The initiation of cell wall synthesis in parasynchronous cultures of Schizosaccharomyces pombe

Summary Schizosaccharomyces pombe has been grown in parasynchronous culture to study the synthesis of cell wall material. After a lag period of 2.5h following inoculation the cells began to grow, as measured by optical density, dry weight and cell size. The cell number remained constant until 4.5h after inoculation when approximately 70% of the population divided synchronously. Immunofluorescence studies of the growing cells have shown that new wall material is inserted at the cell apices from 2.5 h after inoculation; this result is supported by radio-isotope labelling data which indicated that synthesis of new cell wall material also commenced 2.5 h after inoculation. The incorporation experiments also demonstrated an interruption in cell wall synthesis during the cell separation stage. The composition of the cell wall material varied during the growth cycle, with maximum nitrogen levels at inoculation and following cell division. No serological differences could be detected in the cell walls during the growth cycle.     

Fluctuations during growth of the plasma membrane H(+)-ATPase activity of Saccharomyces cerevisiae and Schizosaccharomyces pombe

The plasma membrane H⁺-ATPase activity was determined under various growth conditions using the yeastsSaccharomyces cerevisiae andSchizosaccharomyces pombe. Under early batch-growth conditions in a rich medium, the budding yeastS. cerevisiae ATPase specific activity increased 2-to 3-fold during exponential growth. During late exponential growth, a peak of ATPase activity, followed by a sudden decrease, was observed and termed “growth-arrest control”. The growth arrest phenomenon ofS. cerevisiae could not be related to the acidification of the culture medium or to glucose exhaustion in the medium or to variation of glucose activation of the H⁺-ATPase. Addition of ammonium to a proline minimum medium also stimulated transiently the ATPase activity ofS. cerevisiae. Specific activity of the fission yeastS. pombe ATPase did not show a similar profile and steadily increased to reach a plateau in stationary growth. Under synchronous mitotic growth conditions, the ATPase activity ofS. cerevisiae increased during the cell division cycle according to the “peak” type cycle, while that ofS. pombe was of the “step” type.     

Stability and refractoriness of the high catalase activity in the oxidative-stress-resistant fission yeastSchizosaccharomyces pombe

Effect of oxygen and metabolic substrates (glucose, ethanol) on the catalase activity of anaerobically grownSchizosaccharomyces pombe cells was assessed, and compared with that ofSaccharomyces cerevisiae in order to determine the catalase activity regulation inS. pombe. In contrast toS. cerevisiae, the total catalase activity of permeabilizedS. pombe anaerobically grown cells is higher than that found in aerobically grown cells, is stable and constant under all circumstances (i.e. it is not induced by oxygen and/or substrates), and only a negligible part (3–5%) of it is contributed byde novo protein synthesis during aeration with or without substrates. The patent catalase activity of intact cells rises 2-fold during 6-h aeration without substrate and 7–8-fold in the presence of glucose or ethanol. The increase is not inhibited by cycloheximide and is thus not due tode novo catalase synthesis, but may reflect enhanced transport of catalase to the cell surface or a permeabilization of the plasma membrane during the aeration.     

On the cell wall composition ofSaccharomycopsis guttulata

Summary Cells ofSaccharomycopsis guttulata were ruptured by sonic oscillation and the resulting cell walls were purified by washing and centrifugation. The walls contained 43.7% carbohydrate (expressed as glucose), 39.6% protein and a trace of chitin. Paper chromatography of hydrolyzed cell walls showed that glucose and an unknown reducing compound make up the bulk of the carbohydrate fraction. Mannose and glucosamine were present in small amounts. The cell wall composition ofS. guttulata appears to differ considerably from that ofS. cerevisiae.     

Transglutaminase-like activity participates in cell wall biogenesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Transglutaminases (TGases) catalyze the cross-linking between protein molecules by formation of an amide bond between γ-carboxyamide group of glutamine and the ε-amine group of lysine under deamination of glutamine. We have demonstrated the participation of transglutaminase-like activity in the isolated cell walls and in the process of cell wall regeneration in protoplasts of the yeast Saccharomyces cerevisiae. A radioactive TGase substrate [³H]putrescine was incorporated into the isolated cell walls and into the TCA-insoluble fraction in regenerating protoplasts. The incorporation was increased by adding exogenous artificial substrate of TGase N,N’-dimethylcasein and was inhibited by TGase inhibitor cystamine and/or EDTA. These results suggest the existence of a TGase-type reaction involved in the formation of covalent cross-links between glycoprotein molecules during cell wall construction in S. cerevisiae.     

Localization of α-galactomannan and of wheat germ agglutinin receptors inSchizosaccharomyces pombe

The location of galactomannan on the surface ofSchizosaccharomyces pombe cells was reexamined by scanning electron microscopy by an indirect but specific method using gold markers. The polysaccharide was found on the cell surface and at the end beginning to grow but not on the wall established by division. Galactomannan was also localized onS. pombe thin sections by transmission electron microscopy using the same method. The polysaccharide was found deposited in two layers in the cell wall, i.e. at the periphery of the wall and near the plasmalemma. The septum was also marked but mainly near the plasmalemma. These results indicated that the polysaccharide is elaborated onto the outside of the wall during extension but not during septum formation. When thin sections ofS. pombe were marked with gold granules labeled with wheat germ agglutinin, marking was found in vacuoles but not in the cell wall. This confirmed thatS. pombe cell wall is devoid of chitin.Non-Standard Abbreviations Au gold colloid - RCA_I Ricinus communis lectin - SEM scanning electron microscopy - TEM transmission electron microscopy - WGA wheat germ agglutinin     

The effect of ethanol on cell wall antigens ofSaccharomyces cerevisiae and specific isolation of high ethanol producing strains of this yeast,making use of a serological technique

Generally, natural isolates of high ethanol producingSaccharomyces cerevisiae obtained by screening are used in alcoholic industries. The methods involved in their isolation and identification are elaborate. Antigenic analysis using antibodies raised against wholeSaccharomyces cells indicated species specificity of cell wall surface thermostable antigens. By affinity purification, the specific antibodies could be obtained and used for specific isolation ofS. cerevisiae. Antigenic studies using antibodies raised against isolated cell walls of fermentatively grownS. cerevisiae indicated the occurrence of thermolabile antigens common toSaccharomyces species. Higher concentrations of these antigens could be detected in thoseS. cerevisiae that had the ability for high ethanol production. The concentrations of these cell wall common antigens increased with increasing culture age and ethanol accumulation in culture broths. In younger yeast cells, the concentration could be increased by growing the cells in a medium containing added ethanol. Using dilutions of cross absorbed antibody specific for common antigens and Ouchterlony test, high ethanol producingS. cerevisiae could be identified.     

Autoradiographic study of hyphal cell wall synthesis of Saprolegnia

Incorporation of radioactive glucose in growing apices of Saprolegnia monoïca hyphae were examined by electron microscopic autoradiography. ³H glucose labelling indicates that dictyosomes and apical vesicles do not contain much polysaccharide and that glucan synthesis occurs at the cell surface. ¹⁴C glucose labelling shows that incorporation was chased from the cell walls during hyphal morphogenesis.     

Delays in prophase induced by adenosine 2-deoxyriboside and their relation with DNA synthesis

The rate of DNA synthesis in the course of the division cycle in root meristem ofAllium cepa growing under constant temperature and aeration conditions has been studied by means of treatment with AdR, as a specific inhibitor of the synthesis, as well as by the incorporation of tritiated thymidine. The one-hour treatment with AdR or tritiated thymidine was given at various hours in the course of the interphase of a synchronous population of binucleate cells induced by caffeine. In the case of AdR, sensitivity to the inhibition of DNA synthesis was studied by recording the delays produced by the treatment in the appearance of biprophases and bitelophases. The selection by the use of caffeine, of spontaneously synchronous populations of cells going through the telophase and becoming binucleate and the detection of the first biprophases in the subsequent mitosis provide a highly synchronized system with which to study the incorporation of tritiated thymidine during the interphase. The curves representing sensitivity to the inhibition of DNA synthesis by AdR and the rate of tritiated thymidine incorporation coincide, so that we can regard the delays, under our conditions, as proportional to the rate of DNA synthesis at the moment of the AdR treatment. This rate, in the S period, was found to be variable by both methods, being higher in the first and the last thirds of the S period (S₁ and S₃) and lower in the middle third (S₂).     

Reduction in the intracellular cAMP level triggers initiation of sexual development in fission yeast

Summary Schizosaccharomyces pombe initiates sexual development in response to nutritional starvation. The level of cAMP inS. pombe cells changed during the transition from exponential growth to stationary phase. It also changed in response to a shift from nitrogen-rich medium to nitrogen-free medium. A decrease of approximately 50% was observed in either case, suggesting thatS. pombe cells contain less cAMP when they initiate sexual development.S. pombe cells that expressed the catalytic domain ofSaccharomyces cerevisiae adenylyl cyclase from theS. pombe adh1 promoter contained 5 times as much cAMP as the wild type and could not initiate mating and meiosis. These observations, together with previous findings that exogenously added cAMP inhibits mating and meiosis and that cells with little cAMP are highly derepressed for sexual development, strongly suggest that cAMP functions as a key regulator of sexual development inS. pombe. Thepde1 gene, which encodes a protein homologous toS. cerevisiae cAMP phosphodiesterase I, was isolated as a multicopy suppressor of the sterility caused by a high cAMP level. Disruption ofpde1 madeS. pombe cells partially sterile and meiosis-deficient, indicating that this cAMP phosphodiesterase plays an important role in balancing the cAMP level in vivo.     

Mating reaction in yeast protoplasts

Protoplasts prepared from complementary haploid strains ofSaccharomyces cerevisiae were studied with regard to their ability of conjugating. Neither fresh protoplasts nor the growing protoplasts possessing fibrillar walls exhibited sex specific agglutination or fusion. However, they were capable of inducing sexual activation in normal cells of opposite mating type. After completing the regeneration of cell walls the protoplasts could conjugate either with each other or with cells of opposite sex. The frequency of conjugations was low, about 1%, and was largely dependent on the degree of completition of the wall during regeneration. From the results the following conclusions may be drawn: 1. The initiation of mating is dependent on the integrity of the cell wall. 2. The sex specific morphogenetic changes do not occur in wall-less protoplasts but may happen after the protoplasts have regenerated their cell walls. 3. The lysis of cell walls does not occur until the walls come into close contact. 4. The fusion of plasma membranes in sex-activated protoplasts cannot be induced by artefucial agglutination.     

On the cell wall composition of the apiculate yeasts

Summary Sonic oscillation was used for the purpose of obtaining clean, chemically intact cell walls. The rate of disruption was determined for cells ofHanseniaspora uvarum andSaccharomyces cerevisiae. The carbohydrate fractions of cell walls ofHanseniaspora uvarum, H. valbyensis, Kloeckera apiculata, Saccharomycodes ludwigii andSaccharmyces cerevisiae were shown to be similar. Chromatography of cell wall hydrolysates of all these species demonstrated that glucose and mannose were the only sugars present (in about equal amounts) besides traces of glucosamine. The cell walls ofH. uvarum contained 78.1 per cent carbohydrates, 7 per cent protein and approximately 0.05 per cent of chitin. Fractionation of the polysaccharides lead to a recovery of 83.3 per cent of the carbohydrates present (30.4 per cent glucan and 34.9 per cent mannan). Saccharomyces cerevisiae cell walls were found to have a carbohydrate content of 82.8 per cent, 6.5 per cent protein and a trace of chitin (0.04 per cent). Nadsonia elongata contained a relatively large amount of chitin (ca. 5 per cent) and lacked mannan in its cell walls. It was concluded thatHanseniaspora andSaccharomycodes are closely related to theSaccharomyceteae but they have little in common with species ofNadsonia.     

Characterization of glucose transport inSchizosaccharomyces pombe

Conclusions GHT1 was isolated as suppressor ofd-glucose uptake deficiency ofS. pombe mutant YGS-5. The correspondingS. pombe DNA encodes a putative protein with significant amino acid sequence identity to theS. cerevisiae HXT transporters. Heterologous expression ofGHT1 inS. cerevisiae hxt mutant RE700A (strain HLY709) enabled the mutant to grow ond-glucose as the sole carbon source. HLY709 cells take up hexoses with similar specificity toS. pombe wild strain and accumulate the non-metabolizable analogues of glucose (2DG and 6DG) intracellularly, thus matchingS. pombe wild strain. Southern blot analysis revealed the existence of other putative glucose transporters inS. pombe and the search for related transporter genes inS. pombe genome is in progress.     

Quality control in biosynthetic pathways of N-linked glycoproteins in the yeast endoplasmic reticulum

Summary The initial steps in N-glycosylation involve the synthesis of dolichol-linked Glc₃Man₉GlcNAc₂ oligosaccharides and the transfer of these oligosaccharides to nascent polypeptides. These processes take place at the membrane of the endoplasmic reticulum (ER) and are conserved among eukaryotes. Once transferred to the protein the N-linked oligosaccharides are immediately trimmed by glycosidases located in the ER. This review focuses on the N-linked glycosylation pathway in the ER ofSaccharomyces cerevisiae andSchizosaccharomyces pombe. In particular, we outline how yeast cells ensure that only completely assembled lipid-linked oligosaccharides are transferred to nascent polypeptides. We will discuss the oligosaccharide trimming of glycoproteins with respect to glycoprotein quality control and degradation, focusing on the two different quality control mechanisms ofS. cerevisiae andS. pombe.Abbreviations CPY carboxypeptidase Y - ER endoplasmic reticulum - LLO lipid-linked oligosaccharide - NLO protein-linked oligosaccharide - OTase oligosaccharyltransferase     

Changes in the rate of synthesis of wall polysaccharides during the cell cycle of yeast

Reevaluation and comparison of seemingly contradictory literature data on the mode of synthesis of wall polysaccharides during the cell cycle ofSaccharomyces cerevisiae explained the source of discrepancies and demonstrated their general consonance in the following points: 1. The rate of synthesis of glucan and mannan is not constant and does not increase continuously throughout the entire cell cycle. 2. The rate of synthesis of both polysaccharides is considerably reduced at the time of cell division and in the prebudding phase.     

Inhibition of Protein Translocation in Permeabilized Cells of Schizosaccharomyces pombe by Puromycin

To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe. The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-α-factor, was translocated across the endoplasmic reticulum (ER) of S. pombe posttranslationally, and glycosylated to the same extent as in the ER of S. cerevisiae. This suggested that the size of N-linked core-oligosaccharide in the ER of S. pombe is similar to that in S. cerevisiae. This translocation into the ER of S. pombe was inhibited by puromycin, but the translocation in the P-cells of S. cerevisiae was not inhibited. This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction. Our results suggested that the translocation machinery of S. pombe was sensitive to puromycin and different from that of S. cerevisiae.     

Zero order kinetics of cell wall turnover inStaphylococcus aureus

Cells exponentially grown from four strains ofS. aureus (SG 511, H, 52A5G, and248 PN-1) and uniformly labeled in their walls with³H-N-acetylglucosamine, were found to turn over their old walls at constant rates of up to 25% per generation. Wall turnover was not observed to follow first order kinetics, thus ruling out the implication that maintenance of normal wall thickness was achieved by a random distribution of new wall components in the old wall. Instead, wall turnover in all cases strictly followed zero order kinetics, indicating that newly synthesized wall material was placed layer by layer beneath the inner surface of the old cell wall. This finding correlates with evidence obtained from earlier electron microscopic investigations into the regeneration of the staphylococcal cell wall after chloramphenicol treatment. Based on the experimental data presented, a simplified model for wall turnover of the growing staphylococcal cell was proposed. The model also takes into account the finding, derived from additional experiments with strainSG 511, that the total cell wall turned over at a somewhat higher rate than the old portions of the wall. The rates of cell wall turnover found inS. aureus SG 511 are the highest reported to date for pathogenic bacteria. The medical implications of this finding were discussed.     

Changes in the lanthanum distribution following decapitation of the sporophytes of an aquatic fern,Marsilea drummondii A. Br.

Summary The active terminal bud and the quiescent lateral buds and corresponding nodes inserted at different levels on the main rhizome ofMarsilea drummondii were examined with the EM afterin vivo feeding with lanthanum nitrate. These tracer experiments demonstrate that all the buds are fed by their phloem cells. In the lateral bud axis the labelling of the sieve elements apoplast indicates that a solute transfer took place in the node between xylem and phloem via xylem transfer cells. La³⁺ deposits are completely absent from the apical dome of inhibited buds indicating that the walls of the quiescent meristematic cells are not permeated by the tracer. The removal of the terminal bud has two effects. It rapidly (in 2 hours) allows the lanthanum to penetrate the lateral bud tip walls at a stage when no fine structural changes are discernable and to bind to the outer surface of the plasmalemma as it does in the active terminal bud. This study including inhibited buds and buds released from apical dominance support the view that changes in the state of the cell surface (cell wall and plasma membrane) may be a prerequisite for the resumption growth activity.This study was supported in part by a grant from the Centre National de la Recherche Scientifique to L.Sossountzov (AI 031275).     

Cell wall and cytoskeleton reorganization as the response to hyperosmotic shock in Saccharomyces cerevisiae

Transfer of exponentially growing cells of the yeast Saccharomyces cerevisiae to hyperosmotic growth medium containing 0.7-1 M KCl, 1 M mannitol, and/or 1 M glycerol caused cessation of yeast growth for about 2 h; thereafter, growth resumed at almost the original rate. During this time, formation of fluorescent patches on the inner surface of cell walls stained with Primulin or Calcofluor white was observed. The fluorescent patches also formed in solutions of KCl or when synthesis of the cell wall was blocked with cycloheximide and/or 2-deoxyglucose. The patches gradually disappeared as the cells resumed growth, and the new buds had smooth cell walls. Electron microscopy of freeze-etched replicas of osmotically stressed cells revealed deep plasma membrane invaginations filled from the periplasmic side with an amorphous cell wall material that appeared to correspond to the fluorescent patches on the cell surface. The rate of incorporation of D-[U-14C]glucose from the growth medium into the individual cell wall polysaccharides during osmotic shock followed the growth kinetics. No differences in cell wall composition between osmotically stressed yeast and control cells were found. Hyperosmotic shock caused changes in cytoskeletal elements, as demonstrated by the disappearance of microtubules and actin microfilaments. After 2-3 h in hyperosmotic medium, both microtubules and microfilaments regenerated to their original polarized forms and the actin patches resumed their positions at the apices of growing buds. The response of S. cerevisiae strains with mutations in the osmosensing pathway genes hog1 and pbs2 to hyperosmotic shock was similar to that of the wild-type strain. We conclude that, besides causing a temporary disassembling of the cytoskeleton, hyperosmotic shock induces a change in the organization of the cell wall, apparently resulting from the displacement of periplasmic and cell wall matrix material into invaginations of the plasma membrane created by the plasmolysis.     

Isolation and characterization of prosthecae ofAsticcacaulis biprosthecum

The budding process of the yeast form of Mucor rouxii was examined by electron microscopy of thin sections with particular reference to wall ontogeny. In most instances the bud wall is seen as a continuation of the inner layers of the parent cell wall. As the bud emerges it ruptures the outer layers of the parent wall. The bud wall is much thinner than the parent wall and remains so while the bud grows into a sphere of about one half the diameter of the parent cell. Then a septum begins to form centripetally, at the neck, by invagination of the plasmalemma. Before the neck canal is completely occuluded, electron-dense wall material is deposited into the septum space. Two separate septum walls are deposited, one on the parent side and one on the bud side of the invaginating plasmalemma. Septum wall formation extends to the surrounding neck walls. In this manner, the parent and bud cytoplasms become fully separated and each is surrounded by a continuous wall. The two cells remain attached to each other by the original neck wall; eventually, the bud abscisses leaving a birth scar on the bud cell and a more pronounced bud scar on the parent cell. In general, the mechanism of budding in this zygomycetous fungus resembles that of an ordinary ascomycetous yeast such as Saccharomyces cerevisiae.