Proteinase activities of Saccharomyces cerevisiae during sporulation.

Sporulation in Saccharomyces cerevisiae occurs in the absence of a exogenous nitrogen source. Thus, the internal amino acid pool and the supply of nitrogen compounds from protein and nucleic acid turnover must be sufficient for new protein synthesis. Since sporulation involves an increased rate of protein turnover, an investigation was conducted of the changes in the specific activity of various proteinases. A minimum of 30% of the vegetative proteins was turned over during the course of sporulation. There was a 10- to 25-fold increase in specific activity of various proteinases, with a maximum activity around 20 h after transfer into the sporulation medium. The increase in activities was due to de novo synthesis since inhibition of protein synthesis by cycloheximide blocks both an increase in proteinase activities and sporulation. There was no increase observed in proteinase activities of nonsporogenic cultures (a and alpha/alpha strains) inoculated into the sporulation medium, suggesting that the increase in proteinase activities is "sporulation specific" and not a consequence of step-down conditions. The elution patterns through diethylaminoethyl-Sephadex chromatography of various proteinases extracted from T0 and T18 cells were similar, and no new species was observed.     

Metabolic rates during sporulation of Saccharomyces cerevisiae on acetate

We have quantified yeast carbon and oxygen consumption fluxes and estimated anabolic fluxes through glyoxylate and gluconeogenic pathways under various conditions of sporulation on acetate. The percentage of sporulation reached a maximum of 55% to 60% after 48 h in sporulation medium, for cells harvested from logarithmic growth in acetate minimal medium. When cells were harvested in the stationary phase of growth before transfer to sporulation medium, the maximum percentage of sporulation decreased to 40% along with the occurrence of meiosis as could be judged by counting of bi- and tetra-nucleated cells. In both experiments, the rates of acetate and oxygen consumption decreased as a function of time when exposed to sporulation medium. Apparently, the decrease of metabolic rates was not due to alkalinization. By systematically varying the cell concentration in sporulation medium from 1.4×10⁷ to 20×10⁷ cell ml^-1, the percentage of sporulating cells was found to decrease in parallel with the rate of acetate consumption. When the sporulation efficiency attained under the different experimental conditions was plotted as a function of the rate of acetate consumption, a linear correlation was found. Anabolic fluxes estimation revealed a decrease of the rate through gluconeogenic and glyoxylate pathways occurring during sporulation progression. The pattern of metabolic fluxes progressively evolved toward a predominance of more oxidative catabolic fluxes than those exhibited under growth conditions. The results obtained are discussed in terms of a characteristic pattern of metabolic fluxes and energetics, associated to the development of yeast sporulation.Abbreviations DAPI 4,6-diamidino-2-phenylindole - dw dry weight - OD₅₄₀ optical density at 540 nm - SEM standard error of the mean - RQ respiratory quotient     

Metabolism of myo-inositol during sporulation of myo-inositol-requiring Saccharomyces cerevisiae.

We investigated the sporulation properties of a series of diploid Saccharomyces cerevisiae strains homozygous for inositol auxotrophic markers. The strains required different amounts of inositol for the completion of sporulation. Shift experiments revealed two phases of inositol requirement during sporulation which coincided with the two phases of lipid synthesis found by earlier workers. Phase I was at the beginning and during premeiotic deoxyribonucleic acid synthesis; phase II immediately preceded the appearance of mature asci. Of the inositol taken up by sporulating cells, 90% was incorporated into inositol phospholipids. By two-dimensional thin-layer chromatography, eight compounds were resolved, one of which was sporulation specific. The majority of the inositol phospholipids were, however, identical to those found in vegetatively growing cells. In the absence of inositol, the cells did not sporulate but, after a certain time, were unable to return to vegetative growth. These nonsporulating cells did, however, incorporate acetate into lipids and double their deoxyribonucleic acid content in the premeiotic phase. We believe that it is this lack of coordination of biosynthetic events which causes inositol-less death on sporulation media without inositol.     

Alternative modes of organellar segregation during sporulation in Saccharomyces cerevisiae

Formation of ascospores in the yeast Saccharomyces cerevisiae is driven by an unusual cell division in which daughter nuclei are encapsulated within de novo-formed plasma membranes, termed prospore membranes. Generation of viable spores requires that cytoplasmic organelles also be captured along with nuclei. In mitotic cells segregation of mitochondria into the bud requires a polarized actin cytoskeleton. In contrast, genes involved in actin-mediated transport are not essential for sporulation. Instead, efficient segregation of mitochondria into spores requires Ady3p, a component of a protein coat found at the leading edge of the prospore membrane. Other organelles whose mitotic segregation is promoted by actin, such as the vacuole and the cortical endoplasmic reticulum, are not actively segregated during sporulation but are regenerated within spores. These results reveal that organellar segregation into spores is achieved by mechanisms distinct from those in mitotic cells.     

Synthesis of beta-glucanases during sporulation in Saccharomyces cerevisiae: formation of a new, sporulation-specific 1,3-beta-glucanase.

A biphasic synthesis of 1,3-beta-glucanase occurred when cells of Saccharomyces cerevisiae AP-1 (a/alpha) were incubated in sporulation medium. The capacity to degrade laminarin increased very slowly during the first 7 h but at a much faster rate thereafter. Changes occurring during the first period were not sporulation specific since the moderate increase in activity against laminarin was insensitive to glutamine and hydroxyurea and also took place in the nonsporulating strain S. cerevisiae AP-1 (alpha/alpha). However, the changes taking place after 7 h must be included in the group of sporulation-specific events since they were inhibited by glucose, glutamine, and hydroxyurea and did not occur in the nonsporulating diploid. Consequently, only when the cells had been incubated for at least 7 h in sporulation medium did full induction of activity against laminarin take place upon shift to a medium which favored vegetative growth. Changes in the relative proportions of the vegetative glucanases, namely, endo- and exo-1,3-beta-glucanase, and the formation of a new sporulation-specific 1,3-beta-glucanase account for the observed events and are the consequence of the expression of the sporulation program.     

Fluorescent measurement of the intracellular pH during sporulation of Saccharomyces cerevisiae

This work reports the intracellular pH (pH_i) dynamics of Saccharomyces cerevisiae cells in sporulation medium. Cells loaded with the pH-sensitive dye carboxy-seminaphthorhodafluor-1 (C.SNARF-1) exhibited an alkalization of the pH_i following the extracellular pH during sporulation in the absence of buffer and almost no change in pH_i or ΔpH when sporulation was carried out in buffered medium. The results indicate that the pH gradient does not appear to be directly involved in the regulation of acetate uptake during sporulation. However, the alkalization of pH_i by eliciting a decrease in metabolic fluxes could account for a lower demand for acetate.     

Two-dimensional protein patterns during growth and sporulation in Saccharomyces cerevisiae.

Proteins synthesized by Saccharomyces cerevisiae in presporulation and sporulation media were compared by using sporulating (a/alpha) and nonsporulating (a/a and alpha/alpha) yeast strains. Total cellular proteins were labeled with [35S]methionine and analyzed by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms and/or fluorograms showed some 700 spots per gel. Nine proteins were synthesized by a/alpha cells which were specific to vegetative, log-phase conditions. During incubation in sporulation medium, sporulating (a/alpha) cells synthesized 11 proteins not present in vegetatively growing cell. These same 11 proteins, however, were synthesized by nonsporulating (a/a and alpha/alpha) cells on sporulation medium as well. Nonsporulating diploids (a/a and alpha/alpha) were also examined with the electron microscope at various times during their incubation in sporulation medium. Certain cellular responses found to be unique to meiotic yeast cells in previous studies were exhibited by the nonsporulating controls. The degree to which all cell types (a/alpha, a/a, and alpha/alpha) were committed to sporulation was also determined by shifting cells from sporulation medium to vegetative medium. Some commitment to the meiotic pathway was observed in both the a/alpha and the a/a, alpha/alpha cells.     

Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae.

Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate. Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml). The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants). Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains. The major source of these compounds was degradation of RNA. The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs. Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density. Adenine enhanced sporulation of yeast prototrophs at low cell density. The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.     

Promotion of sporulation by caffeine pretreatment in Saccharomyces cerevisiae

Changes in RNase activity during sporulation of a homothallic diploid strain of Saccharomyces cerevisiae were measured in caffeine-treated and non-treated cells.

Inhibiton by sulfanilamide of sporulation in Saccharomyces cerevisiae.

The antimetabolite sulfanilamide inhibits sporulation in Saccharomyces cerevisiae strain AP1. Cells exposed to sulfanilamide at various times during the sporulation process become progressively insensitive to the drug, although accumulation of sulfanilamide by the cells increases with time. Vegetative growth of AP1 is practically unaffected by sulfanilamide; pregrowth of the cells in the presence of the drug does not prevent sporulation. Thus, inhibition is confined to the meiotic phase of the cell cycle. Sensitivity to sulfanilamide is independent of pH. Increasing the time cells are exposed to sulfanilamide results in a progressive reduction of ascus formation; however, the inhibition is reversible since sporulation can occur in cells exposed to the drug for greater than 24 h. The drug arrests the cells at a point before commitment to sporulation, since yeast cells exposed to sulfanilamide for 12 h do not complete the sporulation process when returnedto vegetative medium, but resume mitotic growth instead. Meiotic nuclear division is largely prevented by sulfanilamide, and synthesis of RNA and protein is severely retarded. DNA synthesis is inhibited up to 50%; glycogen synthesis is approximately 90% inhibited. Other yeast strains showed varying sensitivity to sulfanilamide; homothallic strains were generally less affected.     

Characterization and localization of the sporulation glucoamylase of Saccharomyces cerevisiae

Glucoamylase (SGA) was purified approximately 250-fold from sporulating Saccharomyces cerevisiae cells. The partially purified enzyme was active against glycogen, starch, maltotriose and maltose. It exhibited maximum catalytic activity against glycogen at pH 5.5. The enzyme appears to be glycosylated, because it bound to lentil-lectin Sepharose. SGA was expressed in vegetatively growing cells under the control of the GAL1 promoter, and the cellular location of the enzymatic activity determined by fractionation techniques. SGA was preferentially recovered in fractions which were enriched for the vacuolar hydrolases, carboxypeptidase Y and alpha-mannosidase.     

Controlled fed-batch fermentation of recombinant Saccharomyces cerevisiae to produce hepatitis B surface antigen

We have performed controlled fed-batch fermentation experiments to compare the production level of hepatitis B surface antigen (HBsAg) by recombinant yeast Saccharomyces cerevisiae strains (YNN27/pYBH-1, YNN27/ p2mu-S11, YNN27/pDCB-S2) containing plasmid vector with alcohol dehydrogenase (ADH1), acid phosphatase (PHO5), and glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, respectively. Yeast cell concentrations of 15-35 g dry cell weight/L were obtained. By limiting phosphorous concentration, HBsAg expression level for the YNN27/p2mu-S11 strain with inducible PHO5 promoter reached 0.2-0.3 mg/L. By controlling nutrient addition rate and dissolved oxygen concentration, HBsAg concentrations of 3-10 mg/L were achieved in 60-70 h fermentation using the YNN27/pDCB-S2 strain with the constitutive GPD promoter.     

Changes in regulation of ribosomal protein synthesis during vegetative growth and sporulation of Saccharomyces cerevisiae.

When diploid Saccharomyces cerevisiae cells logarithmically growing in acetate medium were placed in sporulation medium, the relative rates of synthesis of 40 or more individual ribosomal proteins (r-proteins) were coordinately depressed to approximately 20% of those of growing cells. These new depressed rates remained constant for at least 10 h into sporulation. If yeast nitrogen base was added 4 yh after the beginning of sporulation to shift the cells back to vegetative growth, the original relative rates of r-protein synthesis were rapidly reestablished. this upshift in the rates occurred even in diploids homozygous for the regulatory mutation rna2 at the restrictive temperature for this mutation (34 degrees C). However, once these mutant cells began to bud and grow at 34 degrees C, the phenotype of rna2 was expressed and the syntheses of r-proteins were again coordinately depressed. At least one protein whose rate of synthesis was not depressed by rna2 in vegetative cells did have a decreased rate of synthesis during sporulation. Another r-protein whose synthesis was depressed by rna2 maintained a high rate of synthesis at the beginning of sporulation. These data suggest that the mechanism responsible for coordinate control of r-protein synthesis during sporulation does not require the gene product of RNA2 and thus defines a separate mechanism by which r-proteins are coordinately controlled in S. cerevisiae.