The mei4؉ gene of the fission yeast Schizosaccharomyces pombe was cloned by functional complementation. The mei4 disruptant failed to complete meiosis-I but could proliferate normally. Meiosis is required for the formation of germ cells which transmit genetic information from generation to generation. This specialized nuclear division is characterized by a reduction in the chromosome number and frequent genetic recombination, both of which have contributed to the evolution of eukaryotes. Although meiosis has basically the same machinery, including spindles, centrosomes, and kinetochores, as mitosis in somatic cells, these two nuclear divisions are different in many aspects. Meiosis-specific gene products must be responsible for the various different features of meiosis, especially those of meiosis-I, such as the synapsis of homologous chromosomes, nondisjunction of sister chromatids, crossing over, and chiasmata formation.

Formation of the forespore membrane, which becomes the plasma membrane of spores, is an intriguing step in the sporulation of the fission yeast Schizosaccharomyces pombe. Here we report two novel proteins that localize to the forespore membrane. spo3 ϩ encodes a potential membrane protein, which was expressed only during sporulation. Green fluorescent protein (GFP) fusion revealed that Spo3 localized to the forespore membrane. The spo3 disruptant was viable and executed meiotic nuclear divisions as efficiently as the wild type but did not form spores. One of the spo3 alleles, spo3-KC51, was dose-dependently suppressed by psy1 ϩ , which encodes a protein similar to mammalian syntaxin-1A, a component of the plasma membrane docking/fusion complex. psy1 ϩ was essential for vegetative growth, and its transcription was enhanced during sporulation. As expected, Psy1 localized to the plasma membrane during vegetative growth. Interestingly, Psy1 on the plasma membrane disappeared immediately after first meiotic division and relocalized to the forespore membrane as the second division initiated. In the spo3 null mutant, the forespore membrane was initiated but failed to develop a normal morphology. Electron microscopy revealed that membrane vesicles were accumulated in the cytoplasm of immature spo3⌬ asci. These results suggest that Spo3 is a key component of the forespore membrane and is essential for its assembly acting in collaboration with the syntaxin-like protein.

The complete nucleotide sequence of the mei2 gene of Schizosaccharomyces pombe, which is essential for initiation of meiosis, is presented and four transcriptional start sites assigned. Transcription of mei2 and other genes involved in life cycle control of S. pombe, which is inducible by nitrogen starvation, is inhibited by addition of cAMP, suggesting that cAMP can mediate the signal of nitrogen supply in S.pombe. mei2 is the furthest downstream among target genes regulated by cAMP and genetic or physiological factors so far shown to block uncontrolled meiosis in S.pombe, which is provoked by inactivation of the part1 gene product, are either mutations at the mei2 locus or inhibitors of its expression. Cooperation of two regulatory pathways, one leading to the inactivation of pat1 activity and the other to the supply of the mei2 product, appears to commit cells to meiosis in S.pombe.

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G1-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G1-phase. The role ofste9 + in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G1-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G1/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants ofste9 cdc10 ts, cells arrested in G1-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G1-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G1 progression possibly by controlling the amount of the mitotic cyclin in the G1-phase.

The fission yeast Schizosaccharomyces pombe has two mating‐types, h+ (P) and h‐ (M). The mam2 mutant exhibits an h(−)‐specific sterile phenotype. Nucleotide sequencing of the mam2 gene isolated from an S. pombe genomic library revealed an open reading frame composed of 348 amino acids. The deduced mam2 product is a hydrophobic protein of 39 kDa that has significant sequence similarity (26.3% for identical amino acids) with the transmembrane domains of the Saccharomyces cerevisiae STE2 product, the alpha‐pheromone receptor. Hydropathicity analysis suggests that the Mam2 protein contains seven possible membrane‐spanning domains and a carboxy‐terminal hydrophilic region. The mam2 gene was disrupted and found to be non‐essential for growth. An h‐ haploid strain harbouring this disrupted null allele failed to respond to the pheromone of h+ cells, P‐factor. These observations imply that the mam2 gene encodes a receptor for P‐factor. Transcription of mam2 was induced only when strains containing functional mat1‐M allele were cultured under conditions of nitrogen starvation. The mam2 gene was also transcribed in h+/h‐ diploid strains. The fact that the map1/mam2 homozygous diploid cells are incapable of sporulation implies that the pheromone signalling system is necessary for sporulation in diploid cells.

In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, sporulation involves de novo synthesis of forespore membrane (FSM) within the cytoplasm of mother cells. The FSM ultimately becomes the plasma membrane of the developing ascospores. Several protein components of the FSM have been identified. Visualization of these proteins has demonstrated the dynamic nature of the genesis and development of the FSM. It begins to develop at the differentiated outer plaque of the spindle pole bodies (SPBs) and extends outwards, encapsulating each of the haploid nuclei produced by meiosis. Several coiled-coil proteins are specifically recruited to the SPBs and play indispensable roles in FSM assembly. Temporal and spatial coordination of meiotic nuclear divisions and membrane assembly is of special importance. Comparison of the processes of FSM assembly in these yeasts shows that the basic mechanism has been conserved, even though the individual proteins involved are often different. Understanding these dynamic aspects of yeast sporulation will help to elucidate a general mechanism for the cellularization of cytoplasm containing multiple nuclei.

Cullin-4 forms a scaffold for multiple ubiquitin ligases. In Schizosaccharomyces pombe, the Cullin-4 homologue (Pcu4) physically associates with Ddb1 and the COP9 signalosome (CSN). One target of this complex is Spd1. Spd1 regulates ribonucleotide reductase (RNR) activity. Spd1 degradation during S phase, or following DNA damage of G2 cells, results in the nuclear export of the small RNR subunit. We demonstrate that Cdt2, an unstable WD40 protein, is a regulatory subunit of Pcu4-Ddb1-CSN ubiquitin ligase. cdt2 deletion stabilises Spd1 and prevents relocalisation of the small RNR subunit from the nucleus to the cytoplasm. cdt2 þ is periodically transcribed by the Cdc10/DSC1 transcription factor during S phase and transiently transcribed following DNA damage of G2 cells, corresponding to Spd1 degradation profiles. Cdt2 co-precipitates with Spd1, and Cdt2 overexpression results in constitutive Spd1 degradation. We propose that Cdt2 incorporation into the Pcu4-Ddb1-CSN complex prompts Spd1 targeting and subsequent degradation and that Cdt2 is a WD40 repeat adaptor protein for Cullin-4-based ubiquitin ligase.

The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20(+) is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20(+) gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.