A novel role of Dma1 in regulating forespore membrane assembly and sporulation in fission yeast

In fission yeast Schizosaccharomyces pombe, a diploid mother cell differentiates into an ascus containing four haploid ascospores following meiotic nuclear divisions, through a process called sporulation. Several meiosis-specific proteins of fission yeast have been identified to play essential roles in meiotic progression and sporulation. We report here an unexpected function of mitotic spindle checkpoint protein Dma1 in proper spore formation. Consistent with its function in sporulation, expression of dma1(+) is up-regulated during meiosis I and II. We showed that Dma1 localizes to the SPB during meiosis and the maintenance of this localization at meiosis II depends on septation initiation network (SIN) scaffold proteins Sid4 and Cdc11. Cells lacking Dma1 display defects associated with sporulation but not nuclear division, leading frequently to formation of asci with fewer spores. Our genetic analyses support the notion that Dma1 functions in parallel with the meiosis-specific Sid2-related protein kinase Slk1/Mug27 and the SIN signaling during sporulation, possibly through regulating proper forespore membrane assembly. Our studies therefore revealed a novel function of Dma1 in regulating sporulation in fission yeast.     

Changes in the adenylate energy charge and the induction of sporulation in Saccharomyces diastaticus

The induction of sporulation in yeast is generally accompanied by a sharp increase in energy metabolism which is evidenced by a rise of the adenylate energy charge by that time. The energy charge can be held at a low level by limitation of the phosphate supply in the growth medium. Ascus formation remains unaffected by this treatment. This suggests that the rise in ATP production normally encountered during early sporulation is not essential for the initiation of sporulation.     

Natural isolates of Saccharomyces cerevisiae display complex genetic variation in sporulation efficiency

Sporulation is a well-studied process executed with varying efficiency by diverse yeast strains. We developed a high-throughput method to quantify yeast sporulation efficiency and used this technique to analyze a line cross between a high-efficiency oak tree isolate and a low-efficiency wine strain. We find that natural variation in sporulation efficiency mirrors natural variation in higher eukaryotes: it shows divergence between isolated populations, arises from loci of major effect, and exhibits epistasis. We show that the lower sporulation efficiency of the wine strain results from a failure to initiate sporulation, rather than from slower kinetics of meiosis and spore formation. The two strains differentially regulate many genes involved in aerobic respiration, an essential pathway for sporulation, such that the oak tree strain appears better poised to generate energy from this pathway. We also report that a polymorphism in RME1 that affects sporulation efficiency in laboratory strains also cosegregates with significant phenotypic differences in our cross of natural isolates. These results lay the groundwork for the study of variation in sporulation efficiency among natural isolates of yeast.     

Peptidyl-tRNA hydrolase in Bacillus subtilis,encoded by spoVC,is essential to vegetative growth,whereas the homologous enzyme in Saccharomyces cerevisiae is dispensable

Peptidyl-tRNA hydrolase in Escherichia coli, encoded by pth, is essential for recycling tRNA molecules sequestered as peptidyl-tRNA as a result of pre-mature dissociation from the ribosome during translation. Genes homologous to pth are present in other bacteria, yeast and man, but not in archaea. The homologous gene in Bacillus subtilis, spoVC, was first identified as a gene involved in sporulation. A second copy of spoVC, under the control of the xyl promoter, was integrated into B. subtilis at the amy locus. In this background, interruption of the original gene was possible provided that expression of the copy at the amy locus was induced. When spoVC was interrupted, both vegetative growth and sporulation were dependent on xylose, showing that SpoVC is essential. The role of SpoVC in sporulation is discussed and appears to be consistent with previous hypotheses that a relaxation of translational accuracy may occur during sporulation. The homologous gene in Saccharomyces cerevisiae, yHR189W, has been interrupted in both haploid and diploid strains. The mutant haploid strains remain viable, as do the yHR189W mutant spores obtained by tetrad dis-section, with either glucose or glycerol as carbon source, showing that the yHR189W gene product is dispensable for cell growth and for mitochondrial respiration.     

FACTORS CONTROLLING THE SPORULATION OF YEASTS. I. THE PRESPORULATION PHASE

SUMMARY: Yeasts tend to dissociate into mixtures of cell types with different powers of sporulation; hence single cell isolates are recommended for sporulation studies. The ability of yeasts to produce 4-spored asci can be improved by single cell selection. Cells from actively fermenting cultures sporulate much better than those grown under aerobic conditions. Sporulating ability depends on fermentation 'age', reaching a maximum when 85–90% of the CO₂ has been evolved. Carbon dioxide assimilation in the presporulation phase appears essential for maximal sporulation, but complete anaerobiosis in this phase is detrimental to sporulating ability. Malt wort cultures of a baker's yeast have given remarkably constant figures, in successive tests, for sporulation; but some batches of wort have an adverse effect on sporulating ability. The same yeast, grown on Lodder-Rij's synthetic medium containing 4 or 8% (w/v) of glucose, is capable of 80% sporulation (proportion of cells forming asci) on sodium acetate agar, comparable to that obtainable with malt wort cultures. Sporulation is depressed by excess storage of fat, while storage of glycogen does not affect sporulating ability.     

Manipulation of Major Membrane Lipid Synthesis and Its Effects on Sporulation in Saccharomyces cerevisiae

During the sporulation process of Saccharomyces cerevisiae, meiotic progression is accompanied by de novo formation of the prospore membrane inside the cell. However, it remains to be determined whether certain species of lipids are required for spore formation in yeast. In this study, we analyzed the requirement of the synthesis of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and ergosterol for spore formation using strains in which the synthesis of these lipids can be controlled. When synthesis of PE and PC was repressed, sporulation efficiency decreased. This suggests that synthesis of these phospholipids is vital to proper sporulation. In addition, sporulation was also impaired in cells with a lowered sterol content, raising the possibility that sterol content is also important for spore formation.     

Expression, stability, and replacement of glucan-remodeling enzymes during developmental transitions in Saccharomyces cerevisiae

Sporulation is a developmental variation of the yeast life cycle whereby four spores are produced within a diploid cell, with proliferation resuming after germination. The GAS family of glycosylphosphatidylinositol-anchored glucan-remodeling enzymes exemplifies functional interplay between paralogous genes during the yeast life cycle. GAS1 and GAS5 are expressed in vegetative cells and repressed during sporulation while GAS2 and GAS4 exhibit a reciprocal pattern. GAS3 is weakly expressed in all the conditions and encodes an inactive protein. Although Gas1p functions in cell wall formation, we show that it persists during sporulation but is relocalized from the plasma membrane to the epiplasm in a process requiring End3p-mediated endocytosis and the Sps1 protein kinase of the p21-activated kinase family. Some Gas1p is also newly synthesized and localized to the spore membrane, but this fraction is dispensable for spore formation. By way of contrast, the Gas2-Gas4 proteins, which are essential for spore wall assembly, are rapidly degraded after spore formation. On germination, Gas1p is actively synthesized and concentrated in the growing part of the spore, which is essential for its elongation. Thus Gas1p is the primary glucan-remodeling enzyme required in vegetative growth and during reentry into the proliferative state. The dynamic interplay among Gas proteins is crucial to couple glucan remodeling with morphogenesis in developmental transitions.     

Snc1p v-SNARE transport to the prospore membrane during yeast sporulation is dependent on endosomal retrieval pathways

Vesicular traffic is essential for sporulation in Saccharomyces cerevisiae. The Golgi-associated retrograde protein (GARP) tethering complex is required for retrograde traffic from both the early and late endosomes to the Golgi. Analyses of GARP complex mutants in sporulation reveal defects in meiotic progression and spore formation. In contrast, inactivation of the retromer complex, which mediates vesicle budding and cargo selection from the late endosome, or Snx4p, which is involved in retrieval of proteins from the early endosome, has little effect on sporulation. A retromer GARP double mutant is defective in the formation of the prospore membrane (PSM) that surrounds the haploid nuclei. In the retromer GARP double mutant, PSM precursor vesicles carrying the cargo, Dtr1p, are transported to the spindle pole body (SPB), where PSM formation is initiated. However, the v-SNARE Snc1p is not transported to the SPB in the double mutant, suggesting that the defect in PSM formation is because of the failure to retrieve Snc1p, and perhaps other proteins, from the endosomal pathway. Taken together, these results indicate that retrograde trafficking from the endosome is essential for sporulation by retrieving molecules important for PSM and spore wall formation.     

Essential functions of protein tyrosine phosphatases PTP2 and PTP3 and RIM11 tyrosine phosphorylation in Saccharomyces cerevisiae meiosis and sporulation

Tyrosine phosphorylation plays a central role in eukaryotic signal transduction. In yeast, MAP kinase pathways are regulated by tyrosine phosphorylation, and it has been speculated that other biochemical processes may also be regulated by tyrosine phosphorylation. Previous genetic and biochemical studies demonstrate that protein tyrosine phosphatases (PTPases) negatively regulate yeast MAP kinases. Here we report that deletion of PTP2 and PTP3 results in a sporulation defect, suggesting that tyrosine phosphorylation is involved in regulation of meiosis and sporulation. Deletion of PTP2 and PTP3 blocks cells at an early stage of sporulation before premeiotic DNA synthesis and induction of meiotic-specific genes. We observed that tyrosine phosphorylation of several proteins, including 52-, 43-, and 42-kDa proteins, was changed in ptp2Deltaptp3Delta homozygous deletion cells under sporulation conditions. The 42-kDa tyrosine-phosphorylated protein was identified as Mck1, which is a member of the GSK3 family of protein kinases and previously known to be phosphorylated on tyrosine. Mutation of MCK1 decreases sporulation efficiency, whereas mutation of RIM11, another GSK3 member, specifically abolishes sporulation; therefore, we investigated regulation of Rim11 by Tyr phosphorylation during sporulation. We demonstrated that Rim11 is phosphorylated on Tyr-199, and the Tyr phosphorylation is essential for its in vivo function, although Rim11 appears not to be directly regulated by Ptp2 and Ptp3. Biochemical characterizations indicate that tyrosine phosphorylation of Rim11 is essential for the activity of Rim11 to phosphorylate substrates. Our data demonstrate important roles of protein tyrosine phosphorylation in meiosis and sporulation     

Roles of phosphoinositides and of Spo14p (phospholipase D)-generated phosphatidic acid during yeast sporulation

During yeast sporulation, internal membrane synthesis ensures that each haploid nucleus is packaged into a spore. Prospore membrane formation requires Spo14p, a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-stimulated phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. We found that both meiosis and spore formation also require the phosphatidylinositol (PtdIns)/PtdCho transport protein Sec14p. Specific ablation of the PtdIns transport activity of Sec14p was sufficient to impair spore formation but not meiosis. Overexpression of Pik1p, a PtdIns 4-kinase, suppressed the sec14-1 meiosis and spore formation defects; conversely, pik1-ts diploids failed to undergo meiosis and spore formation. The PtdIns(4)P 5-kinase, Mss4p, also is essential for spore formation. Use of phosphoinositide-specific GFP-PH domain reporters confirmed that PtdIns(4,5)P2 is enriched in prospore membranes. sec14, pik1, and mss4 mutants displayed decreased Spo14p PLD activity, whereas absence of Spo14p did not affect phosphoinositide levels in vivo, suggesting that formation of PtdIns(4,5)P2 is important for Spo14p activity. Spo14p-generated PtdOH appears to have an essential role in sporulation, because treatment of cells with 1-butanol, which supports Spo14p-catalyzed PtdCho breakdown but leads to production of Cho and Ptd-butanol, blocks spore formation at concentrations where the inert isomer, 2-butanol, has little effect. Thus, rather than a role for PtdOH in stimulating PtdIns(4,5)P2 formation, our findings indicate that during sporulation, Spo14p-mediated PtdOH production functions downstream of Sec14p-, Pik1p-, and Mss4p-dependent PtdIns(4,5)P2 synthesis.     

Application of a simple yeast extract from spent brewer's yeast for growth and sporulation of Bacillus thuringiensis subsp. kurstaki: a Physiological Study

The suitability of using a simple brewer's yeast extract (BYE), prepared by autolysis of complete beer slurry, for growth and sporulation of Bacillus thuringiensis kurstaki was studied in baffled shake flasks. In a standard buffered medium with 2.5% (w/v) glucose and 1% (w/v) brewer's yeast extract, growth of B. t. kurstaki resulted in a low biomass production with considerable byproduct formation, including organic acids and a concomitant low medium pH, incomplete glucose utilization and marginal sporulation, whereas growth in the same medium with a commercial laboratory-grade yeast extract (Difco) resulted in a high biomass concentration, complete glucose utilization, relatively low levels of byproducts and complete sporulation (2.6 × 10⁹ spores/ml). When glucose was left out of the medium, however, growth parameters and sporulation were comparable for BYE and commercial yeast extract, but absolute biomass levels and spore counts were low. Iron was subsequently identified as a limiting factor in BYE. After addition of 3 mg iron sulphate/l, biomass formation in BYE-medium more than doubled, low byproduct formation was observed, and complete sporulation occurred (2.8 × 10⁹spores/ml). These data were slightly lower than those obtained in media with commercial yeast extract (3.6 × 10⁹spores/ml), which also benefited, but to a smaller extent, from addition of iron.     

Manipulation of major membrane lipid synthesis and its effects on sporulation in Saccharomyces cerevisiae

During the sporulation process of Saccharomyces cerevisiae, meiotic progression is accompanied by de novo formation of the prospore membrane inside the cell. However, it remains to be determined whether certain species of lipids are required for spore formation in yeast. In this study, we analyzed the requirement of the synthesis of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and ergosterol for spore formation using strains in which the synthesis of these lipids can be controlled. When synthesis of PE and PC was repressed, sporulation efficiency decreased. This suggests that synthesis of these phospholipids is vital to proper sporulation. In addition, sporulation was also impaired in cells with a lowered sterol content, raising the possibility that sterol content is also important for spore formation.     

The Arf-GTPase-activating protein Gcs1p is essential for sporulation and regulates the phospholipase D Spo14p

SPO14, encoding the major Saccharomyces cerevisiae phospholipase D (PLD), is essential for sporulation and mediates synthesis of the new membrane that encompasses the haploid nuclei that arise through meiotic divisions. PLD catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid (PA) and choline. PA stimulates Arf-GTPase-activating proteins (Arf-GAPs), which are involved in membrane trafficking events and actin cytoskeletal function. To determine if Spo14p-generated PA mediates its biological response through Arf-GAPs, we analyzed the sporulation efficiencies of cells deleted for each of the five known and potential yeast Arf-GAPs. Only gcs1delta mutants display a sporulation defect similar to that of spo14 mutants: cells deleted for GCS1 initiate the sporulation program but are defective in synthesis of the prospore membrane. Endosome-to-vacuole transport is also impaired in gcs1delta cells during sporulation. Furthermore, Arf-GAP catalytic activity, but not the pleckstrin homology domain, is required for both prospore membrane formation and endosome-to-vacuole trafficking. An examination of Gcs1p-green fluorescent protein revealed that it is a soluble protein. Interestingly, cells deleted for GCS1 have reduced levels of Spo14p-generated PA. Taken together, these results indicate that GCS1 is essential for sporulation and suggest that GCS1 positively regulates SPO14.     

Positive control of sporulation-specific genes by the IME1 and IME2 products in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, meiosis and spore formation require the induction of sporulation-specific genes. Two genes are thought to activate the sporulation program: IME1 and IME2 (inducer of meiosis). Both genes are induced upon entry into meiosis, and IME1 is required for IME2 expression. We report here that IME1 is essential for expression of four sporulation-specific genes. In contrast, IME2 is not absolutely essential for expression of the sporulation-specific genes, but contributes to their rapid induction. Expression of IME2 from a heterologous promoter permits the expression of these sporulation-specific genes, meiotic recombination, and spore formation in the absence of IME1. We propose that the IME1 and IME2 products can each activate sporulation-specific genes independently. In addition, the IME1 product stimulates sporulation-specific gene expression indirectly through activation of IME2 expression.     

SPORULATION OF THE YEAST, HANSENULA SATURNUS: I. EFFECT OF MAGNESIUM AND CALCIUM IONS

Sporulation in a strain of the wild yeast, Hansenula saturnus,was investigated. The yeast was found to form spores even indistilled water. The sporulation rate (percentage of ascus-bearingindividuals) in this case was found to be markedly affectedby the cell concentration adopted in the test. The addition of inorganic nutrients to the sporulation mediumstimulates sporulation. The yeast requires either magnesiumor calcium for growth and sporulation. Higher concentrationsof these ions are required for sporulation than for growth.In both cases magnesium is effective at more dilute concentrationsthan calcium. Under the conditions of the experiments, in which the yeastforms a pellicle, the sporulation rate in the pellicle far exceedsthat in the sediment. The effects of environmental factors on the sporulation wasconsidered in relation to growth. It was found that, under theconditions of poor growth in the sporulation culture, no exogenousmagnesium and calcium are required for sporulation. In suchcases, the yeast cells are inferred to have an endogenous stockof magnesium and calcium enough for the sporulation. ¹ Present address: Laboratory of Microbiology, Department ofAgriculture, Tôhoku University, Sendai. (Received May 4, 1961; )     

Improvement of sporulation in the yeastYarrowia lipolytica

Strains ofYarrowia lipolytica forming exclusively spherical ascospores were developed through inbreeding. These strains are more suitable for micromanipulation than other inbred strains forming helm-shaped ascospores. External factors affecting sporulation frequency and tetrad formation in this yeast were investigated. Optimal formation of complete tetrads occurred at a narrow range of pH values around 6.0. Citrate was found to stimulate sporulation strongly. A synthetic medium containing citrate was developed to obtain standard conditions for maximum sporulation.     

Effect of light on sexual flocculation in Schizosaccharomyces japonicus

A fission yeast, Schizosaccharomyces japonicus can sporulateabundantly under conditions of vegetative growth. Prior to sporulation,strong flocculation was observed. When the culture medium containeda sufficient amount of yeast extract, sporulation was completelyinhibited. When the medium was cultured under light, flocculationoccurred, but not zygote formation. Neither flocculation norzygote formation was observed in the culture under completedarkness. The effect of light occurred even with short-periodillumination at the early to mid logarithmic growth phase. Thepresence of a definite amount of yeast extract was essentialfor the phenomenon in question. (Received July 20, 1976; )     

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.     

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.