Yoshiyuki Imai

Meiotic nuclear reorganization: switching the position of centromeres and telomeres in the fission yeast Schizosaccharomyces pombe

In fission yeast meiotic prophase, telomeres are clustered near the spindle pole body (SPB; a centrosome‐equivalent structure in fungi) and take the leading position in chromosome movement, while centromeres are separated from the SPB. This telomere position contrasts with mitotic nuclear organization, in which centromeres remain clustered near the SPB and lead chromosome movement. Thus, nuclear reorganization switching the position of centromeres and telomeres must take place upon entering meiosis. In this report, we analyze the nuclear location of centromeres and telomeres in genetically well‐characterized meiotic mutant strains. An intermediate structure for telomere‐centromere switching was observed in haploid cells induced to undergo meiosis by synthetic mating pheromone; fluorescence in situ hybridization revealed that in these cells, both telomeres and centromeres were clustered near the SPB. Further analyses in a series of mutants showed that telomere‐centromere switching takes place in two steps; first, association of telomeres with the SPB and, second, dissociation of centromeres from the SPB. The first step can take place in the haploid state in response to mating pheromone, but the second step does not take place in haploid cells and probably depends on conjugation‐related events. In addition, a linear minichromosome was also co‐localized with authentic telomeres instead of centromeres, suggesting that telomere clustering plays a role in organizing chromosomes within a meiotic prophase nucleus.

Schizosaccharomyces pombe map3+ encodes the putative M-factor receptor.

A defect in the map3 gene of the fission yeast Schizosaccharomyces pombe causes h+ mating-type-specific sterility. This gene was cloned by complementation. Nucleotide sequence analysis showed that it has a coding capacity of 365 amino acids. The deduced map3 gene product is a putative seven-transmembrane protein and has 20.0% amino acid identity with the a-factor receptor of Saccharomyces cerevisiae, encoded by STE3. It is also homologous with the Ustilago maydis mating pheromone receptors. The map3 gene is expressed in h+ cells but not in h- cells, and the transcripts are induced in response to nitrogen starvation. h+ cells defective in map3 do not respond to purified M-factor. When map3 is expressed ectopically in h- cells, they apparently acquire the ability to respond to the M-factor produced by themselves. The gpa1 gene, which encodes the alpha-subunit of a G-protein presumed to couple with the mating pheromone receptors, is essential for this function of map3. These observations strongly suggest that map3 encodes the M-factor receptor. Furthermore, this study provides strong support for the notion that pheromone signaling is essential for initiation of meiosis in S. pombe and that either M-factor signaling or P-factor signaling alone is sufficient.

Schizosaccharomyces pombe sxa1+ and sxa2+ encode putative proteases involved in the mating response.

The Schizosaccharomyces pombe sxa1 and sxa2 mutants showed an exaggerated response to mating pheromones, producing excessively long conjugation tubes and exhibiting mating deficiency. This phenotype was similar to phenotypes of cells bearing an activated allele of ras1, such as ras1Val-17 or ras1Leu-66, and phenotypes of cells defective in gap1. However, genetic evidence suggested that the sxa1 and sxa2 gene products are not directly involved in the Ras1 pathway. The gene products of sxa1 and sxa2, as deduced from their nucleotide sequences, were homologous to aspartyl proteases and serine carboxypeptidases, respectively. The sxa1 gene function was required for efficient mating only in h+ cells, although even disruption of sxa1 did not completely abolish the mating ability. Conversely, the sxa2 gene function was required only in h- cells. Wild-type cells produced a diffusible substance, which may be the sxa2 gene product itself, that could confer fertility to sxa2 mutant cells placed at a distance. These observations are consistent with the possibility that the sxa gene products are involved in degradation or processing of the mating pheromones and that their loss cause a persistent response to the pheromones.

Interviral homologies of the 30k proteins of tobamoviruses

The 30K protein of tobacco mosaic virus was recently confirmed to be involved in cell-to-cell movement of the virus. To characterize common structural features of the 30K protein, the nucleotide sequence of the 30K protein gene of cucumber green mottle mosaic virus (CGMMV, a member of the tobamoviruses) RNA has been determined. The CGMMV 30K protein is composed of 264 amino acids with a calculated molecular weight of 28,800. Comparisons among the 30K proteins of tobamoviruses show that the 30K proteins are composed of a rather conserved N-terminal two-thirds and a less-conserved C-terminal one-third. The N-terminal two-thirds contain two particularly well-conserved sequences, one of which contains amino acid substitutions that result in temperature-sensitive cell-to-cell movement. The C-terminal region is further divided into three subregions by distribution of charged amino acid residues.

Identification of a GTPase-activating protein homolog in Schizosaccharomyces pombe.

Loss of function of the Schizosaccharomyces pombe gap1 gene results in the same phenotypes as those caused by an activated ras1 mutation, i.e., hypersensitivity to the mating factor and inability to perform efficient mating. Sequence analysis of gap1 indicates that it encodes a homolog of the mammalian Ras GTPase-activating protein (GAP). The predicted gap1 gene product has 766 amino acids with relatively short N- and C-terminal regions flanking the conserved core sequence of GAP. Genetic analysis suggests that S. pombe Gap1 functions primarily as a negative regulator of Ras1, like S. cerevisiae GAP homologs encoded by IRA1 and IRA2, but is unlikely to be a downstream effector of the Ras protein, a role proposed for mammalian GAP. Thus, Gap1 and Ste6, a putative GDP-GTP-exchanging protein for Ras1 previously identified, appear to play antagonistic roles in the Ras-GTPase cycle in S. pombe. Furthermore, we suggest that this Ras-GTPase cycle involves the ra12 gene product, another positive regulator of Ras1 whose homologs have not been identified in other organisms, which could function either as a second GDP-GTP-exchanging protein or as a factor that negatively regulates Gap1 activity.

Isolation and characterization of krp, a dibasic endopeptidase required for cell viability in the fission yeast Schizosaccharomyces pombe.

The activation of pro‐hormones and many precursor proteins involves cleavage by endopeptidases belonging to the subtilisin‐like family of enzymes. Here we describe the isolation and characterization of the first member of this family from the fission yeast Schizosaccharomyces pombe. The enzyme, which has been named krp for KEX2‐related protease, is a type I membrane‐bound endopeptidase that cleaves substrates after pairs of dibasic residues. It appears to be synthesized as a pre‐pro‐protein that is likely to undergo processing following translocation into the endoplasmic reticulum. Processing has been characterized in a cell‐free translation/translocation system prepared from Xenopus eggs. Krp is N‐glycosylated on all five of its potential sites and both the pre‐sequence and the pro‐sequence are quickly removed following translocation, the latter probably by autocatalytic cleavage. The inhibitor profile of krp broadly reflects the known properties of the eukaryotic subtilisin proteases, while its pH and Ca2+ dependence are consistent with it being active within the secretory pathway. One of its physiological substrates is likely to be the pheromone precursor pro‐P‐factor, which it is shown to process in an in vitro system, but identification of other substrates is complicated because, unlike other members of this family, krp is essential for cell viability.

Schizosaccharomyces pombe Spk1 is a tyrosine-phosphorylated protein functionally related to Xenopus mitogen-activated protein kinase.

Mitogen-activated protein kinase (MAPK) and its direct activator, MAPK kinase (MAPKK), have been suggested to play a pivotal role in a variety of signal transduction pathways in higher eukaryotes. The fission yeast Schizosaccharomyces pombe carries a gene, named spk1, whose product is structurally related to vertebrate MAPK. Here we show that Spk1 is functionally related to Xenopus MAPK. (i) Xenopus MAPK partially complemented a defect in the spk1- mutant. An spk1- diploid strain could not sporulate, but one carrying Xenopus MAPK could. (ii) Both Spk1 and Xenopus MAPK interfered with sporulation if overexpressed in S. pombe cells. (iii) Spk1 underwent tyrosine phosphorylation as does Xenopus MAPK. Tyrosine phosphorylation of Spk1 appeared to be dependent upon mating signals because it occurred in homothallic cells but not in heterothallic cells. Furthermore, this phosphorylation was diminished in a byr1 disruptant strain, suggesting that spk1 lies downstream of byr1, which encodes a MAPKK homolog in S. pombe. Taken together, the MAPKK-MAPK cascade may be evolutionarily conserved in signaling pathways in yeasts and vertebrates.

Genes encoding farnesyl cysteine carboxyl methyltransferase in Schizosaccharomyces pombe and Xenopus laevis.

The mam4 mutation of Schizosaccharomyces pombe causes mating deficiency in h- cells but not in h+ cells. h- cells defective in mam4 do not secrete active mating pheromone M-factor. We cloned mam4 by complementation. The mam4 gene encodes a protein of 236 amino acids, with several potential membrane-spanning domains, which is 44% identical with farnesyl cysteine carboxyl methyltransferase encoded by STE14 and required for the modification of a-factor in Saccharomyces cerevisiae. Analysis of membrane fractions revealed that mam4 is responsible for the methyltransferase activity in S. pombe. Cells defective in mam4 produced farnesylated but unmethylated cysteine and small peptides but no intact M-factor. These observations strongly suggest that the mam4 gene product is farnesyl cysteine carboxyl methyltransferase that modifies M-factor. Furthermore, transcomplementation of S. pombe mam4 allowed us to isolate an apparent homolog of mam4 from Xenopus laevis (Xmam4). In addition to its sequence similarity to S. pombe mam4, the product of Xmam4 was shown to have a farnesyl cysteine carboxyl methyltransferase activity in S. pombe cells. The isolation of a vertebrate gene encoding farnesyl cysteine carboxyl methyltransferase opens the way to in-depth studies of the role of methylation in a large body of proteins, including Ras superfamily proteins.

Tropomyosin is required for the cell fusion process during conjugation in fission yeast

Background:  Tropomyosin is an actin‐binding protein, which is thought to stabilize actin filaments and influence many aspects of F‐actin. In fission yeast, the cdc8 gene encodes tropomyosin, and the gene product Cdc8p is known to be essential for the formation of the F‐actin contractile ring and hence for cytokinesis in the mitotic cell cycle.

Molecular cloning and characterization of mouse testis poly(A) binding protein II encoded by the Pabp3 gene, which transcomplements meiotic mutant sme2 of S. pombe.

A cDNA clone from a mouse testis cDNA library was isolated by the transcomplementation method using a Schizosaccharomyces pombe meiotic mutant (sme2) that is defective in meiosis I. The cDNA clone isolated has an open reading frame encoding 302 amino acids constituting a protein with a strong similarity to mouse poly(A) binding protein II (mPABII) and bovine poly(A) binding protein II (PABII). PABII is known to bind to the growing poly(A) tail and stimulates poly(A) polymerase, which catalyzes the polymerization of the mRNA poly(A) tail. Northern blot analysis of the cDNA clone identified as mPABII revealed a single transcript of 1.2 kb. This was detectable exclusively in adult testis. Immunohistochemical analysis using a polyclonal antibody demonstrated that mPABII protein was expressed in the nucleus at specific stages from late pachytene spermatocytes to round spermatids. Genetic mapping showed the Pabp3 gene encoding mPABII to be located near position 19.5 on mouse chromosome 14. These results suggest that mPABII might be involved in specific spermatogenetic cell differentiation.