Myoinositol gets incorporated into numerous membrane glycoproteins of Saccharomyces cerevisiae; incorporation is dependent on phosphomannomutase (sec53).

We recently described a 125 kd membrane glycoprotein in Saccharomyces cerevisiae which is anchored in the lipid bilayer by an inositol-containing phospholipid. We now find that when S. cerevisiae cells are metabolically labeled with [3H]myoinositol, many glycoproteins become labeled more strongly than the 125 kd protein. Myoinositol is attached to these glycoproteins as part of a phospholipid moiety which resembles glycophospholipid anchors of other organisms. Labeling of proteins with [3H]myoinositol for short times and in secretion mutants blocked at various stages of the secretory pathway shows that these phospholipid moieties can be added to proteins in the endoplasmic reticulum and that these proteins are transported to the Golgi by the regular secretory pathway. sec53, a mutant which cannot produce GDP-mannose at 37 degrees C, does not incorporate myoinositol or palmitic acid into membrane glycoproteins at this temperature, suggesting that GDP-mannose is required for the biosynthesis of these phospholipid moieties. All other secretion and glycosylation mutants tested add phospholipid moieties to proteins normally.     

Inhibition of biosynthesis of Saccharomyces cerevisiae sugar transport system by tunicamycin.

Tunicamycin apparently inhibited the biosynthesis of glucose, galactose, and maltose transport systems in Saccharomyces cerevisiae. Under the conditions used, the antibiotic also blocked the biosynthesis of invertase, a well-known yeast glycoprotein, as well as the glycosylation of a marker mannoprotein of the yeast cell wall. However, the antibiotic did not affect certain proteins which did not contain carbohydrate. It seems, therefore, that these sugar carriers are glycoproteins.     

Localization of acid phosphatase in protoplasts from Saccharomyces cerevisiae.

The localization of acid phosphatase (EC 3.1.3.2) in secreting protoplasts prepared from Saccharomyces cerevisiae is reported for the first time. Using a Gomori technique we were able to show acid phosphatase at those organelles in the protoplasts which are generally involved in the processes of biosynthesis and secretion of glycoproteins in eukaryotic cells.     

Characterization of lipid-linked octa- through undecasaccharides implicated in the biosynthesis of Saccharomyces cerevisiae mannoproteins.

The lipid-linked oligosaccharide Glc3-Man9(GlcNAc)2 (Glc, glucose; Man, mannose; GlcNAc, N-acetylglucosamine) serves as a precursor for the biosynthesis of the inner core portion of the asparagine-linked polysaccharide of Saccharomyces cerevisiae mannoproteins. It has been shown previously that incubation of a microsomal preparation from this organism with UDP-N-acetylglucosamine and GDP-[14C]mannose gives rise to a series of lipid-linked oligosaccharides of the general structure Mann(GlcNAc)2, with n from 1 to 9. A structural characterization of Man1- to Man5(GlcNAc)2 oligosaccharides indicated that the major structures among these were identical to the intermediates proposed for the biosynthesis of animal glycoproteins (C. Prakash and I. K. Vijay, Biochemistry 21:4810-4818, 1982). In the present study, the structural characterization of the Man6- through Man9(GlcNAc)2 species was conducted. The Man6- through Man8(GlcNAc)2 species have two isomers, whereas Man9(GlcNAc)2 is monoisomeric. One isomer each of Man6- through Man8(GlcNAc)2 and the monoisomeric Man9(GlcNAc)2 are identical to the intermediates for the biosynthesis of asparagine-linked glycoproteins in animal systems. It is proposed that the steps of the lipid-linked assembly of the carbohydrate precursor for S. cerevisiae mannoproteins are identical to those of the major pathway in animal systems. A lack of acceptor substrate specificity by the mannosyltransferases, as observed with in vitro studies with animal systems, also might be responsible for the biosynthesis of multiple isomers reported here.     

Biosorption of cadmium ions by different yeast species

Toxicity and accumulation of Cd2+ in yeasts were studied in eight different yeast species. The adaptation to toxic concentration of this metal was dependent on the production of extracellular yeast glycoproteins. The highest concentration of Cd2+ ions in the growth medium was tolerated by a Hansenula anomala, strain while the lowest tolerance was found by the strain of species Saccharomyces cerevisiae. Extracellular glycoproteins of Hansenula anomala absorbed nearly 90% of the total content of Cd2+ ions bound by yeast cells, while extracellular glycoproteins of Saccharomyces cerevisiae bound only 6% of the total amount of cadmium. This difference is caused by the variable composition of the saccharide moiety in the extracellular glycoproteins. The composition of extracellular glycoproteins changed during the adaptation of the yeast cells to the presence of Cd2+ ions.     

Disruption of the MNN10 gene enhances protein secretion in Kluyveromyces lactis and Saccharomyces cerevisiae

Screening for genes affecting super-secreting phenotype of the over-secreting mutant of Kluyveromyces lactis resulted in isolation of the gene named KlMNN10, sharing high homology with Saccharomyces cerevisiae MNN10. The disruption of the KlMNN10 in Kluyveromyces lactis, as well as of MNN10 and MNN11 in Saccharomyces cerevisiae, conferred the super-secreting phenotype. MNN10 isolated from Saccharomyces cerevisiae suppressed the super-secretion phenotype in Kluyveromyces lactis klmnn10, as did the homologous KlMNN10. The genes MNN10 and MNN11 of Saccharomyces cerevisiae encode mannosyltransferases responsible for the majority of the alpha-1,6-polymerizing activity of the mannosyltransferase complex. These data agree with the view that the structure of glycoproteins in a yeast cell wall strongly influences the release of homologous and heterologous proteins in the medium. The set of genes namely the suppressors of the over-secreting phenotype, could be attractive for further analysis of gene functions, over-secreting mechanisms and for construction of new strains optimized for heterologous protein secretion. KlMNN10 has EMBL accession no. AJ575132.     

Two different types of lipid moieties are present in glycophosphoinositol-anchored membrane proteins of Saccharomyces cerevisiae.

Numerous glycoproteins of Saccharomyces cerevisiae are anchored in the lipid bilayer by a glycophosphatidylinositol (GPI) anchor. Mild alkaline hydrolysis reveals that the lipid components of these anchors are heterogeneous in that both base-sensitive and base-resistant lipid moieties can be found on most proteins. The relative abundance of base-resistant lipid moieties is different for different proteins. Strong alkaline or acid hydrolysis of the mild base-resistant lipid component liberates C18-phytosphingosine indicating the presence of a ceramide. Two lines of evidence suggest that proteins are first attached to a base-sensitive GPI anchor, the lipid moiety of which subsequently gets exchanged for a base-resistant ceramide: (i) an early glycolipid intermediate of GPI biosynthesis only contains base-sensitive lipid moieties; (ii) after a pulse with [3H]myo-inositol the relative abundance of base-sensitive GPI anchors decreases significantly during chase. This decrease does not take place if GPI-anchored proteins are retained in the ER.     

Control of fatty-acid synthetase levels by exogeneous long-chain fatty acids in the yeasts Candida lipolytica and Saccharomyces cerevisiae.

Endogeneous fatty acid biosynthesis in the two yeast species, Saccharomyces cerevisiae and Candida lipolytica is completely repressed by the addition of long-chain fatty acids to the growth medium. In Candida lipolytica, this repression is accompanied by a corresponding loss of fatty acid synthetase activity in the cell homogenate, when the cells were grown on fatty acids as the sole carbon source. The activity of the Saccharomyces cerevisiae fatty acid synthetase, however, remains unaffected by the addition of fatty acids to a glucose-containing growth medium. From fatty-acid-grown Candida lipolytica cells no fatty acid synthetase complex can be isolated, nor is there any immunologically cross-reacting fatty acid synthetase protein detectable in the crude cell extract. From this it is concluded that Candida lipolytica, but not Saccharomyces cerevisiae, is able to adapt to the growth on fatty acids either by repression of fatty acid synthetase biosynthesis or by a fatty-acid-induced proteolytic degradation of the multienzyme complex. Similarly, the fatty acid synthetase complex disappears rapidly from stationary phase Candida lipolytica cells even after growth in fatty-acid-free medium. Finally, it was found that the fatty acid synthetase complexes from Saccharomyces cerevisiae and Candida lipolytica, though very similar in size and subunit composition, were immunologically different and had no common antigenic determinants.     

Biosynthesis of glyoxylate from glycine in Saccharomyces cerevisiae

Glyoxylate biosynthesis in Saccharomyces cerevisiae is traditionally mainly ascribed to the reaction catalyzed by isocitrate lyase (Icl), which converts isocitrate to glyoxylate and succinate. However, Icl is generally reported to be repressed by glucose and yet glyoxylate is detected at high levels in S. cerevisiae extracts during cultivation on glucose. In bacteria there is an alternative pathway for glyoxylate biosynthesis that involves a direct oxidation of glycine. Therefore, we investigated the glycine metabolism in S. cerevisiae coupling metabolomics data and (13)C-isotope-labeling analysis of two reference strains and a mutant with a deletion in a gene encoding an alanine:glyoxylate aminotransferase. The strains were cultivated on minimal medium containing glucose or galactose, and (13)C-glycine as sole nitrogen source. Glyoxylate presented (13)C-labeling in all cultivation conditions. Furthermore, glyoxylate seemed to be converted to 2-oxovalerate, an unusual metabolite in S. cerevisiae. 2-Oxovalerate can possibly be converted to 2-oxoisovalerate, a key precursor in the biosynthesis of branched-chain amino acids. Hence, we propose a new pathway for glycine catabolism and glyoxylate biosynthesis in S. cerevisiae that seems not to be repressed by glucose and is active under both aerobic and anaerobic conditions. This work demonstrates the great potential of coupling metabolomics data and isotope-labeling analysis for pathway reconstructions.     

A murine monoclonal antibody directed against a yeast cell wall glycoprotein antigen of the yeast genus Saccharomyces

Abstract Murine monoclonal antibodies (mAbs) were selected against a cell wall glycoprotein of Saccharomyces cerevisiae . One of the mAbs (92-276/018) specifically identified S. cerevisiae and the sibling species S. paradoxus, S. pastorianus and S. bayanus in immunofluorescence studies and immunoblot analyses, while no other yeast genera except Saccharomyces were recognized. Further analysis indicated that the mAb 92-276/018 reacts with an epitope in the carbohydrate chain of the cell wall glycoproteins.     

Membrane-mediated killing of Saccharomyces cerevisiae by glycoproteins from Torulopsis glabrata.

Cell-free supernatants from cultures of Torulopsis glabrata contained glycoprotein toxins that killed sensitive and killer strains of Saccharomyces cerevisiae with single-hit kinetics. Growing S. cerevisiae treated with the toxins showed a leakage of cellular potassium, partial dissipation of the adenosine triphosphate pool, and a coordinate shutdown of macromolecular synthesis. These pool efflux-stimulating toxins have been partially purified and at least three toxic glycoproteins have been separated. Pool efflux-stimulating toxin activity was stable from pH 3 through 7, though killing was maximal close to pH 4.     

Glycosylation defects corrected by the changes in GDPmannose level.

GDPMan is a key substrate in glycoprotein formation. This is especially true for lower eukaryotes where, in addition to the involvement in N-glycan biosynthesis and GPI-anchor formation, GDPMan takes part in the process which is unique for yeast and fungi i.e. O-mannosylation. Several lines of evidence have been presented that the level of GDPMan affects the process occurring in the Golgi compartment i.e. the elongation of outer mannose chain of glycoproteins in Saccharomyces cerevisiae. Results from our laboratory indicate that the availability of GDPMan affects also the early steps of glycoprotein formation ascribed to the endoplasmic reticulum, i.e. assembly of the dolichol-linked oligosaccharide as well as mannosyl-phosphodolichol (MPD) formation. The biochemical basis of carbohydrate deficient glycoprotein syndrome, a severe neurological disorder related to the GDPMan deficiency, is also discussed.     

高等植物赤霉素生物合成及其调节研究进展

主要介绍近年来高等植物中生物活性GAs的生物合成,拟南芥GA生物合成途径中关键酶基因（GA1－GA5）的克隆和GA3基因CYP701A3的母（Saccharomyces cerevisiae）中的成功表达。评述了活性GAs对赤霉不生物合成的反馈抑制作用和反馈调节中信号的传递和接收问题。高等植物中光周期对GA生物合成的调节主要是在20－氧化和／或3β－羟基化步骤。     

Cloning and expression of the 3-isopropylmalate dehydrogenase gene from Candida albicans

Abstract A variety of Saccharomyces cerevisiae genes e.g. HIS3, LEU2, TRP1, URA3 , are expressed in Escherichia coli and have been isolated by complementation of mutations in the corresponding E. coli genes [1]. The LEU2 gene was one of the first S. cerevisiae genes to be isolated in this way [2], and its isolation led to the development of transformation systems for S. cerevisiae [3,4]. The leuB gene in E. coli [5] and the LEU2 gene in S. cerevisiae [6] both code for 3-isopropylmalate dehydrogenase (3-IMDH; EC 1.1.1.85) which is essential for the biosynthesis of leucine in both organisms. This paper describes the cloning of a fragment of C. albicans DNA carrying the gene for 3-IMDH which will be useful in the development of transformation methods in C. albicans .     

The intermediary role of a steroid 8,14-dien-3β-ol system in ergosterol biosynthesis

1. A mechanism for the removal of the 14alpha-methyl group in ergosterol biosynthesis that involves the intermediacy of an 8,14-diene system is outlined. 2. In accordance with the requirements of this scheme, it is shown that 5alpha-ergosta-8,14-dien-3beta-ol is converted into ergosterol by Saccharomyces cerevisiae.     

Heterologous production of secondary metabolites as pharmaceuticals in <Emphasis Type="Italic">Saccharomyces</Emphasis> <Emphasis Type="Italic">cerevisiae</Emphasis>

Heterologous expression of genes involved in the biosynthesis of various products is of increasing interest in biotechnology and in drug research and development. Microbial cells are most appropriate for this purpose. Availability of more microbial genomic sequences in recent years has greatly facilitated the elucidation of metabolic and regulatory networks and helped gain overproduction of desired metabolites or create novel production of commercially important compounds. Saccharomyces cerevisiae, as one of the most intensely studied eukaryotic model organisms with a rich density of knowledge detailing its genetics, biochemistry, physiology, and large-scale fermentation performance, can be capitalized upon to enable a substantial increase in the industrial application of this yeast. In this review, we describe recent efforts made to produce commercial secondary metabolites in Saccharomyces cerevisiae as pharmaceuticals. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, such as naringenin, coumarate, artemisinin, taxol, amorphadiene and vitamin C, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties.     

A method of determining the level of provitamin D in Saccharomyces cerevisiae with altered sterol metabolism

A method is proposed that allows to choose among the polyene-resistant mutants of the yeasts Saccharomyces cerevisiae with changed biosynthesis of ergosterol promising producers of provitamin D4 and structural analogs of provitamin D3. The method involves both UV-spectrophotometry and thin-layer chromatography. The results obtained are supported by the data of mass-spectrometry of sterols.     

Evidence for the occurrence of nucleotide-activated oligosaccharides in Saccharomyces cerevisiae

Recently, nucleotide-activated oligosaccharides have been found to be involved in the biosynthesis of certain glycoconjugates in archaeal and bacterial procaryotes. This paper describes the isolation and partial chemical characterization of nucleotide-activated oligosaccharides from the eucaryotic microbe Saccharomyces cerevisiae. We purified four different nucleotide-activated oligosaccharides from cell extracts of Saccharomyces cerevisiae. Three of the oligosaccharides were UDP, and one was TDP-activated. D-Glucose was the only carbohydrate constituent, except for one oligosaccharide, which also contained glucosamine. The chain length varied between two and four sugar residues.     

Utilization by Saccharomyces cerevisiae of 5'-methylthioadenosine as a source of both purine and methionine.

Cells of the yeast Saccharomyces cerevisiae are normally impermeable to the purine nucleosides adenosine and 5'-deoxy-5'-methylthioadenosine (MTA), a product of polyamine biosynthesis. cordycepin-sensitive, adenosine-utilizing strains of S. cerevisiae were able to use MTA to fulfill an auxotrophic requirement for purine. Cordycepin-sensitive strains carrying a met5 mutation were also able to use MTA as a source of methionine. These MTA-utilizing strains of S. cerevisiae should be useful for metabolic studies of the fate of MTA.