast protein.     

Ascospore formation in the yeast Saccharomyces cerevisiae.

Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.     

Regulation and intracellular localization of Saccharomyces cerevisiae strand exchange protein 1 (Sep1/Xrn1/Kem1), a multifunctional exonuclease.

The Saccharomyces cerevisiae strand exchange protein 1 (Sep1; also referred to as Xrn1, Kem1, Rar5, or Stp beta) catalyzes the formation of hybrid DNA from model substrates in vitro. The protein is also a 5'-to-3' exonuclease active on DNA and RNA. Multiple roles for the in vivo function of Sep1, ranging from DNA recombination and cytoskeleton to RNA turnover, have been proposed. We show that Sep1 is an abundant protein in vegetative S. cerevisiae cells, present at about 80,000 molecules per diploid cell. Protein levels were not changed during the cell cycle or in response to DNA-damaging agents but increased twofold during meiosis. Cell fractionation and indirect immunofluorescence studies indicated that > 90% of Sep1 was cytoplasmic in vegetative cells, and indirect immunofluorescence indicated a cytoplasmic localization in meiotic cells as well. The localization supports the proposal that Sep1 has a role in cytoplasmic RNA metabolism. Anti-Sep1 monoclonal antibodies detected cross-reacting antigens in the fission yeast Schizosccharomyces pombe, in Drosophila melanogaster embryos, in Xenopus laevis, and in a mouse pre-B-cell line.     

End3p-mediated endocytosis is required for spore wall formation in Saccharomyces cerevisiae

During sporulation in Saccharomyces cerevisiae, vesicles transported to the vicinity of spindle pole bodies are fused to each other to generate bilayered prospore membranes (PSMs). PSMs encapsulate the haploid nuclei that arise from the meiotic divisions and serve as platforms for spore wall deposition. Membrane trafficking plays an important role in supplying vesicles for these processes. The endocytosis-deficient mutant, end3Delta, sporulated poorly and the spores produced lost resistance to ether vapor, suggesting that END3-mediated endocytosis is important for sporulation. End3p-GFP localized to cell and spore peripheries in vegetative and sporulating cells and colocalized with actin structures. Correspondingly, the actin cytoskeleton appeared aberrant during sporulation in end3Delta. Analysis of meiosis in end3Delta mutants revealed that the meiotic divisions occurred with wild-type kinetics. Furthermore, PSMs were assembled normally. However, the levels of proteins required for spore wall synthesis and components of the spore wall layers at spores were reduced, indicating that end3Delta mutants are defective in spore wall synthesis. Thus, END3-mediated endocytosis is important for spore wall formation. Additionally, cytological analyses suggest that trafficking between the plasma membrane and PSMs is important earlier during sporulation.     

Effect of sterol metabolism in the yeast-Drosophila system on the frequency of radiation-induced aneuploidy in Drosophila melanogaster oocytes

The influence of sterol metabolism upon mutagenesis in Drosophila melanogaster was investigated using ecological-genetic yeast - drosophila system. Sterol deficiency in the organism of Drosophila was caused by using the strain of Saccharomyces cerevisiae 9-2P712 with a mutation in the nysr1 locus which blocks synthesis of ergosterol as a nutrition substrate for flies. It was concluded that maintenance of females on the mutant yeast strain causes an increase of radiation-induced X-chromosome loss in mature oocytes. Resistance of oocytes to X-ray irradiation is restored, reaching the control level, when 0,1% cholesterol solution in 10% ethanol is added to the yeast biomass. The possible membrane and hormonal mechanisms of elevation of induced aneuploidy and the role of sterol metabolism in ensuring resistance of insects to damaging factors are discussed.     

Sporulation in the budding yeast Saccharomyces cerevisiae

In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae.     

Isolation and characterization of Saccharomyces cerevisiae mutants resistant to Calcofluor white.

Calcofluor is a fluorochrome that exhibits antifungal activity and a high affinity for yeast cell wall chitin. We isolated Saccharomyces cerevisiae mutants resistant to Calcofluor. The resistance segregated in a Mendelian fashion and behaved as a recessive character in all the mutants analyzed. Five loci were defined by complementation analysis. The abnormally thick septa between mother and daughter cells caused by Calcofluor in wild-type cells were absent in the mutants. The Calcofluor-binding capacity, observed by fluorescence microscopy, in a S. cerevisiae wild-type cells during alpha-factor treatment was also absent in some mutants and reduced in others. Staining of cell walls with wheat germ agglutinin-fluorescein complex indicated that the chitin uniformly distributed over the whole cell wall in vegetative or in alpha-factor-treated cells was almost absent in three of the mutants and reduced in the two others. Cell wall analysis evidenced a five- to ninefold reduction in the amount of chitin in mutants compared with that in the wild-type strain. The total amounts of cell wall mannan and beta-glucan in wild-type and mutant strains were similar; however, the percentage of beta-glucan that remained insoluble after alkali extraction was considerably reduced in mutant cells. The susceptibilities of the mutants and the wild-type strains to a cell wall enzymic lytic complex were rather similar. The in vitro levels of chitin synthase 2 detected in all mutants were similar to that in the wild type. The significance of these results is discussed in connection with the mechanism of chitin synthesis and cell wall morphogenesis in S. cerevisiae.     

Free proline content and sensitivity to desiccation and heat during yeast sporulation and spore germination.

Ascospores of a strain of Saccharomyces cerevisiae Hansen were less sensitive to desiccation and heat than vegetative cells. Desiccation resistance was acquired earlier during sporulation and lost later during spore germination than heat resistance. As spores matured, resistance to both stresses increased. With the exception of the first few hours in sporulation medium, when proline appeared to be utilized, the intracellular free proline content increased during sporulation and decreased during spore germination. Not all the proline lost could be detected in the germination medium, indicating that some was metabolically utilized by the germinating spores. Since exogenous proline supplied to vegetative or sporulating cells before desiccation increased their survival, it is suggested that the high level of free proline in mature spores may protect against desiccation stress.     

Homologous subunits of 1,3-beta-glucan synthase are important for spore wall assembly in Saccharomyces cerevisiae

During sporulation in Saccharomyces cerevisiae, the four haploid nuclei are encapsulated within multilayered spore walls. Glucan, the major constituent of the spore wall, is synthesized by 1,3-beta-glucan synthase, which is composed of a putative catalytic subunit encoded by FKS1 and FKS2. Although another homolog, encoded by FKS3, was identified by homology searching, its function is unknown. In this report, we show that FKS2 and FKS3 are required for spore wall assembly. The ascospores of fks2 and fks3 mutants were enveloped by an abnormal spore wall with reduced resistance to diethyl ether, elevated temperatures, and ethanol. However, deletion of the FKS1 gene did not result in a defective spore wall. The construction of fusion genes that expressed Fks1p and Fks2p under the control of the FKS2 promoter revealed that asci transformed with FKS2p-driven Fks1p and Fks2p were resistant to elevated temperatures, which suggests that the expression of FKS2 plays an important role in spore wall assembly. The expression of FKS1p-driven Fks3p during vegetative growth did not affect 1,3-beta-glucan synthase activity in vitro but effectively suppressed the growth defect of the temperature-sensitive fks1 mutant by stabilizing Rho1p, which is a regulatory subunit of glucan synthase. Based on these results, we propose that FKS2 encodes the primary 1,3-beta-glucan synthase in sporulation and that FKS3 is required for normal spore wall formation because it affects the upstream regulation of 1,3-beta-glucan synthase.     

The stress response protein Hsp12p increases the flexibility of the yeast Saccharomyces cerevisiae cell wall

The yeast S. cerevisiae cell wall comprising a 10 nm thick layer of polysaccharides, predominantly beta(1,3)-glucan and proteins, is the interface between the cell and the neighbouring environment. As such it is not a static entity but rather one that is dynamically remodelled in response to changes in the environmental conditions. We have recently proposed from studies using yeast cells lacking the gene encoding Hsp12p (Deltahsp12 yeast) and from incorporation of Hsp12p into agarose, used as a model system for the beta-glucan layer of the cell wall, that the hydrophilic stress response cell wall protein Hsp12p acts as a cell wall plasticizer. In this report we have used force spectroscopy to confirm that Deltahsp12 yeast are indeed less flexible than the wild type strain. The spring constant of the cell wall of Deltahsp12 yeast, kcw was determined to be 72+/-3 mN m-1 as compared to 17+/-5 mN m-1 obtained for the wild type strain. A similar result was found on the basis of a quantitative analysis of the electrophoretic mobilities measured for the two yeast strains. Those indicated that the hydrodynamic permeability quantified through the softness parameter of the external layer of Deltahsp12 cells was smaller than the one of wild type cells. We proposed from surface infrared spectroscopy measurements that yeast compensate for the lack of Hsp12p by reducing the carbohydrate/proteins ratio of the cell wall or increasing the cell wall chitin content.     

Composition of the Cellular Envelopes of Anabaena cylindrica

Comparative chemical analyses were made of the walls of vegetative cells, heterocysts, and spores, and of the mucilage of Anabaena cylindrica. The wall of the vegetative cell is composed predominantly of amino compounds, with a mannose-rich carbohydrate component comprising only 18% of the dry weight. In contrast, 62% of the heterocyst wall and 41% of the spore wall is carbohydrate. The carbohydrate moieties of the heterocyst wall and spore wall are similar in that the ratio of glucose, mannose, galactose, and xylose is approximately 75:20:3:4 in both walls. It appears that, during the differentiation of a vegetative cell into either a spore or a heterocyst, a glucose-rich wall polysaccharide is produced that is different from the polysaccharide component of the wall of the vegetative cell and of the sheath. In the case of the heterocyst, the wall was estimated to account for approximately 52% of the dry weight of the whole cell.     

Centromere-proximal crossovers are associated with precocious separation of sister chromatids during meiosis in Saccharomyces cerevisiae

In most organisms, meiotic chromosome segregation is dependent on crossovers (COs), which enable pairs of homologous chromosomes to segregate to opposite poles at meiosis I. In mammals, the majority of meiotic chromosome segregation errors result from a lack of COs between homologs. Observations in Homo sapiens and Drosophila melanogaster have revealed a second class of exceptional events in which a CO occurred near the centromere of the missegregated chromosome. We show that in wild-type strains of Saccharomyces cerevisiae, most spore inviability is due to precocious separation of sister chromatids (PSSC) and that PSSC is often associated with centromere-proximal crossing over. COs, as opposed to nonreciprocal recombination events (NCOs), are preferentially associated with missegregation. Strains mutant for the RecQ homolog, SGS1, display reduced spore viability and increased crossing over. Much of the spore inviability in sgs1 results from PSSC, and these events are often associated with centromere-proximal COs, just as in wild type. When crossing over in sgs1 is reduced by the introduction of a nonnull allele of SPO11, spore viability is improved, suggesting that the increased PSSC is due to increased crossing over. We present a model for PSSC in which a centromere-proximal CO promotes local loss of sister-chromatid cohesion.