Synthesis of 1,3-β-Glucanases in Saccharomyces cerevisiae During the Mitotic Cycle, Mating, and Sporulation

Upon fractionating Saccharomyces cerevisiae asynchronous cultures by sucrose density gradient centrifugation in a zonal rotor and examining the exo-1,3-beta-glucanase and deoxyribonucleic acid content of the cells, a periodic step increase in the activity of this enzyme was observed, indicating a discontinuous pattern of synthesis or activation of exo-1,3-beta-glucanase during the mitotic cycle at the transition from the S to the G(2) phase. Similar results were obtained for endo-1,3-beta-glucanase by assaying activity against oxidized laminarin in permeabilized cells, suggesting that the synthesis of endo-1,3-beta-glucanase is controlled in the same way. When a and alpha strains were mated, the specific activity of cell extracts against laminarin, oxidized laminarin, and pustulan remained constant while zygote formation was taking place. However, when growth resumed, active synthesis of 1,3-beta-glucanases took place as shown by the occurrence of a significant increase in the specific activity against the three substrates. Specific changes in the level of glucan degradative enzymes, not observed in a haploid parental strain, occurred when the diploid S. cerevisiae AP-1 was induced to sporulate. The sporulation process triggered the activation of first the pustulan degradative capacity and then the capacity to hydrolyze oxidized laminarin. The specific activity against this substrate was 10 times higher than that against pustulan.     

The Saccharomyces cerevisiae SPR1 gene encodes a sporulation-specific exo-1,3-beta-glucanase which contributes to ascospore thermoresistance.

A number of genes have been shown to be transcribed specifically during sporulation in Saccharomyces cerevisiae, yet their developmental function is unknown. The SPR1 gene is transcribed during only the late stages of sporulation. We have sequenced the SPR1 gene and found that it has extensive DNA and protein sequence homology to the S. cerevisiae EXG1 gene which encodes an exo-1,3-beta-glucanase expressed during vegetative growth (C. R. Vasquez de Aldana, J. Correa, P. San Segundo, A. Bueno, A. R. Nebrada, E. Mendez, and F. del Ray, Gene 97:173-182, 1991). We show that spr1 mutant cells do not hydrolyze p-nitrophenyl-beta-D-glucoside or laminarin in a whole-cell assay for exo-1,3-beta-glucanases. In addition to the absence of this enzymatic activity, spr1 mutant spores exhibit reduced thermoresistance relative to isogenic wild-type spores. These observations are consistent with the notion that SPR1 encodes a sporulation-specific exo-1,3-beta-glucanase.     

Nature and timing of some sporulation-specific protein changes in Saccharomyces cerevisiae.

During meiosis and spore formulation in Saccharomyces cerevisiae, changes that occur in a/alpha diploids, but not in isogenic nonsporulating a/a diploids, have been detected in cellular polypeptides. These were found by the technique of prelabeling growing cells with 35SO4(2-) and suspending them in sulfur-free sporulation medium. Under the conditions used, about 400 polypeptides were detected by two-dimensional gel electrophoresis, and 45 were altered during sporulation; of these, 21 changes were specific to a/alpha strains. These alterations were mainly due to the appearance of new polypeptides or to marked increases in the concentrations of a few polypeptides produced during vegetative growth. They could have been due either to modifications of existing polypeptides present in growing cells or to de novo synthesis of new gene products. They occurred at characteristic times during sporulation; whereas the majority of changes took place early (within the first 6 h in sporulation conditions), there were several changes characterizing the later stages of sporulation. Ten of the 35SO4(2-)-labeled polypeptides were also labeled with 32P in the presence of [32P]orthophosphate; of these, three were previously found to be sporulation specific. One of these was phosphorylated at all stages of sporulation and was labeled when [32P]orthophosphate was added either during growth of the culture of 1 h after transfer to sporulation medium. Another was labeled in the same way by adding 32P at either time, so that by 7 h in sporulation medium it was phosphorylated, but was dephosphorylated by 24 h. The third sporulation-specific peptide was labeled in extracts prepared at 7 h in sporulation medium (but not at 24 h) when [32P]-orthophosphate was added during presporulation growth, but not when [32P]-orthophosphate was added 1 h after transfer of the culture to sporulation medium. This polypeptide appeared early during sporulation; it is probably phosphorylated as it appears and is dephosphorylated at some time between 7 h and 24 h of sporulation.     

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.     

Developmental regulation of a sporulation-specific enzyme activity in Saccharomyces cerevisiae.

An alpha-glucosidase activity (SAG) occurs in a/alpha Saccharomyces cerevisiae cells beginning at about 8 to 10 h after the initiation of sporulation. This enzyme is responsible for the rapid degradation of intracellular glycogen which follows the completion of meiosis in these cells. SAG differs from similar activities present in vegetative cells and appears to be a sporulation-specific enzyme. Cells arrested at various stages in sporulation (DNA replication, recombination, meiosis I, and meiosis II) were examined for SAG activity; the results show that SAG appearance depends on DNA synthesis and some recombination events but not on the meiotic divisions.     

Macromolecule Synthesis and Breakdown in Relation to Sporulation and Meiosis in Yeast

The time course of synthesis and breakdown of various macromolecules has been compared for sporulating (a/alpha) and nonsporulating (a/a and alpha/alpha) yeast cells transferred to potassium acetate sporulation medium. Both types of cells incorporate label into ribonucleic acid and protein. The gel electrophoresis patterns of proteins synthesized in sporulation medium are identical for sporulating and nonsporulating diploids; both are different from electropherograms of vegetative cells. Sporulating and nonsporulating strains differ with respect to deoxyribonucleic acid synthesis; no deoxyribonucleic acid is synthesized in the latter case, whereas the deoxyribonucleic acid complement is doubled in the former. Glycogen breakdown occurs only in sporulating strains. Breakdown of preexisting vegetative ribonucleic acid and protein molecules occurs much more extensively in sporulating than in nonsporulating cells. A timetable of these data is presented.     

Involvement of mitochondrial protein synthesis in sporulation: effects of erythromycin on macromolecular synthesis, meiosis, and ascospore formation in Saccharomyces cerevisiae.

Cells of strain Z270 (MAT alpha/MAT alpha) of Saccharomyces cerevisiae did not undergo ascospore formation in buffered or unbuffered acetate sporulation medium in the presence of erythromycin. The drug inhibited sporulation when added within the first 6 to 8 h and affected to different extents some of the metabolic and sporulation-specific events that normally occur during this period. In sporulation medium, protein synthesis was highly sensitive to erythromycin, whereas RNA synthesis was unaffected and premeiotic DNA synthesis was partially inhibited. Intragenic recombination occurred at normal rates for the various heteroallelic loci tested, but rates of intergenic recombination were markedly reduced, and commitment to haploidization did not occur; hence, development was evidently arrested between intragenic and intergenic recombination. Cells kept for 8 h in acetate sporulation medium that were ready for sporulation in water without erythromycin failed to sporulate in water containing the drug, indicating that erythromycin can inhibit sporulation independent of acetate utilization.     

Relationship of Glycolytic Intermediates, Glycolytic Enzymes, and Ammonia to Glycogen Metabolism During Sporulation in the Yeast Saccharomyces cerevisiae

To identify the factors which control glycogen synthesis in Saccharomyces cerevisiae, we have studied the regulation of glycogen metabolism during sporulation, since in vivo glycogen has been reported to undergo significant changes in concentration during this process. We examined the concentration of a number of key glycolytic intermediates and enzymes in strains that sporulate at different rates and those that are deficient in sporulation. There were no significant changes found in the adenylate energy charge or cyclic AMP levels throughout sporulation. Although significant alterations occurred in the levels of glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, and ATP during sporulation, only the fourfold increase in fructose-1,6-bisphosphate appeared to correlate with glycogen synthesis in all of the strains examined. Only limited changes occurred in the level of a number of glycolytic and gluconeogenic enzymes which were examined during this process. Intracellular glucose content underwent a dramatic 30- to 40-fold increase in sporulating cells. Comparison of strains with different rates of sporulation demonstrated that this increase in glucose content coincides with the time of glycogen degradation in each strain. Both the increase in glucose content and the degradation of accumulated glycogen were not observed in nonsporulating alpha/alpha strains, or in cells incubated in NH(4) (+) supplemented sporulation medium. Although glucose appears to be the direct product of glycogen degradation, a 10-fold increase in a nonspecific alkaline phosphatase occurs at this time, which may be degrading phosphorylated sugars to glucose. All of the strains examined released extracellular glucose while suspended in acetate sporulation medium. It is concluded that most of the changes in the glycolytic pathway that occur during sporulation, with the exception of glycogen degradation and the concomitant increase in intracellular glucose pools, are a response to the transfer to sporulation medium and are independent of sporulation-specific processes. Inhibition of sporulation with ammonium ions resulted in a different pattern of change in all of the glycolytic intermediates examined, including a twofold increase in cyclic AMP levels. Ammonia did not interfere with glycogen synthesis, but prevented sporulation-specific glycogen degradation. The levels of the glycolytic enzymes examined were not affected by ammonia.     

Lipid Synthesis During Sporulation of Saccharomyces cerevisiae

Lipid synthesis was studied in both sporulating (diploid) and nonsporulating (haploid) cells of Saccharomyces cerevisiae. Two phases of lipid synthesis occur in diploid cells transferred to sporulation medium. Phase I, which occurs during the first 12 h of exposure to sporulation medium, was also observed in the haploid strains. Phase II, occurring from the 20th to the 25th h, coincided with the appearance of mature asci and was observed only in the diploid cells. The majority of phospholipid synthesis took place during period I, whereas neutral lipid synthesis occurred during both periods. Phospholipid synthesis was virtually identical in both type and quantity in the sporulating and nonsporulating strains.     

Variation of (1 leads to 3)-beta-glucanases in Saccharomyces cerevisiae during vegetative growth, conjugation, and sporulation.

The total (1 leads to 3)-beta-glucanase activities associated with cell extracts and cell walls of Saccharomyces cerevisiae were measured during vegetative growth, conjugation, and sporulation. Using a system of column chromatography, we resolved (1 leads to 3)-beta-glucanase activity into six different enzymes (namely, glucanases I, II, IIIA, IIIB, IV, and V). The contributions of the individual enzymes to the total activity at the different stages of the life cycle were determined. Total glucanase activity increased during exponential growth and decreased in stationary resting-phase cells. Glucanase IIIA was the predominant enzyme in stationary resting-phase cells. Glucanases I, II, IIIB, and IV were either absent or present at low levels in stationary phase cells, but their individual activities (in particular, glucanase IIIB activity) increased substantially during exponential growth. Total (1 leads to 3)-beta-glucanase activity did not change significantly during conjugation of two haploid mating strains, S. cerevisiae 2180A and 2180B, and no notable changes were detected in the activities of the individual enzymes. Sporulation was accompanied by a rapid increase and then a decrease in total glucanase activity. Most of the increase was due to a dramatic rise in the activity of glucanase V, which appeared to be a sporulation-specific enzyme. Glucanase activity was not derepressed by lowering the glucose concentration in the growth medium.     

Regulation of gene expression during meiosis in Saccharomyces cerevisiae: SPR3 is controlled by both ABFI and a new sporulation control element.

The SPR3 gene encodes a sporulation-specific homolog of the yeast Cdc3/10/11/12 family of bud neck filament proteins. It is expressed specifically during meiosis and sporulation in Saccharomyces cerevisiae. Analysis of the sporulation-specific regulation of SPR3 has shown that it is strongly activated under sporulating conditions but shows low levels of expression under nonsporulating conditions. A palindromic sequence located near the TATA box is essential to the developmental regulation of this gene and is the only element directly activating SPR3 at the right time during sporulation. Within the palindrome is a 9-bp sequence, gNCRCAAA(A/T) (midsporulation element [MSE]), found in the known control regions of three other sporulation genes. A previously identified ABFI element is also needed for activation. The MSE has been shown to activate a heterologous promoter (CYC1) in a sporulation-specific manner. Related sequences, including an association of MSE and ABFI elements, have been found upstream of other genes activated during the middle stage of S. cerevisiae sporulation. One group of these may be involved in spore coat formation or maturation.     

Structure of beta-glucan oligomer from laminarin and its effect on human monocytes to inhibit the proliferation of U937 cells

We analyzed the human monocyte-stimulating ability of laminarin from Eisenia bicyclis, lichenan from Cetraria islandica, and their oligomers depolymerized with endo-1,3-beta-glucanase from Arthrobacter sp. The respective beta-glucan oligomers with different degrees of polymerization (DP) were fractionated from hydrolytic products of laminarin and lichenan using gel-filtration chromatography. The monocyte-conditioned medium pre-cultured in the presence of a fraction of beta-glucan oligomer (DP>/=8) from laminarin exhibited inhibitory activity against the proliferation of human myeloid leukemia U937 cells, while those pre-cultured with other beta-glucan oligomers and the original laminarin and lichenan showed little or no activity. NMR analysis indicated that the beta-glucan oligomer (DP>/=8) has an average DP value of 13, and its ratio of beta-1,3- to beta-1,6-linkages in glucopyranose units was estimated to be 1.3:1. These results indicate that the beta-1,3-glucan oligomer with a higher content of beta-1,6-linkage stimulates monocytes to inhibit the proliferation of U937 cells.     

Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell-wall biogenesis as determined by a novel fluorescent assay

BGTs [β-(1,3)-glucanosyltransglycosylases; EC 2.4.1.-] of the GH72 (family 72 of glycosylhydrolases) are GPI (glycosylphosphatidylinositol)-anchored proteins that play an important role in the biogenesis of fungal cell walls. They randomly cleave glycosidic linkages in β-(1,3)-glucan chains and ligate the polysaccharide portions containing newly formed reducing ends to C(3)(OH) at non-reducing ends of other β-(1,3)-glucan molecules. We have developed a sensitive fluorescence-based method for the assay of transglycosylating activity of GH72 enzymes. In the new assay, laminarin [β-(1,3)-glucan] is used as the glucanosyl donor and LamOS (laminarioligosaccharides) fluorescently labelled with SR (sulforhodamine) serve as the acceptors. The new fluorescent assay was employed for partial biochemical characterization of the heterologously expressed Gas family proteins from the yeast Saccharomyces cerevisiae. All the Gas enzymes specifically used laminarin as the glucanosyl donor and a SR-LamOS of DP (degree of polymerization) ≥5 as the acceptors. Gas proteins expressed in distinct stages of the yeast life cycle showed differences in their pH optima. Gas1p and Gas5p, which are expressed during vegetative growth, had the highest activity at pH 4.5 and 3.5 respectively, whereas the sporulation-specific Gas2p and Gas4p were most active between pH 5 and 6. The novel fluorescent assay provides a suitable tool for the screening of potential glucanosyltransferases or their inhibitors.     

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.