End3p-mediated endocytosis is required for spore wall formation in Saccharomyces cerevisiae

During sporulation in Saccharomyces cerevisiae, vesicles transported to the vicinity of spindle pole bodies are fused to each other to generate bilayered prospore membranes (PSMs). PSMs encapsulate the haploid nuclei that arise from the meiotic divisions and serve as platforms for spore wall deposition. Membrane trafficking plays an important role in supplying vesicles for these processes. The endocytosis-deficient mutant, end3Delta, sporulated poorly and the spores produced lost resistance to ether vapor, suggesting that END3-mediated endocytosis is important for sporulation. End3p-GFP localized to cell and spore peripheries in vegetative and sporulating cells and colocalized with actin structures. Correspondingly, the actin cytoskeleton appeared aberrant during sporulation in end3Delta. Analysis of meiosis in end3Delta mutants revealed that the meiotic divisions occurred with wild-type kinetics. Furthermore, PSMs were assembled normally. However, the levels of proteins required for spore wall synthesis and components of the spore wall layers at spores were reduced, indicating that end3Delta mutants are defective in spore wall synthesis. Thus, END3-mediated endocytosis is important for spore wall formation. Additionally, cytological analyses suggest that trafficking between the plasma membrane and PSMs is important earlier during sporulation.     

Ascospore formation in the yeast Saccharomyces cerevisiae.

Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.     

Identification of yeast meiosis-specific genes by differential display

Meiosis, a specialized cell division process, occurs in all sexually reproducing organisms. During this process a diploid cell undergoes a single round of DNA replication followed by two rounds of nuclear division to produce four haploid gametes. In yeast, the meiotic products are packaged into four spores that are enclosed in a sac known as an ascus. To enhance our understanding of the meiotic developmental pathway and spore formation, we followed differential expression of genes in meiotic versus vegetatively growing cells in the yeast Saccharomyces cerevisiae. Such comparative analyses have identified five different classes of genes that are expressed at different stages of the sporulation program. We identified several meiosis-specific genes including some already known to be induced during meiosis. Here we describe one of these previously uncharacterized genes, SSP1, which plays an essential role in meiosis and spore formation. SSP1 is induced midway through meiosis, and the homozygous mutant-diploid cells fail to sporulate. In ssp1 cells, meiosis is delayed, nuclei fragment after meiosis II, and viability declines rapidly. The ssp1 defect is not related to a microtubule-cytoskeletal-dependent event and is independent of two rounds of meiotic divisions. Our results suggest that Ssp1 is likely to function in a pathway that controls meiotic nuclear divisions and coordinates meiosis and spore formation. Functional analysis of other uncharacterized genes is underway.     

Ady3p links spindle pole body function to spore wall synthesis in Saccharomyces cerevisiae

Spore formation in Saccharomyces cerevisiae requires the de novo synthesis of prospore membranes and spore walls. Ady3p has been identified as an interaction partner for Mpc70p/Spo21p, a meiosis-specific component of the outer plaque of the spindle pole body (SPB) that is required for prospore membrane formation, and for Don1p, which forms a ring-like structure at the leading edge of the prospore membrane during meiosis II. ADY3 expression has been shown to be induced in midsporulation. We report here that Ady3p interacts with additional components of the outer and central plaques of the SPB in the two-hybrid assay. Cells that lack ADY3 display a decrease in sporulation efficiency, and most ady3Delta/ady3Delta asci that do form contain fewer than four spores. The sporulation defect in ady3Delta/ady3Delta cells is due to a failure to synthesize spore wall polymers. Ady3p forms ring-like structures around meiosis II spindles that colocalize with those formed by Don1p, and Don1p rings are absent during meiosis II in ady3Delta/ady3Delta cells. In mpc70Delta/mpc70Delta cells, Ady3p remains associated with SPBs during meiosis II. Our results suggest that Ady3p mediates assembly of the Don1p-containing structure at the leading edge of the prospore membrane via interaction with components of the SPB and that this structure is involved in spore wall formation.     

Stage-Specific Effects of X-Irradiation on Yeast Meiosis

Previous work has shown that cdc13 causes meiotic arrest of Saccharomyces cerevisiae following DNA replication by a RAD9-dependent mechanism. In the present work, we have further investigated the implicit effects of chromosomal lesions on progression through meiosis by exposing yeast cells to X-irradiation at various times during sporulation. We find that exposure of RAD9 cells to X-irradiation early in meiosis prevents sporulation, arresting the cells at a stage prior to premeiotic DNA replication. rad9 meiotic cells are much less responsive to X-irradiation damage, completing sporulation after treatment with doses sufficient to cause arrest of RAD9 strains. These findings thereby reveal a RAD9-dependent checkpoint function in meiosis that is distinct from the G(2) arrest previously shown to result from cdc13 dysfunction. Analysis of the spores that continued to be produced by either RAD9 or rad9 cultures that were X-irradiated in later stages of sporulation revealed most spores to be viable, even after exposure to radiation doses sufficient to kill most vegetative cells. This finding demonstrates that the lesions induced by X-irradiation at later times fail to trigger the checkpoint function revealed by cdc13 arrest and suggests that the lesions may be subject to repair by serving as intermediates in the recombination process. Strains mutant for chromosomal synapsis and recombination, and therefore defective in meiotic disjunction, were tested for evidence that X-ray-induced lesions might alleviate inviability by promoting recombination. Enhancement of spore viability when spo11 (but not hop1) diploids were X-irradiated during meiosis indicates that induced lesions may partially substitute for SPO11-dependent functions that are required for the initiation of recombination.     

Importance of mitochondrial dynamics during meiosis and sporulation

Opposing fission and fusion events maintain the yeast mitochondrial network. Six proteins regulate these membrane dynamics during mitotic growth-Dnm1p, Mdv1p, and Fis1p mediate fission; Fzo1p, Mgm1p, and Ugo1p mediate fusion. Previous studies established that mitochondria fragment and rejoin at distinct stages during meiosis and sporulation, suggesting that mitochondrial fission and fusion are required during this process. Here we report that strains defective for mitochondrial fission alone, or both fission and fusion, complete meiosis and sporulation. However, visualization of mitochondria in sporulating cultures reveals morphological defects associated with the loss of fusion and/or fission proteins. Specifically, mitochondria collapse to one side of the cell and fail to fragment during presporulation. In addition, mitochondria are not inherited equally by newly formed spores, and mitochondrial DNA nucleoid segregation defects give rise to spores lacking nucleoids. This nucleoid inheritance defect is correlated with an increase in petite spore colonies. Unexpectedly, mitochondria fragment in mature tetrads lacking fission proteins. The latter finding suggests either that novel fission machinery operates during sporulation or that mechanical forces generate the mitochondrial fragments observed in mature spores. These results provide evidence of fitness defects caused by fission mutations and reveal new phenotypes associated with fission and fusion mutations.     

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.     

A Gip1p-Glc7p phosphatase complex regulates septin organization and spore wall formation.

Sporulation of Saccharomyces cerevisiae is a developmental process in which a single cell is converted into four haploid spores. GIP1, encoding a developmentally regulated protein phosphatase 1 interacting protein, is required for spore formation. Here we show that GIP1 and the protein phosphatase 1 encoded by GLC7 play essential roles in spore development. The gip1Delta mutant undergoes meiosis and prospore membrane formation normally, but is specifically defective in spore wall synthesis. We demonstrate that in wild-type cells, distinct layers of the spore wall are deposited in a specific temporal order, and that gip1Delta cells display a discrete arrest at the onset of spore wall deposition. Localization studies revealed that Gip1p and Glc7p colocalize with the septins in structures underlying the growing prospore membranes. Interestingly, in the gip1Delta mutant, not only is Glc7p localization altered, but septins are also delocalized. Similar phenotypes were observed in a glc7-136 mutant, which expresses a Glc7p defective in interacting with Gip1p. These results indicate that a Gip1p-Glc7p phosphatase complex is required for proper septin organization and initiation of spore wall formation during sporulation.     

Dynamic organization of the actin cytoskeleton during meiosis and spore formation in budding yeast

During sporulation in Saccharomyces cerevisiae, the four daughter cells (spores) are formed inside the boundaries of the mother cell. Here, we investigated the dynamics of spore assembly and the actin cytoskeleton during this process, as well as the requirements for filamentous actin during the different steps of spore formation. We found no evidence for a polarized actin cytoskeleton during sporulation. Instead, a highly dynamic network of non-polarized actin cables is present underneath the plasma membrane of the mother cell. We found that a fraction of prospore membrane (PSM) precursors are transported along the actin cables. The velocity of PSM precursors is diminished if Myo2p or Tpm1/2p function is impaired. Filamentous actin is not essential for meiotic progression, for shaping of the PSMs or for post-meiotic cytokinesis. However, actin is essential for spore wall formation. This requires the function of the Arp2/3p complex and involves large carbohydrate-rich compartments, which may be chitosome analogous structures.     

Differential requirement for phospholipase D/Spo14 and its novel interactor Sma1 for regulation of exocytotic vesicle fusion in yeast meiosis

During sporulation and meiosis of budding yeast a developmental program determines the formation of the new plasma membranes of the spores. This process of prospore membrane (PSM) formation leads to the formation of meiotic daughter cells, the spores, within the lumen of the mother cell. It is initiated at the spindle pole bodies during meiosis II. Spore formation, but not meiotic cell cycle progression, requires the function of phospholipase D (PLD/Spo14). Here we show that PLD/Spo14 forms a complex with Sma1, a meiotically expressed protein essential for spore formation. Detailed analysis revealed that both proteins are required for early steps of prospore membrane assembly but with distinct defects in the respective mutants. In the Deltaspo14 mutant the initiation of PSM formation is blocked and aggregated vesicles of homogenous size are detected at the spindle pole bodies. In contrast, initiation of PSM formation does occur in the Deltasma1 mutant, but the enlargement of the membrane is impaired. During PSM growth both Spo14 and Sma1 localize to the membrane, and localization of Spo14 is independent of Sma1. Biochemical analysis revealed that Sma1 is not necessary for PLD activity per se and that PLD present in a complex with Sma1 is highly active. Together, our results suggest that yeast PLD is involved in two distinct but essential steps during the regulated vesicle fusion necessary for the assembly of the membranous encapsulations of the spores.     

Differential regulation of Saccharomyces cerevisiae phospholipase D in sporulation and Sec14-independent secretion

Saccharomyces cerevisiae Spo14, a phosphatidylcholine-specific, phosphatidylinositol (4,5) bisphosphate-activated phospholipase D (PLD), is essential for meiosis and spore formation. Spo14 is also required for secretion in the absence of the phosphatidylinositol/phosphatidylcholine transfer protein Sec14 (i.e., Sec14-independent secretion). In sporulating cells Spo14 is phosphorylated and relocalized within the cell. In contrast, Spo14 does not relocalize and is not phosphorylated in Sec14-independent secretion. Analysis of a partially phosphatidylinositol (4,5) bisphosphate-activated Spo14 mutant, spo14(R894G), revealed that Spo14 function in Sec14-independent secretion, unlike the situation in meiosis, requires fully stimulated PLD activity. Consistent with the differential regulation of Spo14 function during sporulation and secretion, we isolated a mutant allele, spo14-S251P, the product of which is improperly phosphorylated and fails to relocalize and rescue the sporulation phenotype of homozygous spo14 diploids, but supports Sec14-independent secretion. Furthermore, we show that the N-terminal domain of Spo14 is both phosphorylated and sufficient for prospore membrane localization during sporulation. These data indicate that Spo14 phosphorylation and relocalization are essential for the process of sporulation, but dispensable for Sec14-independent secretion. Finally, we demonstrate that Spo14 phosphorylation and relocalization are initiated by nitrogen and glucose limitation and occur independently of the process of meiosis.     

Prospore membrane formation: how budding yeast gets shaped in meiosis

During meiosis in Saccharomyces cerevisiae four daughter cells, called spores, are generated within the boundaries of the mother cell. This cell differentiation process requires de novo synthesis of prospore membranes (PSMs), which are the precursors of the spore plasma membranes. Assembly of these membranes is initiated at the spindle pole bodies (SPBs) during meiosis II. At this stage of the cell cycle, 4 SPBs are present. Two different meiosis-specific structures are known to be required for PSM formation. At the SPBs, specialized attachments, called the meiotic plaques, provide the required functionality necessary for the recruitment and assembly of the membranes. During subsequent membrane elongation, a second structure becomes important. This proteinaceous assembly forms a coat, called the leading edge protein coat (LEP coat), which covers the boundaries of the membranes. Assembly of the coat occurs at sites next to the SPBs, whereas its disassembly is concomitant to the closure of the membranes. This mini review discusses our current understanding of how the meiotic plaque and the LEP coat might function during biogenesis of the prospore membrane.     

SPO71 Mediates Prospore Membrane Size and Maturation in Saccharomyces cerevisiae

The mechanisms that control the size and shape of membranes are not well understood, despite the importance of these structures in determining organelle and cell morphology. The prospore membrane, a double lipid bilayer that is synthesized de novo during sporulation in S. cerevisiae, grows to surround the four meiotic products. This membrane determines the shape of the newly formed spores and serves as the template for spore wall deposition. Ultimately, the inner leaflet of the prospore membrane will become the new plasma membrane of the cell upon germination. Here we show that Spo71, a pleckstrin homology domain protein whose expression is induced during sporulation, is critical for the appropriate growth of the prospore membrane. Without SPO71, prospore membranes surround the nuclei but are abnormally small, and spore wall deposition is disrupted. Sporulating spo71Δ cells have prospore membranes that properly localize components to their growing leading edges yet cannot properly localize septin structures. We also found that SPO71 genetically interacts with SPO1, a gene with homology to the phospholipase B gene that has been previously implicated in determining the shape of the prospore membrane. Together, these results show that SPO71 plays a critical role in prospore membrane development.     

Polyamine biosynthesis during germination of yeast ascospores.

The role of the diamine putrescine during germination and outgrowth of ascospores of Saccharomyces cerevisiae was examined. Ornithine decarboxylase activity increased and declined rapidly during germination and outgrowth; peak activity was attained after the cells had proceeded through the G1 interval of the cell cycle, whereas minimal activity was present at the completion of the first cell division. alpha-Methylornithine inhibited both ornithine decarboxylase activity and the in vivo accumulation of putrescine. In the presence of alpha-methylornithireak dormancy and proceed through one cell division. Subsequent cellular growth, however, was retarded but not completely inhibited. The supplementation of Methylglyoxal bis(guanylhydrazone) to sporulation medium greatly inhibited this sexual process. These data suggest that the synthesis of putrescine is not required for the breaking of spore dormancy, but that polyamine biosynthesis may be essential for meiosis and sporulation.     

RSC Nucleosome-remodeling complex plays prominent roles in transcriptional regulation throughout budding yeast gametogenesis

RSC is a nucleosome-remodeling complex of Saccharomyces cerevisiae essential for growth that can alter histone-DNA interaction by using the energy of ATP hydrolysis. Nps1p/Sth1p is an ATPase subunit of RSC. A mutation in the conserved ATPase domain of Nps1p causes a sporulation defect with decreased expression of early meiotic genes, especially IME2. This defect is partially suppressed by the overexpression of either IME1 or IME2. A homozygous diploid of a novel temperature-sensitive nps1 mutation, nps1-13, harboring amino acid substitutions within the bromodomain, was unable to sporulate. Overexpression of IME, IME2, or both of these genes allowed the completion of meiosis I and meiosis II in nps1-13 but not the formation of mature asci. In nps1-13 carrying YEpIME1, the expression of a group of sporulation-specific genes, which express at the middle stages of sporulation and are required for spore-wall formation, notably diminished, and several late sporulation genes expressed at the early stages of sporulation. These results suggest that Nps1p/RSC plays important roles during the spore development process by controlling gene expression for initiating both meiosis and spore morphogenesis, and ensures proper expression timing of late meiotic genes.     

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.     

<Emphasis Type="Italic">SSP2</Emphasis>, a sporulation-specific gene necessary for outer spore wall assembly in the yeast<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

Sporulation in yeast consists of two highly coordinated processes. First, a diploid cell that is heterozygous at the mating-type locus undergoes meiosis, in which one round of DNA replication is followed by two rounds of nuclear division. Second, the meiotic products are packaged into spore cells that remain within the mother cell. A large number of genes are induced specifically during sporulation, and their products carry out different sporulation-specific events. Expression of these sporulation-specific genes is controlled by several regulators which function at different stages of the sporulation program, resulting in a cascade of gene expression following induction of meiosis. Here we describe one sporulation-specific gene, SSP2, which is induced midway through meiosis. Ssp2 shows significant homology to the predicted product of a hypothetical ORF in Candida albicans. Homozygous mutant ssp2 diploid cells fail to sporulate. In the mutant background, meiotic recombination and nuclear divisions remain normal; however, viability declines rapidly. Following meiosis, ssp2 cells form the prospore membrane, but fail to form the outer layer of the spore wall. The Ssp2 protein localizes to the spore wall after meiosis II. In addition, the ssp2 defect is also associated with delayed and reduced expression of late sporulation-specific genes. Our results suggest that SSP2 function is required after meiosis II and during spore wall formation.     

A role for MMS4 in the processing of recombination intermediates during meiosis in Saccharomyces cerevisiae.

The MMS4 gene of Saccharomyces cerevisiae was originally identified due to its sensitivity to MMS in vegetative cells. Subsequent studies have confirmed a role for MMS4 in DNA metabolism of vegetative cells. In addition, mms4 diploids were observed to sporulate poorly. This work demonstrates that the mms4 sporulation defect is due to triggering of the meiotic recombination checkpoint. Genetic, physical, and cytological analyses suggest that MMS4 functions after the single end invasion step of meiotic recombination. In spo13 diploids, red1, but not mek1, is epistatic to mms4 for sporulation and spore viability, suggesting that MMS4 may be required only when homologs are capable of undergoing synapsis. MMS4 and MUS81 are in the same epistasis group for spore viability, consistent with biochemical data that show that the two proteins function in a complex. In contrast, MMS4 functions independently of MSH5 in the production of viable spores. We propose that MMS4 is required for the processing of specific recombination intermediates during meiosis.     

Subcellular differentiation in sporulating yeast cells

We have previously described the induction of two sets of sporulation-specific mRNAs in Saccharomyces cerevisiae. Herein we correlate the appearance of these RNAs with the major morphogenic events of sporulation, and we analyze the spatial distribution of the RNAs within the ascus. Several observations suggest that the first set of messages is involved in spore wall synthesis. In fractionation experiments, these mRNAs are detected in the ascal cytoplasm but not in developing spores, indicating that the proteinaceous component of the spore wall is synthesized from the external compartment. The second set of messages is induced later in the course of spore maturation. These mRNAs accumulate within the spores and, unlike the first set of mRNAs, are retained in mature asci until the early stages of germination. We conclude that the development of ascospores proceeds through the differentiation of functionally distinct subcellular compartments.