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SUMMARYIn ascomycete fungi, sexual spores, termed ascospores, are formed after meiosis. Ascospore formation is an unusual cell division in which daughter cells are created within the cytoplasm of the mother cell by de novo generation of membranes that encapsulate each of the haploid chromosome sets created by meiosis. This review describes the molecular events underlying the creation, expansion, and closure of these membranes in the budding yeast, Saccharomyces cerevisiae . Recent advances in our understanding of the regulation of gene expression and the dynamic behavior of different membrane-bound organelles during this process are detailed. While less is known about ascospore formation in other systems, comparison to the distantly related fission yeast suggests that the molecular events will be broadly similar throughout the ascomycetes., Competing Interests: The author declares no conflict of interest.

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71-Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation., Competing Interests: The authors have declared that no competing interests exist.

Dolichols are isoprenoid lipids of varying length that act as sugar carriers in glycosylation reactions in the endoplasmic reticulum. In Saccharomyces cerevisiae , there are two cis -prenyltransferases that synthesize polyprenol-an essential precursor to dolichol. These enzymes are heterodimers composed of Nus1 and either Rer2 or Srt1. Rer2-Nus1 and Srt1-Nus1 can both generate dolichol in vegetative cells, but srt1 ∆ cells grow normally while rer2 ∆ grows very slowly, indicating that Rer2-Nus1 is the primary enzyme used in mitotically dividing cells. In contrast, SRT1 performs an important function in sporulating cells, where the haploid genomes created by meiosis are packaged into spores. The spore wall is a multilaminar structure and SRT1 is required for the generation of the outer chitosan and dityrosine layers of the spore wall. Srt1 specifically localizes to lipid droplets associated with spore walls, and, during sporulation there is an SRT1 -dependent increase in long-chain polyprenols and dolichols in these lipid droplets. Synthesis of chitin by Chs3, the chitin synthase responsible for chitosan layer formation, is dependent on the cis -prenyltransferase activity of Srt1, indicating that polyprenols are necessary to coordinate assembly of the spore wall layers. This work shows that a developmentally regulated cis -prenyltransferase can produce polyprenols that function in cellular processes besides protein glycosylation., (Copyright © 2017 by the Genetics Society of America.)

In response to nutrient starvation, diploid cells of the budding yeast Saccharomyces cerevisiae differentiate into a dormant form of haploid cell termed a spore. The dityrosine layer forms the outermost layer of the wall of S. cerevisiae spores and endows them with resistance to environmental stresses. ll-Bisformyl dityrosine is the main constituent of the dityrosine layer, but the mechanism of its assembly remains elusive. Here, we found that ll-bisformyl dityrosine, but not ll-dityrosine, stably associated in vitro with dit1 Δ spores, which lack the dityrosine layer. No other soluble cytosolic materials were required for this incorporation. In several aspects, the dityrosine incorporated in trans resembled the dityrosine layer. For example, dityrosine incorporation obscured access of the dye calcofluor white to the underlying chitosan layer, and ll-bisformyl dityrosine molecules bound to dit1 Δ spores were partly isomerized to the dl-form. Mutational analyses revealed several spore wall components required for this binding. One was the chitosan layer located immediately below the dityrosine layer in the spore wall. However, ll-bisformyl dityrosine did not stably bind to chitosan particles, indicating that chitosan is not sufficient for this association. Several lines of evidence demonstrated that spore-resident proteins are involved in the incorporation, including the Lds proteins, which are localized to lipid droplets attached to the developing spore wall. In conclusion, our results provide insight into the mechanism of dityrosine layer formation, and the in vitro assay described here may be used to investigate additional mechanisms in spore wall assembly., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

In response to starvation, diploid cells of Saccharomyces cerevisiae undergo meiosis and form haploid spores, a process collectively referred to as sporulation. The differentiation into spores requires extensive changes in gene expression. The transcriptional activator Ndt80 is a central regulator of this process, which controls many genes essential for sporulation. Ndt80 induces ∼300 genes coordinately during meiotic prophase, but different mRNAs within the NDT80 regulon are translated at different times during sporulation. The protein kinase Ime2 and RNA binding protein Rim4 are general regulators of meiotic translational delay, but how differential timing of individual transcripts is achieved was not known. This report describes the characterization of two related NDT80 -induced genes, PES4 and MIP6 , encoding predicted RNA binding proteins. These genes are necessary to regulate the steady-state expression, translational timing, and localization of a set of mRNAs that are transcribed by NDT80 but not translated until the end of meiosis II. Mutations in the predicted RNA binding domains within PES4 alter the stability of target mRNAs. PES4 and MIP6 affect only a small portion of the NDT80 regulon, indicating that they act as modulators of the general Ime2/Rim4 pathway for specific transcripts., (Copyright © 2017 American Society for Microbiology.)

During the budding yeast life cycle, a starved diploid cell undergoes meiosis followed by production of four haploid spores, each surrounded by a spore wall. The wall allows the spores to survive in harsh environments until conditions improve. Spores are also more resistant than vegetative cells to treatments such as ether vapor, glucanases, heat shock, high salt concentrations, and exposure to high or low pH, but the relevance of these treatments to natural environmental stresses remains unclear. This protocol describes a method for assaying the yeast spore wall under natural environmental conditions by quantifying the survival of yeast spores that have passed through the digestive system of a yeast predator, the fruit fly., (© 2016 Cold Spring Harbor Laboratory Press.)

Yeast sporulation is a highly regulated developmental program by which diploid cells generate haploid gametes, termed spores. To better define the genetic pathways regulating sporulation, a systematic screen of the set of ~3300 nonessential Schizosaccharomyces pombe gene deletion mutants was performed to identify genes required for spore formation. A high-throughput genetic method was used to introduce each mutant into an h(90) background, and iodine staining was used to identify sporulation-defective mutants. The screen identified 34 genes whose deletion reduces sporulation, including 15 that are defective in forespore membrane morphogenesis. In S. pombe, the total number of sporulation-defective mutants is a significantly smaller fraction of coding genes than in S. cerevisiae, which reflects the different evolutionary histories and biology of the two yeasts., (Copyright © 2014 Ucisik-Akkaya et al.)

During ascospore formation in Saccharomyces cerevisiae, the secretory pathway is reorganized to create new intracellular compartments, termed prospore membranes. Prospore membranes engulf the nuclei produced by the meiotic divisions, giving rise to individual spores. The shape and growth of prospore membranes are constrained by cytoskeletal structures, such as septin proteins, that associate with the membranes. Green fluorescent protein (GFP) fusions to various proteins that associate with septins at the bud neck during vegetative growth as well as to proteins encoded by genes that are transcriptionally induced during sporulation were examined for their cellular localization during prospore membrane growth. We report localizations for over 100 different GFP fusions, including over 30 proteins localized to the prospore membrane compartment. In particular, the screen identified IRC10 as a new component of the leading-edge protein complex (LEP), a ring structure localized to the lip of the prospore membrane. Localization of Irc10 to the leading edge is dependent on SSP1, but not ADY3. Loss of IRC10 caused no obvious phenotype, but an ady3 irc10 mutant was completely defective in sporulation and displayed prospore membrane morphologies similar to those of an ssp1 strain. These results reveal the architecture of the LEP and provide insight into the evolution of this membrane-organizing complex.

The creation of haploid gametes in yeast, termed spores, requires the de novo formation of membranes within the cytoplasm. These membranes, called prospore membranes, enclose the daughter nuclei generated by meiosis. Proper growth and closure of prospore membranes require the highly conserved Vps13 protein. Mutation of SPO71, a meiosis-specific gene first identified as defective in spore formation, was found to display defects in membrane morphogenesis very similar to those seen in vps13Δ cells. Specifically, prospore membranes are smaller than in the wild type, they fail to close, and membrane vesicles are present within the prospore membrane lumen. As in vps13Δ cells, the levels of phophatidylinositol-4-phosphate are reduced in the prospore membranes of spo71Δ cells. SPO71 is required for the translocation of Vps13 from the endosome to the prospore membrane, and ectopic expression of SPO71 in vegetative cells results in mislocalization of Vps13. Finally, the two proteins can be coprecipitated from sporulating cells. We propose that Spo71 is a sporulation-specific partner for Vps13 and that they act in concert to regulate prospore membrane morphogenesis.

The spore wall of Saccharomyces cerevisiae is a multilaminar extracellular structure that is formed de novo in the course of sporulation. The outer layers of the spore wall provide spores with resistance to a wide variety of environmental stresses. The major components of the outer spore wall are the polysaccharide chitosan and a polymer formed from the di-amino acid dityrosine. Though the synthesis and export pathways for dityrosine have been described, genes directly involved in dityrosine polymerization and incorporation into the spore wall have not been identified. A synthetic gene array approach to identify new genes involved in outer spore wall synthesis revealed an interconnected network influencing dityrosine assembly. This network is highly redundant both for genes of different activities that compensate for the loss of each other and for related genes of overlapping activity. Several of the genes in this network have paralogs in the yeast genome and deletion of entire paralog sets is sufficient to severely reduce dityrosine fluorescence. Solid-state NMR analysis of partially purified outer spore walls identifies a novel component in spore walls from wild type that is absent in some of the paralog set mutants. Localization of gene products identified in the screen reveals an unexpected role for lipid droplets in outer spore wall formation., Competing Interests: The authors have declared that no competing interests exist.

The hereditary disorders chorea acanthocytosis and Cohen syndrome are caused by mutations in different members of a family of genes that are orthologs of yeast VPS13. In vegetatively growing yeast, VPS13 is involved in the delivery of proteins to the vacuole. During sporulation, VPS13 is important for formation of the prospore membrane that encapsulates the daughter nuclei to give rise to spores. We report that VPS13 is required for multiple aspects of prospore membrane morphogenesis. VPS13 (1) promotes expansion of the prospore membrane through regulation of phosphatidylinositol phosphates, which in turn activate the phospholipase D, Spo14; (2) is required for a late step in cytokinesis that gives rise to spores; and (3) regulates a membrane-bending activity that generates intralumenal vesicles. These results demonstrate that Vps13 plays a broader role in membrane biology than previously known, which could have important implications for the functions of VPS13 orthologs in humans.

In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae.

Spore formation in Saccharomyces cerevisiae is driven by de novo assembly of new membranes termed prospore membranes. A vesicle-docking complex called the meiosis II outer plaque (MOP) forms on the cytoplasmic faces of the spindle-pole bodies at the onset of meiosis II and serves as the initiation site for membrane formation. In this study, a fluorescence-recovery assay was used to demonstrate that the dynamics of the MOP proteins change coincident with the coalescence of precursor vesicles into a membrane. Proteins within the MOP exchange freely with a soluble pool prior to membrane assembly, but after membranes are formed they remain stably within the MOP. By contrast, constitutive spindle-pole-body proteins display low exchange in both conditions. The MOP component Ady4p plays a role in maintaining the integrity of the MOP complex, but this role differs depending on whether the MOP is associated with docked vesicles or a fully formed membrane. These results suggest an architectural rearrangement of the MOP coincident with vesicle fusion.

The ascospores of Saccharomyces cerevisiae are surrounded by a complex wall that protects the spores from environmental stresses. The outermost layer of the spore wall is composed of a polymer that contains the cross-linked amino acid dityrosine. This dityrosine layer is important for stress resistance of the spore. This work reports that the dityrosine layer acts as a barrier blocking the diffusion of soluble proteins out of the spore wall into the cytoplasm of the ascus. Diffusion of a fluorescent protein out of the spore wall was used as an assay to screen for mutants affecting spore wall permeability. One of the genes identified in this screen, OSW3 (RRT12/YCR045c), encodes a subtilisin-family protease localized to the spore wall. Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer. These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore. OSW3 and other OSW genes identified in this screen are strong candidates to encode enzymes involved in assembly of this protective dityrosine coat.

Sporulation of Saccharomyces cerevisiae is a developmental process in which four haploid spores are generated inside a diploid cell. Gip1, a sporulation-specific targeting subunit of protein phosphatase type 1, together with its catalytic subunit, Glc7, colocalizes with septins along the extending prospore membrane and is required for septin organization and spore wall formation. However, the mechanism by which Gip1-Glc7 phosphatase promotes these events is unclear. We show here that Ysw1, a sporulation-specific coiled-coil protein, has a functional relationship to Gip1-Glc7 phosphatase. Overexpression of YSW1 partially suppresses the sporulation defect of a temperature-sensitive allele of gip1. Ysw1 interacts with Gip1 in a two-hybrid assay, and this interaction is required for suppression. Ysw1 tagged with green fluorescent protein colocalizes with septins and Gip1 along the extending prospore membrane during spore formation. Sporulation is partially defective in ysw1Delta mutant, and cytological analysis revealed that septin structures are perturbed and prospore membrane extension is aberrant in ysw1Delta cells. These results suggest that Ysw1 functions with the Gip1-Glc7 phosphatase to promote proper septin organization and prospore membrane formation.

Ascospore formation in yeast is accomplished through a cell division in which daughter nuclei are engulfed by newly formed plasma membranes, termed prospore membranes. Closure of the prospore membrane must be coordinated with the end of meiosis II to ensure proper cell division. AMA1 encodes a meiosis-specific activator of the anaphase promoting complex (APC). The activity of APC(Ama1) is inhibited before meiosis II, but the substrates specifically targeted for degradation by Ama1 at the end of meiosis are unknown. We show here that ama1Delta mutants are defective in prospore membrane closure. Ssp1, a protein found at the leading edge of the prospore membrane, is stabilized in ama1Delta mutants. Inactivation of a conditional form of Ssp1 can partially rescue the sporulation defect of the ama1Delta mutant, indicating that an essential function of Ama1 is to lead to the removal of Ssp1. Depletion of Cdc15 causes a defect in meiotic exit. We find that prospore membrane closure is also defective in Cdc15 and that this defect can be overcome by expression of a form of Ama1 in which multiple consensus cyclin-dependent kinase phosphorylation sites have been mutated. These results demonstrate that APC(Ama1) functions to coordinate the exit from meiosis II with cytokinesis.

In nature, yeasts are subject to predation by flies of the genus Drosophila. In response to nutritional starvation Saccharomyces cerevisiae differentiates into a dormant cell type, termed a spore, which is resistant to many types of environmental stress. The stress resistance of the spore is due primarily to a spore wall that is more elaborate than the vegetative cell wall. We report here that S. cerevisiae spores survive passage through the gut of Drosophila melanogaster. Constituents of the spore wall that distinguish it from the vegetative cell wall are necessary for this resistance. Ascospores of the distantly related yeast Schizosaccharomyces pombe also display resistance to digestion by D. melanogaster. These results suggest that the primary function of the yeast ascospore is as a cell type specialized for dispersion by insect vectors.

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

In Saccharomyces cerevisiae exit from mitosis requires the Cdc14 phosphatase to reverse CDK-mediated phosphorylation. Cdc14 is released from the nucleolus by the Cdc14 early anaphase release (FEAR) and mitotic exit network (MEN) pathways. In meiosis, the FEAR pathway is essential for exit from anaphase I. The MEN component Cdc15 is required for the formation of mature spores. To analyze the role of Cdc15 during sporulation, a conditional mutant in which CDC15 expression was controlled by the CLB2 promoter was used. Cdc15-depleted cells proceeded normally through the meiotic divisions but were unable to properly disassemble meiosis II spindles. The morphology of the prospore membrane was aberrant and failed to capture the nuclear lobes. Cdc15 was not required for Cdc14 release from the nucleoli, but it was essential to maintain Cdc14 released and for its nucleo-cytoplasmic transport. However, cells carrying a CDC14 allele with defects in nuclear export (Cdc14-DeltaNES) were able to disassemble the spindle and to complete spore formation, suggesting that the Cdc14 nuclear export defect was not the cause of the phenotypes observed in cdc15 mutants.

The GAS multigene family of Saccharomyces cerevisiae is composed of five paralogs (GAS1 to GAS5). GAS1 is the only one of these genes that has been characterized to date. It encodes a glycosylphosphatidylinositol-anchored protein functioning as a beta(1,3)-glucan elongase and required for proper cell wall assembly during vegetative growth. In this study, we characterize the roles of the GAS2 and GAS4 genes. These genes are expressed exclusively during sporulation. Their mRNA levels showed a peak at 7 h from induction of sporulation and then decreased. Gas2 and Gas4 proteins were detected and reached maximum levels between 8 and 10 h from induction of sporulation, a time roughly coincident with spore wall assembly. The double null gas2 gas4 diploid mutant showed a severe reduction in the efficiency of sporulation, an increased permeability of the spores to exogenous substances, and production of inviable spores, whereas the single gas2 and gas4 null diploids were similar to the parental strain. An analysis of spore ultrastructure indicated that the loss of Gas2 and Gas4 proteins affected the proper attachment of the glucan to the chitosan layer, probably as a consequence of the lack of coherence of the glucan layer. The ectopic expression of GAS2 and GAS4 genes in a gas1 null mutant revealed that these proteins are redundant versions of Gas1p specialized to function in a compartment at a pH value close to neutral.

Spore formation in Saccharomyces cerevisiae requires the de novo formation of prospore membranes. The coalescence of secretory vesicles into a membrane sheet occurs on the cytoplasmic surface of the spindle pole body. Spo14p, the major yeast phospholipase D, is necessary for prospore membrane formation; however, the specific function of Spo14p in this process has not been elucidated. We report that loss of Spo14p blocks vesicle fusion, leading to the accumulation of prospore membrane precursor vesicles docked on the spindle pole body. A similar phenotype was seen when the t-SNARE Sso1p, or the partially redundant t-SNAREs Sec9p and Spo20p were mutated. Although phosphatidic acid, the product of phospholipase D action, was necessary to recruit Spo20p to the precursor vesicles, independent targeting of Spo20p to the membrane was not sufficient to promote fusion in the absence of SPO14. These results demonstrate a role for phospholipase D in vesicle fusion and suggest that phospholipase D-generated phosphatidic acid plays multiple roles in the fusion process.

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.

The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation. A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor. The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays. Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly. The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins. The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure.

The Saccharomyces cerevisiae spore wall is a multilaminar coat that surrounds individual spores and protects them from environmental insult. Scanning electron microscopy reveals that the four spores of an ascus are connected by interspore bridges. Transmission electron microscopy of spores indicates that these bridges are continuous with the outer layers of the spore wall. In chs3 mutants, which lack the chitosan and dityrosine layers of the spore wall, bridges are absent. By contrast, in dit1 mutants, which lack only the dityrosine layer, bridges are present, suggesting that the bridges may be composed of chitosan. Interspore bridges are shown to be necessary to hold spores together after release from the ascus. A function for these bridges in the maintenance of heterozygous markers in a homothallic yeast population is proposed.

Spore formation in the yeast Saccharomyces cerevisiae depends on a modification of spindle pole bodies (SPBs) at the onset of meiosis II that allows them to promote de novo membrane formation. Depletion of the environmental carbon source during sporulation results in modification of only one SPB from each meiosis II spindle and formation of a two-spored ascus, called a nonsister dyad (NSD). We have found that mutants impaired in the glyoxylate pathway, which is required for the conversion of acetate to glucose, make NSDs when acetate is the primary carbon source. Wild-type cells make NSDs when the carbon source is glycerol, which is converted to glucose independently of the glyoxylate pathway. During NSD formation in glycerol, only the two SPBs created at the meiosis I/II transition ("daughters") are modified. In these conditions, the SPB components Mpc70p and Spo74p are not recruited to mother SPBs. Moreover, cooverexpression of Mpc70p and Spo74p suppresses NSD formation in glycerol. Our findings indicate that flux through the glyoxylate pathway during sporulation regulates modification of mother SPBs via recruitment of Mpc70p and Spo74p. These results define a cellular response in which the accumulation of an intermediary metabolite serves as a measure of biosynthetic capacity to regulate the number of daughter cells formed.

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.

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.