Scanning electron microscope study of Saccharomyces cerevisiae spheroplast formation.

A suspension of Saccharomyces cerevisiae NCY366 in buffered 1.2 M sorbitol containing Zymolyase-5000 (a beta-glucanase-containing preparation/showed maximum osmotic sensitivity after 30 min of incubation at 30 degrees C. A scanning electron microscope study of spheroplast formation, using a very high resolution (4-nm) machine, revealed several new morphological features. The surface of the plug in bud scars on intact cells appeared warty. The wall, which assumed a beady appearance as digestion proceded, ultimately sloughed off to reveal the furrowed surface of the plasma membrane. Bud scars were resistant to digestion and. as incubation proceeded, they became surrounded by an outer annulus, which may be the seconary septum. Wall material was completely removed from the majority of cells only after 60 min of digestion. The surface of spheroplasts was studded with particles, about 25 to 30 nm in diameter. Many spheroplasts had a single large indentation, which may be in that part of the plasma membrane originally underlying the birth scar.     

Meiotic cytology of Saccharomyces cerevisiae in protoplast lysates

Summary This report describes cytological features of meiosis in Saccharomyces cerevisiae prepared for electron microscopy by lysis of protoplasts or nuclei on an aqueous surface. Whereas the chromatin of cells lysed before or after meiotic prophase was widely dispersed, pachytene bivalents appeared as discrete, elongate masses of compact chromatin. These bivalents were of nearly uniform thickness; they ranged in length from about 0.6 m to 4.0 m, with a median of 1.6–1.8 m. Enzymatic digestion of chromosomal DNA removed the chromatin to reveal the underlying synaptonemal complex. The lysis of partially purified nuclei was less disruptive and thereby revealed the regular association of the telomeres with fragments of the nuclear envelope. In tetraploid cells, pachytene lysates contained quadrivalents characterized by the close apposition of chromatin masses of similar length. One or more points of intimate association appear to represent sites of exchange between pairing partners. The departure of the diploid cells from pachytene was accompanied by the renewed association of spindle microtubules with the chromosomes shortly before the diplotene chromosomes decondensed. Later, the successive meiotic divisions were identified by the appearance of a single spindle for meiosis I and of two spindles for meiosis II.     

Identification of two Saccharomyces cerevisiae cell wall mannan chemotypes

We have obtained evidence for two structurally and antigenically different Saccharomyces cerevisiae cell wall mannans. One, which occurs widely and is found in S. cerevisiae strain 238C, is already known to be a neutral mannan which yields mannose, mannobiose, mannotriose, and mannotetraose on acetolysis of the (1 --> 6)-linked backbone. The other, which was found in S. cerevisiae brewer's strains, is a phosphomannan with a structure very similar to that of Kloeckera brevis mannan. S. cerevisiae (brewer's yeast strain) was agglutinated by antiserum prepared against Kloeckera brevis cells. The mannan, isolated from a proteolytic digest of the cell wall of the former, did not react with S. cerevisiae 238C antiserum, whereas it cross-reacted strongly with K. brevis antiserum. Controlled acetolysis cleaved the (1 --> 6)-linkages in the polysaccharide backbone and released mannose, mannobiose, mannotriose, and mannotriose phosphate. Mild acid treatment of the phosphomannan hydrolyzed the phosphodiester linkage, yielding phosphomonoester mannan and mannose. The resulting phosphomonoester mannan reacted with antiserum prepared against K. brevis possessing monoester phosphate groups on the cell surface. alpha-d-Mannose-1-phosphate completely inhibited the precipitin reaction between brewer's yeast mannan and the homologous antiserum. Flocculent and nonflocculent strains of this yeast were shown to have similar structural and immunological properties.     

Biosynthesis of yeast mannan. Properties of a mannosylphosphate transferase in Saccharomyces cerevisiae.

A homogenate of mechanically broken, freshly grown Saccharomyces cerevisiae X2180 cells catalyzes the transfer of mannosylphosphate units from guanosine diphosphate mannose to reduced alpha1 leads to 2-[3H]mannotetraose to yield reduced mannosylphosphoryl [3H]-mannotetraose. The product is analogous in structure to the phosphorylated mannan side chains, which suggests that the enzymic activity is involved in mannoprotein biosynthesis in the intact cell. The mannosylphosphate transferase activity, localized in a membrane fraction obtained by differential centrifugation at 100,000 x g, was solubilized by Triton X-155 and purified 250-fold by ammonium sulfate precipitation and by ion exchange and gell filtration chromatographies. The enzyme requires MN2+ OR Co2+ ions for activity and is stimulated by various detergents. The mnn2 and mnn3 mannan mutants of S. cerevisiae possess normal levels of mannosylphosphate transferase activity, whereas the mnn4 mutant cells contain very low, if any, activity. This is consistent with a previous conclusion that the mnn4 mutation affects the mannosylphosphate transferase activity, whereas the mnn2 and mnn3 strains possess phosphate-deficient mannans because they are unable to synthesize the appropriate side chain precursors. A new mannan mutant class with the mnn4 chemotype was isolated, but the mutation proved to be recessive and nonallelic with the mnn4 locus. This new locus is designated mnn6.     

The components of the Saccharomyces cerevisiae mannosyltransferase complex M-Pol I have distinct functions in mannan synthesis

The yeast Saccharomyces cerevisiae processes N-linked glycans in the Golgi apparatus in two different ways. Whereas most of the proteins of internal membranes receive a simple core-type structure, a long branched polymer termed mannan is attached to the glycans of many of the proteins destined for the cell wall. The first step in mannan synthesis is the initiation and extension of an alpha-1,6-linked polymannose backbone. This requires the sequential action of two enzyme complexes, mannan polymerases (M-Pol) I and II. M-Pol I contains the proteins Mnn9p and Van1p, although the stoichiometry and individual contributions to enzyme action are unclear. We report here that the two proteins are each present as a single copy in the complex. Both proteins contain a DXD motif found in the active site of many glycosyltransferases, and mutations in this motif in Mnn9p or Van1p reveal that both proteins contribute to mannose polymerization. However, the effects of these mutations on both the in vivo and in vitro activity are distinct, suggesting that the two proteins may have different roles in the complex. Finally, we show that a simple glycoprotein based on hen egg lysozyme can be used as a substrate for modification by purified M-Pol I in vitro.     

Inhibition of protein synthesis in reticulocyte lysates by trichodermin.

1. The effect of trichodermin as an inhibitor of eukaryotic protein synthesis was studied in a reticulocyte cell-free system. 2. Trichodermin at a concentration of 25 mug/ml inhibits total protein synthesis instantaneously and stabilizes polyribosome profiles. Conversely, at a concentration of 0.25 mug/ml the drug inhibits total protein synthesis by only 70-75% and allows 30-35% breakdown of the polyribosomes in the system. These effects were compared with those produced by two other drugs (pactamycin and anisomycin) examined under conditions identical with those used for trichodermin.     

Low ribonuclease activity in cellular lysates of osmotic sensitive Saccharomyces cerevisiae mutants.

Summary Cellular lysates with very low total ribonuclease activities are obtained by lysis of Saccharomyces cerevisiae VY1160 osmotic sensitive mutant cells in 1% sorbitol solution. These lysates could be used for isolation of intact polysomes and messenger RNA molecules, or for studying of specific ribonucleases.     

Characterization of oligosaccharides from an antigenic mannan of Saccharomyces cerevisiae

Mannans of the yeast Saccharomyces cerevisiae have been implicated as containing the allergens to which bakers and brewers are sensitive and also the antigen recognized by patients with Crohn's disease. A fraction of S. cerevisiae mannan, Sc500, having high affinity for antibodies in Crohn's patients has been characterized by NMR spectroscopy followed by fragmentation using alkaline elimination, partial acid hydrolysis and acetolysis. The released oligosaccharides were separated by gel filtration on a Biogel P4 column and analyzed by fluorescence labeling, HPLC and methylation analysis. The relationship between structure and antigen activity was measured by competitive ELISA. The antigenic activity of the original high molecular weight mannan could be ascribed to terminal Man13Man12 sequences which are rarely found in human glycoproteins but were over-represented in Sc500 compared to other yeast mannans.     

Short-chain and medium-chain aliphatic-ester synthesis in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the enzymes which catalyse the synthesis of ethyl acetate, ethyl n-hexanoate and isoamyl acetate were partly resolved from a fraction containing slowly sedimenting lipoproteins released during cell disruption with glass beads. Solubilization with detergents and fractionation by affinity chromatography have demonstrated the presence of at least three, and probably four, ester synthases which differ in their catalytic properties. Isoamyl-acetate synthase was solubilized and extensively purified to apparent homogeneity by successive chromatographies on various columns. On the basis of its specific activity in cell-free extracts, the enzyme was purified 19,000-fold with a 5% activity yield. As judged by SDS/PAGE, it consists of a single polypeptide chain with a molecular mass of 57 +/- 3 kDa and its apparent pI is 5.5. The enzyme acetylates isoamyl alcohol, ethanol and 12-DL-hydroxystearic acid from acetyl-CoA but is unable to use n-hexanoyl-CoA as a cosubstrate. This enzyme, defined as an acetyl-CoA: O-alcohol acetyltransferase, could be the product of one of the anaerobically induced genes in S. cerevisiae.     

Inhibition of protein synthesis by double-stranded RNA in reticulocyte lysates: evidence for activation of an endoribonuclease.

Double-stranded RNA is a potent inhibitor of protein synthesis in rabbit reticulocyte lysates. Three lines of evidence suggest that at least part of this inhibitory activity is due to activation of a nuclease which degrades mRNA: (1) In the presence of emetine reticulocyte polysomes are partially degraded to structures containing 1–3 ribosomes; (2) 34S Mengo-virus RNA is degraded to fragments sedimenting at less than 18S; (3) The template activity of globin mRNA extracted from the lysates is reduced by 90% when compared to appropriate controls. The ability of double-stranded RNA to activate a nuclease in the reticulocyte system is very similar to that observed in extracts from interferon treated cells and probably involves formation of the unusual oligonucleotide pppA^2′ p^5′ A^2′ p^5′ A.     

Effect of inositol starvation on the in vitro syntheses of mannan and N-acetylglucosaminylpyrophosphoryldolichol in Saccharomyces cerevisiae.

An early consequence of starvation for inositol in yeast is inhibition of synthesis of the major cell wall components mannan and glucan. In looking for the mechanism of this inhibition, we found that the activity of the enzyme catalyzing the synthesis of N-acetylglucosaminylpyrophosphoryldolichol was diminished in particular membrane preparations from cells starved for inositol. This loss of reactivity was observed under a variety of in vitro assay conditions and could be restored by the addition of phosphatidylinositol but not by other phosphoinositol-containing sphingolipids known to occur in yeast. When assayed in the presence of high concentrations of Triton X-100, enzyme preparations from both control and inositol-starved cells required phosphatidylinositol for maximal activity. Since this enzyme catalyzed an early step in the synthesis of mannan that is N-linked to protein, a reasonable hypothesis is that inhibition of mannan synthesis in inositol-starved cells results from the depletion of the necessary cofactor phosphatidylinositol.     

Inhibition of mitochondrial protein synthesis in vivo by erythromycin in Schizosaccharomyces pombe and Saccharomyces cerevisiae

Summary In the presence of erythromycin (0.01 mg/ml) growth of Schizosaccharomyces pombe in non-fermentable substrate (glycerol) is reduced to 5–15% of the control without erythromycin, whereas growth in fermentable substrate (5% glucose) is left unaffected by concentrations up to 5 mg/ml. The reduction of growth under derepressed conditions is paralleled by inhibition of the formation of cytochromes a·a₃ and b. Mitochondrial protein synthesis is inhibited to about 50% in Schizosaccharomyces pombe and to about 90% in Saccharomyces cerevisiae. These results support the hypothesis that inhibition of mitochondrial protein synthesis is the primary effect of erythromycin.     

Transformation of Escherichia coli with DNA from Saccharomyces cerevisiae cell lysates

We developed a system to monitor the transfer of heterologous DNA from a genetically manipulated strain of Saccharomyces cerevisiae to Escherichia coli. This system is based on a yeast strain that carries multiple integrated copies of a pUC-derived plasmid. The bacterial sequences are maintained in the yeast genome by selectable markers for lactose utilization. Lysates of the yeast strain were used to transform E. coli. Transfer of DNA was measured by determining the number of ampicillin-resistant E. coli clones. Our results show that transmission of the Amp(r) gene to E. coli by genetic transformation, caused by DNA released from the yeast, occurs at a very low frequency (about 50 transformants per microg of DNA) under optimal conditions (a highly competent host strain and a highly efficient transformation procedure). These results suggest that under natural conditions, spontaneous transmission of chromosomal genes from genetically modified organisms is likely to be rare.     

Inhibition of hemoglobin synthesis by pancreatic deoxyribonuclease in rabbit reticulocyte lysates.

A pancreatic deoxyribonuclease preparation, shown to be free of significant ribonuclease activity, inhibits hemoglobin synthesis in a rabbit reticulocytelysate. Preincubation with DNAase (15 min at 25 degrees C) is required to obtain a marked inhibitory effect (nearly 70%). It has been shown that DNAase does not significantly interfere with the elongation or termination steps of translation, but it seems to prevent reinitiation of globin polypeptide chains allowing polysome run-off. In particular, the formation of the 40S/met-tRNAmetf complex is greatly reduced in the DNAase-treated lysate. At the moment the mechanism by which DNAase inhibits initiation is not clear.