The Role of Amino-Terminal and Carboxy-Terminal Extensions in the Processing and Translocation of a Plant Proteinase (Actinidin) Expressed in the Yeast Saccharomyces cerevisiae

Summary A plant proteinase gene naturally occuring in the Kiwi fruit plant (Actinidia chinensis) has been expressed in a yeast Saccharomyces cerevisiae. Different gene constructions consisting of different portions of the whole actinidin-encoding gene have been created and expressed using an expression-secretion yeast vector. It was observed that the amino- and carboxy-terminal extensions of the actinidin-encoding gene were required for the correct expression of the gene in yeast. A gene construction lacking both amino- and C-terminal extensions did not result in a detectable protein product. Similarly, a gene construction consisting of the amino-terminal extension plus mature actinidin-encoding DNA did not result in a detectable expression. However, intracellular expression was observed when a gene construction consisting of mature actinidin-encoding DNA plus C-terminal extension portion was employed. The expressed polypeptide was found however not to be correctly processed as it had a bigger size than the native actinidin. The correctly processed polypeptide was expressed intracellularly when the full-length actinidin cDNA was expressed in a vacuolar protease-proficient yeast strain. However, when a vacuolar protease-deficient yeast strain was employed, it was found that the precursor protein was not correctly processed, suggesting that the actinidin precursor had entered the vacuole and undergone proteolytic processing. The full-length actinidin cDNA consisted of the amino-terminal extension DNA, mature actinidin-encoding DNA, and C-terminal extension DNA. The results thus suggested that both amino- and C-terminal extensions were required for correct expression and processing of actinidin in yeast. The intracellular expression also suggested that the actinidin-encoding sequences contain intracellular targeting sequences which override the secretion signal included in the expression-secretion vector.     

Alterations in the cell wall ofSaccharomyces cerevisiae induced by the alpha sex factor or a mutation in the cell cycle

We performed experiments in parallel to study the rate of synthesis of cell wall polysaccharides and the activity of glycosyl transferases inSaccharomyces cerevisiae after arrest of acdc 28 mutant in G1 phase by either addition of alpha-factor or transfer to the non-permissive temperature. Both effectors brought about similar time-dependent increases in the rate of synthesis and deposition of the cell wall polysaccharides chitin, glucan and mannan. These changes in cell wall composition were accompanied by an increase in the specific activities of glucan and chitin synthetases. This increase was inhibited by cycloheximide suggesting that it representedde novo enzyme biosynthesis and not enzyme activation. Our data are consistent with the notion that both alpha-factor and thecdc 28 mutation affect the same stage-specific function that controls the temporal expression of glycosyl transferases.Abbreviations GlcNAc N-acetyl glucosamine - UDPGIcNAc uridine-diphosphate-N-acetyl glucosamine - UDPGlc uridine-diphosphate glucose - TCA trichloroacetic acid - EDTA ethylene diamino tetraacetate - TAME tosyl-L-arginyl methyl ester - GTP guanosine triphosphate - WGA wheat germ agglutinin     

An altered ribosomal protein in an edeine-resistant mutant of Saccharomyces cerevisiae

Summary The r-proteins of an edeine-resistant mutant of Saccharomyces cerevisiae were compared to those of the wild-type strain by using two different two-dimensional electrophoretic techniques: (1) the Kaltschmidt-Wittmann method and, (2) the Kaltschmidt-Wittmann system, in the first dimension and the Na Dodecyl-SO₄ system in the second.With the first technique, the results indicate that the patterns of basic ribosomal proteins are similar in the two strains. However, the pattern of acidic ribosomal proteins of the mutant revealed an additional protein band with respect to the normal one. Using the other technique, the patterns of basic and acidic ribosomal proteins of the mutant demonstrated a similarity to the corresponding pattern of the wild-type strain.The data disclose that an acidic ribosomal protein of the mutant may have two forms with different electrophoretic mobilities and similar molecular weights.     

A PMR2 tandem repeat with a modified C-terminus is located downstream from the KRS1 gene encoding lysyl-tRNA synthetase in Saccharomyces cerevisiae

Summary TheKRS1 gene encodes the cytoplasmic form ofSaccharomyces cerevisiae lysyl-tRNA synthetase. TheKRS1 locus has been characterized. The lysyl-tRNA synthetase gene is unique in the yeast genome. The gene is located on the right arm of chromosome IV and disruption of the open reading frame leads to lethality. These results contrast with the situation encountered inEscherichia coli where lysyl-tRNA synthetase is coded by two distinct genes,lysS andlysU, and further address the possible biological significance of this gene duplication. The nucleotide sequence of the 3′-flanking region has been established. It encodes a long open reading frame whose nucleotide and amino acid structures are almost identical toPMR2, a cluster of tandemly repeated genes coding for P-type ion pumps. The sequence alterations relative toPMR2 are mainly located at the C-terminus of the protein.     

Auxin controls Golgi-localized glucan synthetase activity in the maize mesocotyl

Decapitation or red light irradiation (R) inhibited growth and Golgi-localized glucan synthetase (GS I) activity in the mesocotyl of intact maize (Zea mays L.) seedlings. Applied auxin (indole-3-acetic acid) prevented the effects of R and of decapitation on both growth and GS I. Auxin applied several hours after irradiation prevented any further decline in GS I but did not restore it. Mesocotyl segments incubated in solution elongated in response to auxin but lost GS I with time regardless of the presence of exogenous auxin. An attached seed was necessary for maintenance of GS I in the dark-grown mesocotyl.Abbreviations GS glucan synthetase - IAA indole-3-acetic acid - R red light     

An examination of factors affecting the instability of Saccharomyces cerevisiae glucan synthetase in cell free extracts

Yeast (1–3) glucan synthetase is stimulated and stabilized by EDTA. Sucrose protects the enzyme from selfinactivaton. Preincubation of cell free extracts at low sucrose concentrations indicates a slow transition of the enzyme towards dissociation. Transition kinetics at 30° C and 0° C in the presence and in the absence of sucrose are interpreted assuming that a subunit is thermolabile in the free state and that sucrose increases its stability. Magnesium is deletereous for glucan synthetase in cell-free extracts. Chaotropic agents inactivate glucan synthetase according to their capacity to solubilize and depolymerize biological compounds. Fluoride plays a special role in the activation of glucan synthetase. Its action appears to be dependent on the presence of GTP (or other nucleotides). The role of all these agents on the activity and stability of the enzyme is interpreted in a unified scheme.Abbreviations EDTA ethylene diamine tetraacetate - Tris tris-(hydroxymethyl) aminomethane - MMF mixed membrane fraction     

Flocculation of industrial and laboratory strains ofSaccharomyces cerevisiae

Summary A comparative study has been made of different laboratory and industrial wild-type strains ofSaccharomyces cerevisiae in relation to their flocculation behavior. All strains were inhibited by mannose and only one by maltose. In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains. This was to such an extent that it did not allow their inclusion in the Flol or NewFlo phenotypes. Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic toFLO1 found in genetic strains.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Cloning, sequencing and characterization of the Saccharomyces cerevisiae URA7 gene encoding CTP synthetase

Summary The URA7 gene of Saccharomyces cerevisiae encodes CTP synthetase (EC 6.3.4.2) which catalyses the conversion of uridine 5-triphosphate to cytidine 5-triphosphate, the last step of the pyrimidine biosynthetic pathway. We have cloned and sequenced the URA 7 gene. The coding region is 1710 by long and the deduced protein sequence shows a strong degree of homology with bacterial and human CTP synthetases. Gene disruption shows that URA7 is not an essential gene: the level of the intracellular CTP pool is roughly the same in the deleted and the wild-type strains, suggesting that an alternative pathway for CTP synthesis exists in yeast. This could involve either a divergent duplicated gene or a different route beginning with the amination of uridine mono- or diphosphate.     

酿酒酵母表达和纯化功能性的铁载体合成蛋白PchE

【目的】利用酿酒酵母表达系统,通过乙醇脱氢酶启动子异源表达细菌源的铁载体合成蛋白PchE,并与来源于枯草芽孢杆菌的泛酰化酶Sfp同宿主共表达,探索真核表达体系表达具有生化活性的细菌源蛋白。【方法】从大肠杆菌BAP1染色体上扩增sfp基因,将pchE基因及串联的pchE与sfp基因分别构建到酵母-大肠杆菌穿梭质粒pXW55中,各自转化酿酒酵母BJ5464-npg A表达,经过亲和层析和离子交换层析纯化蛋白,利用HPLC检测细菌源与酵母源表达的PchE在体外重构生化反应中的催化活性。【结果】利用酿酒酵母表达系统可以获得高纯度的原核蛋白PchE。真菌源的泛酰化基因NpgA和细菌源的Sfp,均可泛酰化修饰PchE,合成中间产物HPT-Cys。【结论】在酿酒酵母Saccharomyces cerevisiae BJ5464-npgA表达系统中,首次证明真菌源的泛酰化基因NpgA和细菌源的Sfp,均可泛酰化修饰细菌源的非核糖体肽合酶。比较酵母和细菌宿主的目标蛋白表达,证明酵母表达的巨大蛋白PchE的纯度更高,非特异性条带减少,推测酵母宿主可能更适合表达纯化功能性的巨型蛋白质。     

走出青藏高原：拉格啤酒酵母的起源与演化

采用低温底层发酵的拉格(lager)啤酒15世纪开始在德国巴伐利亚地区出现,19世纪初流行至全世界,目前已成为全球产量最高的酒精饮料。目前已阐明,拉格啤酒发酵酵母为巴斯德酿酒酵母(Saccharomyces pastorianus),该种是一个杂交种,由艾尔(ale)啤酒酵母(Saccharomyces cerevisiae)与野生真贝氏酿酒酵母(Saccharomyces eubayanus)杂交而成,后者赋予了拉格啤酒酵母的耐低温能力。近年的群体遗传学和群体基因组学研究表明,拉格啤酒酵母的野生亲本S.eubayanus起源于青藏高原,可能通过丝绸之路传播到了欧洲。比较基因组学研究表明,拉格啤酒酵母包含2个株系,即Ⅰ系/Saaz系和Ⅱ系/Frohberg系,早期分别流行于中欧和西欧地区。前者为近似异源3倍体,后者为近似异源4倍体。2个株系在耐低温、麦芽三糖利用和风味物质产生能力等方面具有明显差异。在中国普通微生物菌种保藏管理中心(China General Microbiological Culture Collection Center,CGMCC)保藏的S.pastorianus...     

Allele-specific suppression of aSaccharomyces cerevisiae prp20 mutation by overexpression of a nuclear serine/threonine protein kinase

The yeast PRP20 protein is homologous to the RCC1 protein of higher eukaryotes and is required for mRNA export and maintenance of nuclear structure. RCC1/PRP20 act as guanine nucleotide exchange factors for the nuclear Ras-like Ran/GSP1 proteins. In a search forprp20-10 allele-specific high-copy-number suppressors, theKSP1 locus, encoding a serine/threonine protein kinase was isolated. Ksp1p is a nuclear protein that is not essential for vegetative growth of yeast. Inactivation of the kinase activity by a mutation affecting the catalytic center of the Ksp1p eliminated the suppressing activity. Based on the isolation of a protein kinase as a high-copy-number suppressor, the phosphorylation of Prp20p was examined. In vivo labeling experiments showed that Prp20p is a phosphoprotein; however, deletion of the KSP1 kinase did not affect Prp20p phosphorylation.     

Comparison of barley malt α-amylase isozymes 1 and 2: construction of cDNA hybrids by in vivo recombination and their expression in yeast

Germinating barley produces two α-amylase isozymes, AMY1 and AMY2, having 80% amino acid (aa) sequence identity and differing with respect to a number of functional properties. Recombinant AMY1 (re-AMYI) and AMY2 (re-AMY2) are produced in yeast, but whereas all re-AMYI is secreted, re-AMY2 accumulates within the cell and only traces are secreted. Expression of AMY1::AMY2 hybrid cDNAs may provide a means of understanding the difference in secretion efficiency between the two isozymes. Here, the efficient homologous recombination system of the yeast, Saccharomyces cerevisiae, was used to generate hybrids of barley AMY with the N-terminal portion derived from AMY1, including the signal peptide (SP), and the C-terminal portion from AMY2. Hybrid cDNAs were thus generated that encode either the SP alone, or the SP followed by the N-terminal 21, 26, 53, 67 or 90 aa from AMY1 and the complementary C-terminal sequences from AMY2. Larger amounts of re-AMY are secreted by hybrids containing, in addition to the SP, 53 or more aa of AMY1. In contrast, only traces of re-AMY are secreted for hybrids having 26 or fewer aa of AMY1. In this case, re-AMY hybrid accumulates intracellularly. Transformants secreting hybrid enzymes also accumulated some re-AMY within the cell. The AMY1 SP, therefore, does not ensure re-AMY2 secretion and a certain portion of the N-terminal sequence of AMY1 is required for secretion of a re-AMYI::AMY2 hybrid.     

Protein overexport in a Saccharomyces cerevisiae mutant depends on mitochondrial genome integrity and function

The wild-type yeast Saccharomyces cerevisiae (S. cerevisiae) is able to export less than 1 percent of the protein to be secreted. The reasons for retention of most of the secretory proteins on the cell surface of S. cerevisiae are unknown. Recently, temperature-sensitive (ts) mutants of S. cerevisiae showing an oversecretion phenotype were isolated. In order to study the influence of the mitochondrial genome status on protein export in yeast cells, we have isolated several types of respiratory impaired mitochondrial mutants of either the parental S. cerevisiae strain or their derivative ts protein-overexporting mutants. In this paper we demonstrate by quantitative analyses of exported proteins and by SDS-PAGE analysis that protein overexport in ts mutants requires mitochondrial genome integrity and function.     

Two Cases of Vaginitis Caused by Itraconazole-Resistant Saccharomyces cerevisiae and a Review of Recently Published Studies

Genitourinary infections caused by non-Candida yeasts are uncommon, and especially due to Saccharomyces cerevisiae. We describe the cases of two adult females with vulvovaginal infections caused by itraconazole-resistant S. cerevisiae who made a full recovery after oral fluconazole therapy. We also provide a concise review of recently published studies on this topic.     

Electron microscopy of germinating ascospores ofSaccharomyces cerevisiae

The wall of mature ascospores ofSaccharomyces cerevisiae showed in sections under the electron microscope a dark outer layer and a lighter inner layer. The latter was composed of a greyish inner part and a light outer part. During germination, the spore grew out at one side and the dark outer layer was broken. Of the light inner layer, the inner greyish part became the wall of the vegetative cell, but the extented part of the cell had a new wall.     

Prevalence and susceptibility of Saccharomyces cerevisiae causing vaginitis in Greek women

Saccharomyces cerevisiae is an ascomycetous yeast, that is traditionally used in wine bread and beer production. Vaginitis caused by S. cerevisiae is rare.The aim of this study was to evaluate the frequency of S. cerevisiae isolation from the vagina in two groups of women and determined the in vitro susceptibility of this fungus.

Subjects and methods

Vaginal samples were collected from a total of262 (asymptomaticandsymptomatic) women with vaginitis attending the centre of family planning of General hospital ofPiraeus. All blastomycetes that isolated from the vaginal samples were examined for microscopic morphological tests and identified by conventional methods: By API 20 C AUX and ID 32 C (Biomerieux). Antifungal susceptility testing for amphotericin B,fluconazole itraconazole,voriconazole, posaconazole and caspofungin was performed by E -test (Ab BIODIKS SWEDEN) against S. cerevisiae.

Results

A total of 16 isolates of S. cerevisiae derived from vaginal sample of the referred women, average 6.10%. Susceptibility of 16 isolates of S. cerevisiae to a variety of antimycotic agents were obtained. So all isolates of S. cerevisiae were resistant to fluconazole, posaconazole and intraconazole, but they were sensitive to voriconazole caspofungin and Amphotericin B which were found sensitive (except 1/16 strains). None of the 16 patients had a history of occupational domestic use of baker’s yeast.

Conclusions

Vaginitis caused by S. cerevisiae occur, is rising and cannot be ignored. Treatment of Saccharomyces vaginitis constitutes a major challenge and may require selected and often prolonged therapy.     

The influence of pH on sulphite formation by yeasts

The influence of the initial pH of the substrate on the sulphite formation of three low-sulphite-and five high-sulphite-forming yeasts is described. Four distinctly different groups become apparent. The need for better evaluation of pure culture wine yeasts is stressed.     

A genome-wide screen in Saccharomyces cerevisiae reveals pathways affected by arsenic toxicity

We have used Saccharomyces cerevisiae to identify toxicologically important proteins and pathways involved in arsenic-induced toxicity and carcinogenicity in humans. We performed a systemic screen of the complete set of 4733 haploid S. cerevisiae single-gene-deletion mutants to identify those that have decreased or increased growth, relative to wild type, after exposure to sodium arsenite (NaAsO₂). IC₅₀ values for all mutants were determined to further validate our results. Ultimately we identified 248 mutants sensitive to arsenite and 5 mutants resistant to arsenite exposure. We analyzed the proteins corresponding to arsenite-sensitive mutants and determined that they belonged to functional categories that include protein binding, phosphate metabolism, vacuolar/lysosomal transport, protein targeting, sorting, and translocation, cell growth/morphogenesis, cell polarity and filament formation. Furthermore, these data were mapped onto a protein interactome to identify arsenite-toxicity-modulating networks. These networks are associated with the cytoskeleton, ubiquitination, histone acetylation and the MAPK signaling pathway. Our studies have potential implications for understanding toxicity and carcinogenesis in arsenic-induced human conditions, such as cancer and aging.