Structure of Saccharomyces cerevisiae alg3, sec18 mutant oligosaccharides

Asparagine-linked oligosaccharides are synthesized by transfer of Glc3Man9GlcNAc2 from dolichol pyrophosphate to nascent polypeptides. Assembly of the precursor proceeds by highly ordered sequential addition of mannose and glucose to form Glc3Man9GlcNAc2-P-P-dolichol. Yeast mutants in asparagine-linked glycosylation (alg), generated by an 3H-Man suicide technique, were assigned to eight complementation groups which define steps in oligosaccharide-lipid synthesis (Huffaker, T.C., and Robbins, P.W. (1982) J. Biol. Chem. 257, 3203-3210). Alg3 invertase oligosaccharides are resistant to endo-beta-N-acetylglucosaminidase H, and the lipid-oligosaccharide pool yields Man5Glc-NAc2, suggesting its structure may be that from mammalian cells lacking Man-P-dolichol (Chapman, A., et al. (1980) J. Biol. Chem. 255, 4441-4446). To test this supposition, the endoplasmic reticulum form of invertase derepressed in alg3,sec18 yeast at 37 degrees C was isolated as a source of oligosaccharides whose processing beyond glucose and/or mannose trimming, if involved, would be prevented. Man8GlcNAc2 and Man5GlcNAc2 were released by peptide-N-glycosidase F from alg3,sec18 invertase in a 1:5 molar ratio. 1H NMR spectroscopy revealed Man8GlcNAc2 to be the alpha 1,2-mannosidase-trimming product described earlier (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666), while Man5GlcNAc2 was Man alpha 1, 2Man alpha 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc beta 1, 4GlcNAc. This provides a structural proof for the lipid-linked Man5GlcNAc2 originally proposed from enzymatic and chemical analyses of the radiolabeled mammalian precursor. Experimental evidence indicates that, unlike the mammalian cell mutants which are unable to synthesize Man-P-dolichol, alg3 yeast accumulate Man5GlcNAc2-P-P-dolichol due to a defective alpha 1,3-mannosyltransferase required for the next step in oligosaccharide-lipid elongation.     

Subcellular location of enzymes involved in the N-glycosylation and processing of asparagine-linked oligosaccharides in Saccharomyces cerevisiae

A particulate translation system isolated from the yeast Saccharomyces cerevisiae was shown to translate faithfully in-vitro-transcribed mRNA coding for a mating hormone precursor (prepro-alpha-factor mRNA) and to N-glycosylate the primary translation product after its translocation into the lumen of the microsomal vesicles. Glycosylation of its three potential sugar attachment sites was found to be competitively inhibited by acceptor peptides containing the consensus sequence Asn-Xaa-Thr, supporting the view that the glycan chains are N-glycosidically attached to the prepro-alpha-factor polypeptide. The accumulation in the presence of acceptor peptides of a membrane-specific, unglycosylated translation product (pp-alpha-F0) differing in molecular mass from a cytosolically located, protease-K-sensitive alpha-factor polypeptide (pp-alpha-Fcyt) by about 1.3 kDa, suggests that, in contrast to previous reports, a signal sequence is cleaved from the mating hormone precursor on/after translocation. This conclusion is supported by the observation that the multiply glycosylated alpha-factor precursor is cleaved by endoglucosaminidase H to a product with a molecular mass smaller than the primary translation product pp-alpha-Fcyt but larger than the membrane-specific pp-alpha-F0. Translation and glycosylation experiments carried out in the presence of various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-1-deoxynojirimyin and 1-deoxymannojirimycin) indicate that the N-linked oligosaccharide chains of the glycosylated prepro-alpha-factor species are extensively processed under the in vitro conditions of translation. From the specificity of the glycosidase inhibitors applied and the differences in the molecular mass of the glycosylated translation products generated in their presence, we conclude that the glycosylation-competent microsomes contain trimming enzymes, most likely glucosidase I, glucosidase II and a trimming mannosidase, which process the prepro-alpha-factor glycans down to the (Man)8(GlcNAc)2 stage. Furthermore, several arguments strongly suggest that these three enzymes, which apparently represent the full array of trimming activities in yeast, are exclusively located in the lumen of microsomal vesicles derived from endoplasmic reticulum membranes.     

Biosynthesis of sulfated asparagine-linked oligosaccharides on bovine lutropin

The asparagine-linked oligosaccharides on bovine lutropin (bLH) are unusual, containing GalNAc and sulfate but no galactose or sialic acid. Oligosaccharides from metabolically radiolabeled or purified bLH consist of non- (neutral), mono- (S-1), and di- (S-2) sulfated structures. We have previously shown that S-2 is a complex type oligosaccharide bearing two peripheral branches with the sequence SO4----GalNAc----GlcNAc attached to a typical Man3GlcNAc2 core (Green, E.D., van Halbeek, H., Boime, I., and Baenziger, J.U. (1985) J. Biol. Chem. 260, 15623-15630). We have now characterized the S-1 oligosaccharides on bLH which, in contrast to S-2, consist of several different structures of both the hybrid and complex types. The sulfate on S-1 oligosaccharides is located exclusively within the peripheral sequence SO4----GalNAc----GlcNAc. The GalNAc bearing hybrid structures, either with or without sulfate, cannot be processed to mono- or disulfated complex oligosaccharides due to the inability of either alpha-mannosidase II or GlcNAc-transferase II to act on GalNAc containing oligosaccharides. Since both Gal and GalNAc are added to oligosaccharides on some pituitary hormones, for example bovine and ovine follitropin and human lutropin, the Gal- and GalNAc-transferases appear to be key elements in regulating the synthesis of sulfated oligosaccharides on bLH and the other pituitary glycoprotein hormones.     

Spontaneous mutation of leu2 in Saccharomyces cerevisiae

The data obtained indicate that spontaneous mutations in Saccharomyces cerevisiae are formed during DNA replication. With no DNA replication in the lag-period, in the stationary growth phase, spontaneous mutations are not formed in cell culture during the G1 phase of cell cycle. Experimental data show the absence of primary spontaneously occurring DNA lesion accumulation in the cell G1 phase. Spontaneous mutations of yeasts are formed in the S phase of cell cycle, apparently as DNA replication errors. It is established that the frequency of spontaneous reversions of the leu2 gene in Saccharomyces cerevisiae strain NA3-24 increases when the cells are cultivated on the culture medium with different concentrations of leucine.     

Adaptive mutation in Saccharomyces cerevisiae

Adaptive mutation is a generic term for processes that allow individual cells of nonproliferating cell populations to acquire advantageous mutations and thereby to overcome the strong selective pressure of proliferation-limiting environmental conditions. Prerequisites for an occurrence of adaptive mutation are that the selective conditions are nonlethal and that a restart of proliferation may be accomplished by some genetic change in principle. The importance of adaptive mutation is derived from the assumption that it may, on the one hand, result in an accelerated evolution of microorganisms and, on the other, in multicellular organisms may contribute to a breakout of somatic cells from negative growth regulation, i.e., to cancerogenesis. Most information on adaptive mutation in eukaryotes has been gained with the budding yeast Saccharomyces cerevisiae. This review focuses comprehensively on adaptive mutation in this organism and summarizes our current understanding of this issue.     

Biosynthesis of natural flavanones in Saccharomyces cerevisiae

A four-step flavanone biosynthetic pathway was constructed and introduced into Saccharomyces cerevisiae. The recombinant yeast strain was fed with phenylpropanoid acids and produced the flavanones naringenin and pinocembrin 62 and 22 times more efficiently compared to previously reported recombinant prokaryotic strains. Microbial biosynthesis of the flavanone eriodictyol was also achieved.     

Regulation of Pyrimidine Biosynthesis in Saccharomyces cerevisiae

Biochemical steps of the pyrimidine pathway have been found to be the same in yeast as in bacteria, and all except one step have been characterized. The activities of the first two enzymes, carbamoyl phosphate synthetase and aspartic transcarbamylase, are simultaneously controlled by feedback inhibition and repression. Moreover, these enzymes are coded by the same genetic region (ura-2) and seem to form a single enzymatic complex. The enzymes that follow later in the pathway are induced in a sequential way by the intermediary products and are insensitive to pyrimidine repression. The corresponding genes (ura-4, ura-1, ura-3) are not linked to each other or to ura-2, the gene for carbamoyl phosphate synthetase and aspartic transcarbamylase. Mutants that have simultaneously lost feedback inhibition by uridine triphosphate for carbamoyl phosphate synthetase and for aspartic transcarbamylase have been found and mapped in the gene ura-2.     

Biosynthesis of Natural Flavanones in Saccharomyces cerevisiae

A four-step flavanone biosynthetic pathway was constructed and introduced into Saccharomyces cerevisiae. The recombinant yeast strain was fed with phenylpropanoid acids and produced the flavanones naringenin and pinocembrin 62 and 22 times more efficiently compared to previously reported recombinant prokaryotic strains. Microbial biosynthesis of the flavanone eriodictyol was also achieved.     

Isolation of new nonconditional Saccharomyces cerevisiae mutants defective in asparagine-linked glycosylation

We describe the isolation and partial characterization of Saccharomycescerevisiae nonconditional mutants that show defects in N-glycosylationof proteins. The selection method is based on the reductionof affinity for the ion exchanger QAE-Sephadex as a consequenceof the decrease in the negative charge of the cell surface.This characteristic reflects a decrease in the incorporationof mannosyl-phosphate units into the N-linked oligosaccharidesof the mannoproteins. The mutants exhibit low affinity for thebasic dye alcian blue and for that reason we have called themldb (low dye binding) mutants. Eight of the complementationgroups seem to be new as shown by complementation studies withpreviously isolated mutants of similar phenotype. Four of thegroups showed a significant reduction in the number and/or sizeof the N-linked oligosaccharides attached to secreted invertase.We have analyzed the N-linked oligosaccharides of ldb1 and ldb2,the mutants that show the most drastic reduction in the affinityfor the alcian blue dye. In both cases, the purified endo H-releasedoligosaccharides from the mannoproteins lacked detectable amountsof phosphate groups as shown by ion exchange chromatographyand the ¹H NMR spectra. In addition, ldb1 synthesizes a truncatedand unbranched outer chain lacking any     

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.     

Biosynthesis of sulphur amino acids in Saccharomyces cerevisiae

A number of strains of Saccharomyces which produce sulphite by sulphate reduction were examined from an enzymatic and genetic point of view.There are a number of mechanisms that regulate this activity. All of these mechanisms involve the sulphite-reducing activity. In the strains examined, reduced function as a result of mutation in the Sr-locus (affecting H₂S-NADP oxidoreductase EC 1.8.1.2), repression of biosynthesis of the enzyme because of a mutation below the specific locus, and inhibition of the enzyme by endogenous factors were found to be responsible. The production of sulphite can also be connected with a complex state of heterozygosity.It is probably this multiplicity of biochemical and genetic mechanisms that accounts for the frequency with which the production of sulphite is observed in wild strains in nature.This investigation was supported by a research grant of C.N.R. (Consiglio Nazionale delle Ricerche, Roma).     

Biosynthesis of cofactor-activatable iron-only nitrogenase in Saccharomyces cerevisiae

Engineering nitrogenase in eukaryotes is hampered by its genetic complexity and by the oxygen sensitivity of its protein components. Of the three types of nitrogenases, the Fe-only nitrogenase is considered the simplest one because its function depends on fewer gene products than the homologous and more complex Mo and V nitrogenases. Here, we show the expression of stable Fe-only nitrogenase component proteins in the low-oxygen mitochondria matrix of S. cerevisiae. As-isolated Fe protein (AnfH) was active in electron donation to NifDK to reduce acetylene into ethylene. Ancillary proteins NifU, NifS and NifM were not required for Fe protein function. The FeFe protein existed as apo-AnfDK complex with the AnfG subunit either loosely bound or completely unable to interact with it. Apo-AnfDK could be activated for acetylene reduction by the simple addition of FeMo-co in vitro, indicating preexistence of the P-clusters even in the absence of coexpressed NifU and NifS. This work reinforces the use of Fe-only nitrogenase as simple model to engineer nitrogen fixation in yeast and plant mitochondria.     

Multivalent Repression of Isoleucine-Valine Biosynthesis in Saccharomyces cerevisiae

Regulation of the biosynthesis of four of the five enzymes of the isoleucine-valine pathway was studied in Saccharomyces cerevisiae. A method is described for limiting the growth of a leucine auxotroph by using valine as a competitor for the permease. Limitation for isoleucine and valine was accomplished by the use of peptides containing these amino acids conjugated with glycine as nutritional supplements for auxotrophs. The enzymes were repressed on synthetic medium containing isoleucine, valine, and leucine, as well as on broth supplemented with these amino acids. Limitation for any of the three branched-chain amino acids led to derepression of the isoleucine-valine biosynthetic pathway. Maximal derepression ranged from 3-fold for threonine deaminase to approximately 10-fold for acetohydroxyacid synthase. (Two of the enzymes, acetohydroxyacid synthase and dihydroxyacid dehydrase, may be controlled by a mechanism different from that regulating threonine deaminase.) Possible molecular mechanisms for multivalent repression are discussed.     

Biosynthesis of sulphur amino acids in Saccharomyces cerevisiae

The hypothesis of an alternative pathway of sulphur amino acid synthesis as the basis of the prototrophy of sulphite reductase negative (Sr^-) strains of Saccharomyces cerevisiae has been rejected. Met^- mutants obtained after phenylmercuric nitrate treatment of Sr^- strains accumulate H₂S as the consequence of a metabolic block which leads to methionine auxotrophy. This mutation has been shown to be independent of the Sr locus. We assume that the molecular basis of the prototrophy of Sr^- strains resides in a leaky missense induced in the Sr gene.     

Biosynthesis of glycophosphoinositol anchors in Saccharomyces cerevisiae.

Numerous membrane glycoproteins of Saccharomyces cerevisiae are posttranslationally modified by the addition of a glycophosphatidylinositol (GPI). These proteins can be detected most easily by metabolic labelling of yeast cells with 3H-myoinositol or 3H-palmitate. This report summarizes what little is known about the identity, biosynthesis and cellular localization of GPI-modified glycoproteins in Saccharomyces cerevisiae as well as what could be learned from the system with respect to the biosynthesis of GPI's in general.     

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.     

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

The wall gives a Saccharomyces cerevisiae cell its osmotic integrity; defines cell shape during budding growth, mating, sporulation, and pseudohypha formation; and presents adhesive glycoproteins to other yeast cells. The wall consists of β1,3- and β1,6-glucans, a small amount of chitin, and many different proteins that may bear N- and O-linked glycans and a glycolipid anchor. These components become cross-linked in various ways to form higher-order complexes. Wall composition and degree of cross-linking vary during growth and development and change in response to cell wall stress. This article reviews wall biogenesis in vegetative cells, covering the structure of wall components and how they are cross-linked; the biosynthesis of N- and O-linked glycans, glycosylphosphatidylinositol membrane anchors, β1,3- and β1,6-linked glucans, and chitin; the reactions that cross-link wall components; and the possible functions of enzymatic and nonenzymatic cell wall proteins.     

The mnn2 mutant of Saccharomyces cerevisiae is affected in phosphorylation of N-linked oligosaccharides

We studied the phosphorylation of the inner core region of N-linked oligosaccharides in the mannan defective mutant Saccharomyces cerevisiae mnn2 which was described as unable to synthesize branches on the outer chain. We performed structural studies of the N-oligosaccharides synthesized by the strains mnn2, mnn1mnn2mnn9 and mnn1mnn9ldb8, and the results are compared with previously published structural data of mnn1mnn2mnn10 and mnn1mnn9 [Hernández, L.M., Ballou, L., Alvarado, E., Tsai, P.-K. and Ballou, C.E. (1989) J. Biol. Chem. 264, 13648-13659]. We conclude that the mnn2/ldb8 mutation is responsible for the inhibition of incorporation of phosphate to mannose A(3) (see below), a particular phosphorylation site of the inner core, while phosphorylation at the other possible site (mannose C(1)) is allowed, although it is also reduced. *Phosphorylation sites in mnn1mnn9. (see structure below)