The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor

In the fission yeast Schizosaccharomyces pombe, cells of opposite mating type communicate via diffusible peptide pheromones prior to mating. We have cloned the S. pombe mam1 gene, which encodes a 1336-amino acid protein belonging to the ATP-binding cassette (ABC) superfamily. The mam1 gene is only expressed in M cells and the gene product is responsible for the secretion of the mating pheromone, M-factor, a nonapeptide that is S-farnesylated and carboxy-methylated on its C-terminal cysteine residue. The predicted Mam1 protein is highly homologous to mammalian multiple drug-resistance proteins and to the Saccharomyces cerevisiae STE6 gene product, which mediates export of a-factor mating pheromone. We show that STE6 can also mediate secretion of M-factor in S. pombe.     

Autocrine response of Schizosaccharomyces pombe haploid cells to mating pheromones

Abstract The mating response of the fission yeast Schizosaccharomyces pombe is mediated by mating pheromones, M-factor and P-factor, produced by h⁻ and h⁺ cells, respectively. When the M-factor receptor (Map3) was ectopically expressed in h⁻ cells lacking the P-factor receptor (Mam2), they acquired mating competence in response to M-factor which they secreted. The autocrine response to P-factor in h⁺ cells was so weak that mating competence was not acquired, although expression of the pheromone-responsive gene mat1-Pm was detected. These observations support the notion that the intensity of cellular response to mating pheromones is different between h⁻ and h⁺ cells, although downstream pathways of the pheromone receptors are shared by the two mating types.     

The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor

In the fission yeast Schizosaccharomyces pombe, cells of opposite mating type communicate via diffusible peptide pheromones prior to mating. We have cloned the S. pombe mam1 gene, which encodes a 1336-amino acid protein belonging to the ATP-binding cassette (ABC) superfamily. The mam1 gene is only expressed in M cells and the gene product is responsible for the secretion of the mating pheromone, M-factor, a nonapeptide that is S-farnesylated and carboxy-methylated on its C-terminal cysteine residue. The predicted Mam1 protein is highly homologous to mammalian multiple drug-resistance proteins and to the Saccharomyces cerevisiae STE6 gene product, which mediates export of a-factor mating pheromone. We show that STE6 can also mediate secretion of M-factor in S. pombe. Received: 20 December 1996 / Accepted: 29 January 1997     

One motif to bind them: A small-XXX-small motif affects transmembrane domain 1 oligomerization,function, localization,and cross-talk between two yeast GPCRs

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in mammals and facilitate a range of physiological responses triggered by a variety of ligands. GPCRs were thought to function as monomers, however it is now accepted that GPCR homo- and hetero-oligomers also exist and influence receptor properties. The Schizosaccharomyces pombe GPCR Mam2 is a pheromone-sensing receptor involved in mating and has previously been shown to form oligomers in vivo. The first transmembrane domain (TMD) of Mam2 contains a small-XXX-small motif, overrepresented in membrane proteins and well-known for promoting helix–helix interactions. An ortholog of Mam2 in Saccharomyces cerevisiae, Ste2, contains an analogous small-XXX-small motif which has been shown to contribute to receptor homo-oligomerization, localization and function. Here we have used experimental and computational techniques to characterize the role of the small-XXX-small motif in function and assembly of Mam2 for the first time. We find that disruption of the motif via mutagenesis leads to reduction of Mam2 TMD1 homo-oligomerization and pheromone-responsive cellular signaling of the full-length protein. It also impairs correct targeting to the plasma membrane. Mutation of the analogous motif in Ste2 yielded similar results, suggesting a conserved mechanism for assembly. Using co-expression of the two fungal receptors in conjunction with computational models, we demonstrate a functional change in G protein specificity and propose that this is brought about through hetero-dimeric interactions of Mam2 with Ste2 via the complementary small-XXX-small motifs. This highlights the potential of these motifs to affect a range of properties that can be investigated in other GPCRs.     

The sxa2-dependent inactivation of the P-factor mating pheromone in the fission yeast Schizosaccharomyces pombe

Haploid cells of the fission yeast Schizosaccharomyces pombe exist in one of two mating types, referred to as M and P. Conjugation occurs between cells of opposite mating type and is controlled by the reciprocal action of diffusible pheromones. Loss of function of the sxa2 gene in M cells causes hypersensitivity to the P-factor mating pheromone and a reduction in mating efficiency. Here we demonstrate the secretion of an sxa2-dependent carboxypeptidase that inactivates P-factor by removal of the C-terminal leucine residue.     

A comparison of three fission yeast mitochondrial genomes

The fission yeasts are members of the fungal order Schizosaccharomycetales, a candidate deep-diverging group within Ascomycota. Although a great deal of molecular information is available from Schizosaccharomyces pombe, a model eukaryote, very little is available from other members of this group. In order to better characterize mitochondrial genome evolution in this fungal lineage, the mitochondrial DNA (mtDNA) of two additional fission yeasts, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus var. japonicus, was sequenced. Whereas the mtDNA of S.pombe is only 19 431 bp, the mtDNA of S.octosporus is 44 227 bp, and that of S.japonicus var. japonicus is over 80 kb. The size variation of these mtDNAs is due largely to non-coding regions. The gene content in the latter two mtDNAs is almost identical to that of the completely sequenced S.pombe mtDNA, which encodes 25 tRNA species, the large and small mitochondrial ribosomal RNAs (rnl and rns), the RNA component of mitochondrial RNaseP (rnpB), mitochondrial small subunit ribosomal protein 3 (rps3), cytochrome oxidase subunits 1, 2 and 3 (cox1, cox2 and cox3) and ATP-synthase subunits 6, 8 and 9 (atp6, atp8 and atp9). However, trnI2(cau) (C modified to lysidine) is absent in the S.octosporus mtDNA, as are corresponding ATA codons in its protein-coding genes, and rps3 and rnpB are not found in the mtDNA of S.japonicus var. japonicus. The mtDNA of S.octosporus contains five double hairpin elements, the first report of these elements in an ascomycete. This study provides further evidence in favor of the mobility of these elements, and supports their role in mitochondrial genome rearrangement. The results of our phylogenetic analysis support the monophyly of the Schizosaccharomycetales, but question their grouping within the Archiascomycota.     

Genome-wide identification and characterization of replication origins by deep sequencing

Background

DNA replication initiates at distinct origins in eukaryotic genomes, but the genomic features that define these sites are not well understood.

Results

We have taken a combined experimental and bioinformatic approach to identify and characterize origins of replication in three distantly related fission yeasts: Schizosaccharomyces pombe, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus. Using single-molecule deep sequencing to construct amplification-free high-resolution replication profiles, we located origins and identified sequence motifs that predict origin function. We then mapped nucleosome occupancy by deep sequencing of mononucleosomal DNA from the corresponding species, finding that origins tend to occupy nucleosome-depleted regions.

Conclusions

The sequences that specify origins are evolutionarily plastic, with low complexity nucleosome-excluding sequences functioning in S. pombe and S. octosporus, and binding sites for trans-acting nucleosome-excluding proteins functioning in S. japonicus. Furthermore, chromosome-scale variation in replication timing is conserved independently of origin location and via a mechanism distinct from known heterochromatic effects on origin function. These results are consistent with a model in which origins are simply the nucleosome-depleted regions of the genome with the highest affinity for the origin recognition complex. This approach provides a general strategy for understanding the mechanisms that define DNA replication origins in eukaryotes.     

Mating pheromone-like diffusible factor released by Schizosaccharomyces pombe

We demonstrate that a diffusible factor is secreted by h⁻ cells of the fission yeast Schizosaccharomyces pombe, whose mating pheromones have not been described. This factor, tentatively named the h⁻-factor, affects h⁺ S. pombe cells and induces their elongation under nitrogen-depleted conditions. Circumstantial evidence suggests its physiological significance in the mating process. Despite their sterility, h⁻ ras1⁻ cells secrete this factor. However, h⁺ ras1⁻ cells have apparently lost the ability to respond to it. This may suggest that the gene product of S. pombe ras1, a homologue of mammalian ras oncogenes, is involved in the mechanism for responding to mating pheromones.     

Mate and fuse: how yeast cells do it

Many cells are able to orient themselves in a non-uniform environment by responding to localized cues. This leads to a polarized cellular response, where the cell can either grow or move towards the cue source. Fungal haploid cells secrete pheromones to signal mating, and respond by growing a mating projection towards a potential mate. Upon contact of the two partner cells, these fuse to form a diploid zygote. In this review, we present our current knowledge on the processes of mating signalling, pheromone-dependent polarized growth and cell fusion in Saccharomyces cerevisiae and Schizosaccharomyces pombe, two highly divergent ascomycete yeast models. While the global architecture of the mating response is very similar between these two species, they differ significantly both in their mating physiologies and in the molecular connections between pheromone perception and downstream responses. The use of both yeast models helps enlighten both conserved solutions and species-specific adaptations to a general biological problem.     

Signal transduction during mating and meiosis in S. pombe

When starved, the fission yeast Schizosaccharomyces pombe responds by producing mating factors or pheromones that signal to cells of the opposite sex to initiate mating. Like its distant relative Saccharomyces cerevisiae, cells of the two mating types of S. pombe each produce a distinct pheromone that binds to receptors on the opposite cell type to induce the morphological changes required for mating. While the pathways are basically very similar in the two yeasts, pheromone signalling in S. pombe differs in several important ways from that of the more familiar budding yeast. In this article, Olaf Nielsen describes the pheromones and their effects in S. pombe, and compares the signalling pathways of the two yeasts.     

Insights into the ecology of Schizosaccharomyces species in natural and artificial habitats

The fission yeast genus Schizosaccharomyces contains important model organisms for biological research. In particular, S. pombe is a widely used model eukaryote. So far little is known about the natural and artificial habitats of species in this genus. Finding out where S. pombe and other fission yeast species occur and how they live in their habitats can promote better understanding of their biology. Here we investigate in which substrates S. pombe, S. octosporus, S. osmophilus and S. japonicus are present. To this end about 2100 samples consisting of soil, tree sap fluxes, fresh fruit, dried fruit, honey, cacao beans, molasses and other substrates were analyzed. Effective isolation methods that allow efficient isolation of the above mentioned species were developed. Based on the frequency of isolating different fission yeast species in various substrates and on extensive literature survey, conclusions are drawn on their ecology. The results suggest that the primary habitat of S. pombe and S. octosporus is honeybee honey. Both species were also frequently detected on certain dried fruit like raisins, mango or pineapple to which they could be brought by the honey bees during ripening or during drying. While S. pombe was regularly isolated from grape mash and from fermented raw cacao beans S. octosporus was never isolated from fresh fruit. The main habitat of S. osmophilus seems to be solitary bee beebread. It was rarely isolated from raisins. S. japonicus was mainly found in forest substrates although it occurs on fruit and in fruit fermentations, too.

     

Mating of the fission yeast occurs independently of pmd1 + gene product,a structural homologue of budding yeast STE6 and mammalian P-glycoproteins

The pmd1 ⁺, a multidrug resistance gene of the fission yeast Schizosaccharomyces pombe, encodes a protein similar to the budding yeast Saccharomyces cerevisiae STE6 gene product and mammalian P-glycoproteins. The STE6 protein is a membrane transporter of a-factor, a mating pheromone of a-type S. cerevisiae, which is structurally related to M-factor of the fission yeast. However, heterothallic or homothallic pmd1 null mutant cells of S. pombe, which were constructed by means of gene disruption, showed no significant decrease in the mating abilities. On the other hand, the multidrug resistance conferred by the pmd1 ⁺ was overcome by the treatment with verapamil, a typical inhibitor of mammalian P-glycoproteins. These results indicate that the pmd1 ⁺ gene product is functionally similar to mammalian P-glycoproteins, rather than to the budding yeast STE6.     

Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating

During the life cycle of heterothallic tetrapolar Agaricomycetes such as Lentinula edodes (Berk.) Pegler, the mating type system, composed of unlinked A and B loci, plays a vital role in controlling sexual development and resulting formation of the fruit body. L. edodes is produced worldwide for consumption and medicinal purposes, and understanding its sexual development is therefore of great importance. A considerable amount of mating type factors has been indicated over the past decades but few genes have actually been identified, and no complete genetic structures of L. edodes B mating-type loci are available. In this study, we cloned the matB regions from two mating compatible L. edodes strains, 939P26 and 939P42. Four pheromone receptors were identified on each new matB region, together with three and four pheromone precursor genes in the respective strains. Gene polymorphism, phylogenetic analysis and distribution of pheromone receptors and pheromone precursors clearly indicate a bipartite matB locus, each sublocus containing a pheromone receptor and one or two pheromone precursors. Detailed sequence comparisons of genetic structures between the matB regions of strains 939P42, 939P26 and a previously reported strain SUP2 further supported this model and allowed identification of the B mating type subloci borders. Mating studies confirmed the control of B mating by the identified pheromone receptors and pheromones in L. edodes.     

An RNA Binding Protein Negatively Controlling Differentiation in Fission Yeast

The fission yeast Schizosaccharomyces pombe starts sexual development when starved for nutrients and simultaneously activated by mating pheromones. We have identified a new gene regulating the onset of this process. This gene, called nrd1⁺, encodes a typical RNA binding protein that preferentially binds poly(U). Deletion of nrd1⁺ causes cells to initiate sexual development without nutrient starvation. We have found that the biological role of nrd1⁺ is to block the onset of sexual development by repressing the Ste11-regulated genes essential for conjugation and meiosis until cells reach a critical level of starvation.     

The model unicellular eukaryote, <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The fission yeast Schizosaccharomyces pombe has long been a model organism for studies of eukaryotic cells, winning renown especially for studies of the cell cycle. Now that its genome has been sequenced, S. pombe is ready to assume its rightful place in the pantheon of small eukaryotic giants.     

The Role of the RACK1 Ortholog Cpc2p in Modulating Pheromone-Induced Cell Cycle Arrest in Fission Yeast

The detection and amplification of extracellular signals requires the involvement of multiple protein components. In mammalian cells the receptor of activated C kinase (RACK1) is an important scaffolding protein for signal transduction networks. Further, it also performs a critical function in regulating the cell cycle by modulating the G₁/S transition. Many eukaryotic cells express RACK1 orthologs, with one example being Cpc2p in the fission yeast Schizosaccharomyces pombe. In contrast to RACK1, Cpc2p has been described to positively regulate, at the ribosomal level, cells entry into M phase. In addition, Cpc2p controls the stress response pathways through an interaction with Msa2p, and sexual development by modulating Ran1p/Pat1p. Here we describe investigations into the role, which Cpc2p performs in controlling the G protein-mediated mating response pathway. Despite structural similarity to Gβ-like subunits, Cpc2p appears not to function at the G protein level. However, upon pheromone stimulation, cells overexpressing Cpc2p display substantial cell morphology defects, disorientation of septum formation and a significantly protracted G₁ arrest. Cpc2p has the potential to function at multiple positions within the pheromone response pathway. We provide a mechanistic interpretation of this novel data by linking Cpc2p function, during the mating response, with its previous described interactions with Ran1p/Pat1p. We suggest that overexpressing Cpc2p prolongs the stimulated state of pheromone-induced cells by increasing ste11 gene expression. These data indicate that Cpc2p regulates the pheromone-induced cell cycle arrest in fission yeast by delaying cells entry into S phase.     

A Minisatellite Sequence within the Propeptide Region of the Vacuolar Carboxypeptidase Y Gene of Schizosaccharomyces pombe

We describe the presence of a minisatellite sequence that displays length polymorphisms in the fission yeast Schizosaccharomyces pombe. The minisatellite sequence was found to reside within the propeptide region of the vacuolar carboxypeptidase Y gene. The minisatellite sequence, which was found only at a single locus, was mitotically stable and displayed length polymorphisms between the two varieties of S. pombe (S. pombe var. pombe and S. pombe var. malidevorans). The minisatellite sequence, however, appeared to be species specific and was absent in other members of the Schizosaccharomyces genus. This report constitutes the first experimental demonstration of the presence of such sequences in yeasts.     

Interspecific protoplast fusion in fission yeasts

Protoplasts of auxotrophic mutants isolated fromSchizosaccharomyces pombe andSchizosaccharomyces octosporus were fused. The fusion products were capable of complementation and growth on minimal medium, but the morphological observations suggested a disturbed physiological balance, which was reflected in low viability, osmotic sensitivity, and the formation of incomplete cell wall. Their asymmetric segregation indicated thatSchizosaccharomyces octosporus might be dominant in the fused cells.