A sex pheromone, protoplast release-inducing protein (PR-IP) inducer, induces sexual cell division and production of PR-IP in Closterium

The sex pheromone protoplast release-inducing protein (PR-IP) inducer and a sexual cell division-inducing pheromone-minus (SCD-IP-minus) that mediates the sexual reproduction of the heterothallic Closterium peracerosum-strigosum-littorale (C. psl) complex were investigated in this study. Recombinant PR-IP inducer produced by yeast cells was prepared and assayed for production of PR-IP and induction of SCD. Both biological activities were observed after treating mating-type plus (mt+) cells with the recombinant pheromone. SCD was induced by exposure to a lower concentration of the same pheromone and by a shorter treatment period with the pheromone than was production of PR-IP. This indicates that the previously characterized PR-IP inducer has both PR-IP-inducing and SCD-inducing activities with mt+ cells, although the inducing mechanisms of the two pheromones differ.     

Inositol is specifically involved in the sexual program of the fission yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a natural auxotroph for inositol and fails to grow in the complete absence of it. It was previously reported that a small concentration of inositol in the culture medium supports vegetative growth, but not mating and sporulation, and a tenfold of that concentration also supports mating and sporulation. The purpose of the present work was to investigate whether a moderate inositol starvation specifically affected events of the sexual program of development. A homothallic culture grown to the stationary phase in medium with a small inositol concentration was sterile but cells in the stationary phase of growth synchronously entered and completed the sexual cycle when inositol was added, without need of previous cell divisions. This suggests the involvement of inositol in a mechanism (or mechanisms) of the sexual program. The events of the program that were affected by inositol starvation were investigated. Commitment to mating and production of pheromone M were shown not to be inositol-dependent. A diploid strain homozygous at the mating-type locus and carrying a pat1-114 temperature-sensitive mutation in homozygous configuration sporulated under inositol starvation at the restrictive temperature; therefore starvation did not directly affect meiosis or sporulation. In contrast, production of pheromone P and the response of cells to pheromones were found to be inositol-dependent. The possibility that inositol or one of its derivative compounds is involved in pheromone P secretion and in pheromone signal reception is discussed.     

Sexual agglutinin of mating-type minus gametes in Chlamydomonas reinhardii : I. Loss and recovery of agglutinability of gametes treated with EDTA

The effect of EDTA on the mating-type-specific agglutinins located on the flagellar surfaces of Chlamydomonas reinhardii gametes was investigated. The mating-type minus (mt-) gametes lost their agglutinability without apparent loss of motility soon after addition of EDTA at low concentrations (1-2 mM). At the same time, the cells released into the medium agglutinins which can elicit agglutinative responses of mating-type plus (mt+) gametes specifically. When EDTA was neutralized with Mg2+ or removed by centrifugation, the mt- cells quickly replaced agglutinins by protein synthesis: the recovery process was sensitive to cycloheximide, but not to tunicamycin. The EDTA-treated mt+ gametes lost their agglutinins much more slowly than the mt- gametes. The replacement of mt+ agglutinins was inhibited by both cycloheximide and tunicamycin.     

INHIBITORY EFFECTS OF CONCANAVALIN A AND TUNICAMYCIN ON SEXUAL ATTACHMENT OF ALEXANDRIUM CATENELLA (DINOPHYCEAE)1

The effect of concanavalin A (a lectin) and tunicamycin (an inhibitor of protein glycosylation) on sexual attachment of gamete pairs of the dinoflagellate Alexandrium catenella Whedon & Kofoid was studied. Concanavalin A inhibited sexual attachment at a concentration of 0.005%, and this inhibition was released by the addition of glucose or mannose at 10 mM. Mating type plus cells of A. catenella treated with tunicamycin for 12 h were not capable of sexual attachment. These results are the first to suggest the existence of a cell–cell recognition system in sexual attachment in A. catenella.     

Behavioural changes induced by the conjugation-inducing pheromones,gamone 1 and 2, in the ciliate Blepharisma japonicum

Preconjugant interactions between complementary mating-type cells in ciliates occur before sexual reproduction. The interactions include retardation of swimming behaviour, courtship dancing, chemoattraction, nuclear activation, cell division, or cell agglutination, depending on ciliate species. In Blepharisma japonicum, chemoattraction of mating-type I by mating-type II has been reported previously. It has been shown that chemoattraction here is caused by a conjugation-inducing substance called gamone 2 secreted by mating-type II cells. In this study, we show that mating-type II cells accumulate near the site where gamone 1 secreted by mating-type I cells is present at a high concentration. We also show that the behaviour of individual cells changes when exposed to the complementary mating-type gamone; cells begin to rotate and swim slowly, thus shortening their minimum path length (final displacement of a cell from its origin). These results suggest that gamones 1 and 2 induce behavioural changes in type II and I cells, respectively, and that gamone-stimulated cells may accumulate at the site with the highest activity of the complementary gamone, after repetition of swimming changes in the gradient of gamone concentration. This reciprocal induction of the changes in behaviour may increase the probability of sexual encounters for conjugation.     

Roles for receptors, pheromones, G proteins, and mating type genes during sexual reproduction in Neurospora crassa

Here we characterize the relationship between the PRE-2 pheromone receptor and its ligand, CCG-4, and the general requirements for receptors, pheromones, G proteins, and mating type genes during fusion of opposite mating-type cells and sexual sporulation in the multicellular fungus Neurospora crassa. PRE-2 is highly expressed in mat a cells and is localized in male and female reproductive structures. Δpre-2 mat a females do not respond chemotropically to mat A males (conidia) or form mature fruiting bodies (perithecia) or meiotic progeny (ascospores). Strains with swapped identity due to heterologous expression of pre-2 or ccg-4 behave normally in crosses with opposite mating-type strains. Coexpression of pre-2 and ccg-4 in the mat A background leads to self-attraction and development of barren perithecia without ascospores. Further perithecial development is achieved by inactivation of Sad-1, a gene required for meiotic gene silencing. Findings from studies involving forced heterokaryons of opposite mating-type strains show that presence of one receptor and its compatible pheromone is necessary and sufficient for perithecial development and ascospore production. Taken together, the results demonstrate that although receptors and pheromones control sexual identity, the mating-type genes (mat A and mat a) must be in two different nuclei to allow meiosis and sexual sporulation to occur.     

A pheromone mutant of Schizosaccharomyces pombe displays nucleolar fragmentation

Stresses and nutritional starvation are two main external signals for the induction of sex pheromones in the fission yeast Schizosaccharomyces pombe. In an attempt to identify the components involved in transduction of starvation signals, we screened 135 temperature-sensitive (ts) mutants and isolated 6 mutants that induced the pheromone even in the presence of a nitrogen source. These mutants exhibited two distinct induction phenotypes: pheromone induction at restrictive but not at permissive temperatures; and pheromone induction at both permissive and restrictive temperatures. The times required for the maximum pheromone induction at the restrictive temperature differed slightly in each mutant. In addition to the pheromone induction phenotype, the ts243 and ts304 mutants exhibited cell-division-cycle defects. The ts304 mutant cells showed an abnormal cytoplasmic DAPI staining pattern. The nucleolus of this mutant seemed to be fragmented, a phenomenon which is typically observed in aged yeast cells. The result of our genetic analysis indicated that the pheromone induction mutants belonged to 6 separate complementation groups. We designated these mutants pws1 to pws6.     

Intercellular Communication During Sexual Reproduction of Closterium (Conjugatophyceae)

Closterium were reviewed. In the case of Closterium peracerosum-strigosum-littorale complex, two sex-specific pheromones and their receptors were involved in sexual reproduction. These pheromones were glycoproteins and the expression of corresponding genes was critically regulated by the sex and environmental conditions. In the case of Closterium ehrenbergii, chemotactic and sexual cell division-inducing activities for mating-type plus cells were detected and characterized. Although many processes remain to be elucidated, the present results will be helpful for understanding not only the mode of sexual reproduction in Closterium but also the variety of intercellular communication in the plant kingdom especially during sexual reproduction. Received 10 June 2000/ Accepted in revised form 6 July 2000     

Saccharomyces cerevisiae mating pheromones specifically inhibit the synthesis of proteins destined to be N-glycosylated

alpha Factor specifically inhibits the synthesis of N-glycosylated proteins in Saccharomyces cerevisiae mating type a cells but not in alpha cells or in a/alpha diploids. a Factor has the same effect of alpha cells. The synthesis of O-glycosylated proteins is not inhibited. Although the mating pheromones act like a 'physiological tunicamycin', the mechanism of inhibition is different: not the glycosylation of proteins as such but rather the synthesis of those proteins destined to be N-glycosylated is inhibited. Thus none of a number of glycosylating enzymes tested in vitro is reduced in activity in alpha-factor-treated cells. The synthesis of the glycoprotein carboxypeptidase Y, on the other hand, is strongly inhibited by tunicamycin as well as by alpha factor; but only in the former case did carbohydrate-free protein accumulate in the cells. alpha Factor causes maximal inhibition of glycoprotein formation after as little as 30 min, long before all cells in the population are arrested in G1; moreover, release from this inhibition precedes the increase in budding index (resumption of cell division). It is postulated, therefore, that N-glycosylated proteins are required for the G1/S-phase transition in the yeast cell cycle. This is supported by previous reports that first cycle arrest in G1 occurs when (a) tunicamycin is added to growing cultures, and (b) a temperature-sensitive N-glycosylation mutant is shifted to its restrictive temperature.     

Effect of tunicamycin on germ tube and yeast bud formation in Candida albicans

Tunicamycin is an antimicrobial agent which inhibits the first reaction of the dolichol pathway leading to N-glycosylation of proteins. The effect of tunicamycin on the growth of the dimorphic fungus Candida albicans differed depending on the growth phase of the organism. Addition of tunicamycin to stationary phase yeast cells inhibited the resumption of growth of those cells in either morphology, as cultures failed to initiate either yeast bud or germ tube formation. When tunicamycin was added to growing cells, growth was inhibited but not immediately. When it was added to germ tube cultures, nuclear division and septum formation continued for some time before ceasing. Addition of the drug to exponential phase yeast cultures resulted in an approximately 45% increase in cell number before cell division ceased and yeast accumulated in both budded and unbudded stages of the cell cycle. Accumulation of trichloroacetic acid precipitable radiolabelled protein and nucleic acid continued unchanged for some time following addition of tunicamycin; however, after a while a reduced rate of accumulation was noted.     

Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells.

Apoptosis as a form of programmed cell death (PCD) in multicellular organisms is a well-established genetically controlled process that leads to elimination of unnecessary or damaged cells. Recently, PCD has also been described for unicellular organisms as a process for the socially advantageous regulation of cell survival. The human Bcl-2 family member Bak induces apoptosis in mammalian cells which is counteracted by the Bcl-x(L) protein. We show that Bak also kills the unicellular fission yeast Schizosaccharomyces pombe and that this is inhibited by coexpression of human Bcl-x(L). Moreover, the same critical BH3 domain of Bak that is required for induction of apoptosis in mammalian cells is also required for inducing death in yeast. This suggests that Bak kills mammalian and yeast cells by similar mechanisms. The phenotype of the Bak-induced death in yeast involves condensation and fragmentation of the chromatin as well as dissolution of the nuclear envelope, all of which are features of mammalian apoptosis. These data suggest that the evolutionarily conserved metazoan PCD pathway is also present in unicellular yeast.     

Deletion of the Fission Yeast Homologue of Human Insulinase Reveals a TORC1-Dependent Pathway Mediating Resistance to Proteotoxic Stress

Insulin Degrading Enzyme (IDE) is a protease conserved through evolution with a role in diabetes and Alzheimer''s disease. The reason underlying its ubiquitous expression including cells lacking identified IDE substrates remains unknown. Here we show that the fission yeast IDE homologue (Iph1) modulates cellular sensitivity to endoplasmic reticulum (ER) stress in a manner dependent on TORC1 (Target of Rapamycin Complex 1). Reduced sensitivity to tunicamycin was associated with a smaller number of cells undergoing apoptosis. Wild type levels of tunicamycin sensitivity were restored in iph1 null cells when the TORC1 complex was inhibited by rapamycin or by heat inactivation of the Tor2 kinase. Although Iph1 cleaved hallmark IDE substrates including insulin efficiently, its role in the ER stress response was independent of its catalytic activity since expression of inactive Iph1 restored normal sensitivity. Importantly, wild type as well as inactive human IDE complemented gene-invalidated yeast cells when expressed at the genomic locus under the control of iph1⁺ promoter. These results suggest that IDE has a previously unknown function unrelated to substrate cleavage, which links sensitivity to ER stress to a pro-survival role of the TORC1 pathway.     

Identification and Characterization of a Mutation Affecting the Division Arrest Signaling of the Pheromone Response Pathway in Saccharomyces Cerevisiae

Mating pheromones, a- and alpha-factors, arrest the division of cells of opposite mating types, alpha and a cells, respectively. I have isolated a sterile mutant of Saccharomyces cerevisiae that is defective in division arrest in response to alpha-factor but not defective in morphological changes and agglutinin induction. The mutation was designated dac2 for division arrest control by mating pheromones. The dac2 mutation was closely linked to gal1 and was different from the previously identified cell type nonspecific sterile mutations (ste4, ste5, ste7, ste11, ste12, ste18 and dac1). Although dac2 cells had no phenotype in the absence of pheromones, they showed morphological alterations and divided continuously in the presence of pheromones. As a result, dac2 cells had a mating defect. The dac2 mutation could suppress the lethality caused by the disruption of the GPA1 gene (previously shown to encode a protein with similarity to the alpha subunit of mammalian G proteins). In addition, dac2 cells formed prezygotes with wild-type cells of opposite mating types, although they could not undergo cell fusion. These results suggest that the DAC2 product may control the signal for G-protein-mediated cell-cycle arrest and indicate that the synchronization of haploid yeast cell cycles by mating pheromones is essential for cell fusion during conjugation.     

A newly identified chemotactic sexual pheromone from Closterium ehrenbergii

 In sexual reproduction of Closterium ehrenbergii, pairing with the sexual partner cells is the first process observed. A cell migration-inducing activity, specific for mating-type plus (mt⁺; NIES-228) cells, was detected in the culture medium of mating-type minus (mt^–; NIES-229) cells. Light was necessary for production of the active substance by mt^– cells and for migration of mt⁺ cells. The active substance was heat-labile and had an apparent molecular mass of 20 kDa, as determined by gel filtration. A protein of 20 kDa was detected in the active fraction of gel filtration after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on these results, it is proposed that a chemotactic sexual pheromone involved in the formation of sexual pairs of cells is secreted by mt^– cells of C. ehrenbergii and is proteinaceous, like other sexual pheromones secreted by Closterium species. Received: 31 July 1997 / Revision accepted: 25 November 1997     

Apoptosis-like yeast cell death in response to DNA damage and replication defects

In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast-reactive oxygen species (ROS)-can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.     

Inhibition of N-linked glycosylation induces early apoptosis in human promyelocytic HL-60 cells

Inhibition of protein N-glycosylation by tunicamycin induced morphological changes characteristic of apoptosis in human promyelocytic HL-60 cells. Internu-cleosomal DMA fragmentation could be detected after short-time incubation (between 6 and 9 h) of HL-60 cells with low doses of tunicamycin (0.05 μg/ml). Under these conditions the synthesis of glycoproteins was reduced to 17% of control values, while no significant changes in the rates of total protein synthesis could be observed. Tunicamycin ability to induce DNA fragmentation was in good correlation with its potency as glycosylation inhibitor in several myeloid cell lines. Tunicamycin-induced apoptosis was potentiated by activation of protein kinease C (PKC) by phorbol esters and partially prevented by the PKC inhibitor staurosporine. Inhibitors of RNA and protein synthesis displayed a protective effect. Treatment of HL-60 cells with tunicamycin did not elicit the expression of cell surface differentiation antigens or their ability to generate superoxide anion. In contrast, tunicamycin significantly inhibited these processes during dimethyl sulfoxide (DMSO)-induced myeloid differentiation. These observations indicate that the main effect of tunicamycin in HL-60 cells is the induction of apoptosis. © 1995 Wiley-Liss, Inc.     

A novel gene, msa1, inhibits sexual differentiation in Schizosaccharomyces pombe

Sexual differentiation in the fission yeast Schizosaccharomyces pombe is triggered by nutrient starvation or by the presence of mating pheromones. We identified a novel gene, msa1, which encodes a 533-aa putative RNA-binding protein that inhibits sexual differentiation. Disruption of the msa1 gene caused cells to hypersporulate. Intracellular levels of msa1 RNA and Msa1 protein diminished after several hours of nitrogen starvation. Genetic analysis suggested that the function of msa1 is independent of the cAMP pathway and stress-responsive pathway. Deletion of the ras1 gene in diploid cells inhibited sporulation and in haploid cells decreased expression of mating-pheromone-induced genes such as mei2, mam2, ste11, and rep1; simultaneous deletion of msa1 reversed both phenotypes. Overexpression of msa1 decreased activated Ras1(Val17)-induced expression of mam2. Phenotypic hypersporulation was similar between cells with deletion of only rad24 and both msa1 and rad24, but simultaneous deletion of msa1 and msa2/nrd1 additively increased hypersporulation. Therefore, we suggest that the primary function of Msa1 is to negatively regulate sexual differentiation by controlling the expression of Ste11-regulated genes, possibly through the pheromone-signaling pathway.