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-DNES) 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. MEIOSIS is a specialized cell division that produces the haploid cells needed for sexual reproduction, and the completion of meiosis is normally coupled to differentiation programs that produce gametes. In Saccharomyces cerevisiae, sporulation is initiated when diploid cells are grown in the absence of nitrogen and the presence of a nonfermentable carbon source such as acetate (Esposito and Klapholtz 1981). When triggered to enter the sporulation program, cells exit the mitotic cycle from the G1 phase. This is followed by one round of S phase and two meiotic divisions that generate four haploid nuclei, which are packaged into individual spores. As a consequence of this process, four haploid gametes (spores) are formed in the cytoplasm of the mother cell (ascus) (reviewed by Neiman 2005). At the onset of meiosis II, the spindle pole body (SPB) is modified by the incorporation of several meiosis-specific proteins, which together make up the meiosis II outer plaque (Knop and Strasser 2000;Bajgier et al. 2001;Nickas et al. 2003). This change in composition converts the function of the cytoplasmic face of the SPB from microtubule nucleation to membrane nucleation. The process of spore construction requires the de novo assembly of two cellular structures, the prospore membrane (PSM) that is generated around the daughter nuclei to create prospores and a protective spore wall that surrounds the prospores. Once the meiosis II outer plaque of the SPB has been assembled, it becomes a site for the docking and coalescence of secretory vesicles to form flattened double-membrane sheets termed PSMs (Moens 1971;Neiman 1998). These nascent PSMs expand during meiosis II, and at the time of nuclear division each PSM completely engulfs the nuclear lobe to which it is anchored via the SPB. At the completion of meiosis II, each PSM fuses with itself so that each daughter nucleus (and assoc...
SummaryMeiosis is the developmental programme by which sexually reproducing diploid organisms generate haploid gametes. In yeast, meiosis is followed by spore morphogenesis. The formation of the Schizosaccharomyces pombe ascospore wall requires the co-ordinated activity of enzymes involved in the biosynthesis and modification of its components, such as glucans. During sporogenesis, the b-1,3-glucan synthase bgs2p synthesizes linear b-1,3-glucans, which remain unorganized and alkali-soluble until covalent linkages are set up between b-1,3-glucans and other cell wall components. Several proteins belonging to the glycoside hydrolase family 72 (GH72) with b-1,3-glucanosyltransferase activity have been described in other organisms, such as the Saccharomyces cerevisiae Gas1p or the Aspergillus fumigatus Gel1p. Here we describe the characterization of gas4 + , a new gene that encodes a protein of the GH72 family. Deletion of this gene does not lead to any apparent defect during vegetative growth, but homozygous gas4D diploids show a sporulation defect. Although meiosis occurs normally, ascospores are unable to mature or to germinate. The expression of gas4 + is strongly induced during sporulation and a yellow fluorescent protein (YFP)-gas4p fusion protein localizes to the ascospore periphery during sporulation. We conclude that gas4p is required for ascospore maturation in S. pombe.
BackgroundThe formation of the cell wall in Schizosaccharomyces pombe requires the coordinated activity of enzymes involved in the biosynthesis and modification of β-glucans. The β(1,3)-glucan synthase complex synthesizes linear β(1,3)-glucans, which remain unorganized until they are cross-linked to other β(1,3)-glucans and other cell wall components. Transferases of the GH72 family play important roles in cell wall assembly and its rearrangement in Saccharomyces cerevisiae and Aspergillus fumigatus. Four genes encoding β(1,3)-glucanosyl-transferases -gas1+, gas2+, gas4+ and gas5+- are present in S. pombe, although their function has not been analyzed.Methodology/Principal FindingsHere, we report the characterization of the catalytic activity of gas1p, gas2p and gas5p together with studies directed to understand their function during vegetative growth. From the functional point of view, gas1p is essential for cell integrity and viability during vegetative growth, since gas1Δ mutants can only grow in osmotically supported media, while gas2p and gas5p play a minor role in cell wall construction. From the biochemical point of view, all of them display β(1,3)-glucanosyl-transferase activity, although they differ in their specificity for substrate length, cleavage point and product size. In light of all the above, together with the differences in expression profiles during the life cycle, the S. pombe GH72 proteins may accomplish complementary, non-overlapping functions in fission yeast.Conclusions/SignificanceWe conclude that β(1,3)-glucanosyl-transferase activity is essential for viability in fission yeast, being required to maintain cell integrity during vegetative growth.
BackgroundIn Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth, where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore membrane.ResultsHere, we demonstrate that nutrient limitation triggers a change in the localization of at least two vegetative septins (Cdc10 and Cdc11) from the bud neck to the microtubules. The association of Cdc10 and Cdc11 with microtubules persists into meiosis, and they are found associated with the meiotic spindle until the end of meiosis II. In addition, the meiosis-specific septin Spr28 displays similar behavior, suggesting that this is a common feature of septins. Septin association to microtubules is a consequence of the nutrient limitation signal, since it is also observed when haploid cells are incubated in sporulation medium and when haploid or diploid cells are grown in medium containing non-fermentable carbon sources. Moreover, during meiosis II, when the nascent prospore membrane is formed, septins moved from the microtubules to this membrane. Proper organization of the septins on the membrane requires the sporulation-specific septins Spr3 and Spr28.ConclusionNutrient limitation in S. cerevisiae triggers the sporulation process, but it also induces the disassembly of the septin bud neck ring and relocalization of the septin subunits to the nucleus. Septins remain associated with microtubules during the meiotic divisions and later, during spore morphogenesis, they are detected associated to the nascent prospore membranes surrounding each nuclear lobe. Septin association to microtubules also occurs during growth in non-fermentable carbon sources.
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