Asymmetric diversification of mating pheromones in fission yeast.

In fungi, mating between partners depends on the molecular recognition of two peptidyl mating pheromones by their respective receptors. The fission yeast Schizosaccharomyces pombe (Sp) has two mating types, Plus (P) and Minus (M). The mating pheromones P-factor and M-factor, secreted by P and M cells, are recognized by the receptors mating type auxiliary minus 2 (Mam2) and mating type auxiliary plus 3 (Map3), respectively. Our recent study demonstrated that a few mutations in both M-factor and Map3 can trigger reproductive isolation in S. pombe. Here, we explored the mechanism underlying reproductive isolation through genetic changes of pheromones/receptors in nature. We investigated the diversity of genes encoding the pheromones and their receptor in 150 wild S. pombe strains. Whereas the amino acid sequences of M-factor and Map3 were completely conserved, those of P-factor and Mam2 were very diverse. In addition, the P-factor gene contained varying numbers of tandem repeats of P-factor (4-8 repeats). By exploring the recognition specificity of pheromones between S. pombe and its close relative Schizosaccharomyces octosporus (So), we found that So-M-factor did not have an effect on S. pombe P cells, but So-P-factor had a partial effect on S. pombe M cells. Thus, recognition of M-factor seems to be stringent, whereas that of P-factor is relatively relaxed. We speculate that asymmetric diversification of the two pheromones might be facilitated by the distinctly different specificities of the two receptors. Our findings suggest that M-factor communication plays an important role in defining the species, whereas P-factor communication is able to undergo a certain degree of flexible adaptation-perhaps as a first step toward prezygotic isolation in S. pombe

ADAM Family Protein Mde10 Is Essential for Development of Spore Envelopes in the Fission Yeast Schizosaccharomyces pombe

We report the identification of Schizosaccharomyces pombe mde10(+) as a gene possessing a FLEX element, which forms a binding site for the meiosis-specific transcription factor Mei4. In fact, mde10(+) is transcribed only in diploid cells that are induced to meiosis in a Mei4-dependent manner. Western blot analysis indicated that the epitope-tagged Mde10 protein accumulates transiently during meiosis and then rapidly decreases. Mde10 is a multidomain protein containing a metalloprotease catalytic domain, a disintegrin domain, a cysteine-rich domain, and membrane-spanning regions, all of which are shared by members of the mammalian ADAM family. A fusion protein of Mde10 and green fluorescent protein localized to the endoplasmic reticulum during meiosis and was located at the peripheral region of spores at the end of meiosis. An mde10Δ deletion mutant showed no apparent defects in meiosis, sporulation, or spore germination. However, the mutant spores exhibited an aberrant surface appearance, in which the ragged outer spore wall was lost to a large extent. Furthermore, mde10Δ spores were found to be less tolerant to ethanol and diethyl ether than were wild-type spores. The mutagenic replacement of the conserved glutamic acid in the putative protease active site with an alanine residue did not affect the surface morphology or the resistance of spores to environmental stress. Our observations indicate that Mde10 is important in the development of the spore envelope, although this function of Mde10 seems to be independent of its metalloprotease activity

Ectopic Overproduction of a Sporulation-Specific Transcription Factor Induces Assembly of Prespore-Like Membranous Compartments in Vegetative Cells of Fission Yeast

Mei4 is a key sporulation-specific transcription factor in fission yeast. Ectopic expression of Mei4 in vegetative cells caused formation of nucleated membranous compartments, which shared common features with normal forespore membranes, thereby perturbing nuclear division. These results suggest why expression of development-specific transcription factors must be strictly controlled

Distal and Proximal Actions of Peptide Pheromone M-Factor Control Different Conjugation Steps in Fission Yeast

<div><p>Mating pheromone signaling is essential for conjugation between haploid cells of P-type (P-cells) and haploid cells of M-type (M-cells) in <i>Schizosaccharomyces pombe</i>. A peptide pheromone, M-factor, produced by M-cells is recognized by the receptor of P-cells. An M-factor-less mutant, in which the M-factor-encoding genes are deleted, is completely sterile. In liquid culture, sexual agglutination was not observed in the mutant, but it could be recovered by adding exogenous synthetic M-factor, which stimulated expression of the P-type-specific cell adhesion protein, Map4. Exogenous M-factor, however, failed to recover the cell fusion defect in the M-factor-less mutant. When M-factor-less cells were added to a mixture of wild-type P- and M-cells, marked cell aggregates were formed. Notably, M-factor-less mutant cells were also incorporated in these aggregates. In this mixed culture, P-cells conjugated preferentially with M-cells secreting M-factor, and rarely with M-factor-less M-cells. The kinetics of mating parameters in liquid culture revealed that polarized growth commenced from the contact region of opposite mating-type cells. Taken together, these findings indicate that M-factor at a low concentration induces adhesin expression, leading to initial cell-cell adhesion in a type of “distal pheromone action”, but M-factor that is secreted directly in the proximity of the adhered P-cells may be necessary for cell fusion in a type of “proximal pheromone action”.</p> </div

Expression of mating-type-specific adhesin proteins, Map4 and Mam3 (A, left).

<p>A heterothallic <i>h</i>⁺ strain (FS103) carrying a <i>map4</i>-<i>GFP</i> fusion gene integrated at the authentic chromosomal locus was precultured in SSL+N for 18 hr, then shifted to SSL−N with or without 200 nM of M-factor, and cultured for 4 hr. (A, right) A heterothallic <i>h</i>⁻<i>mam2Δ</i> strain (SS1463) carrying a <i>mam3-mCherry</i> fusion gene integrated at the original chromosomal locus was incubated for 4 hr in SSL−N (−N). Scale bar, 50 µm. (B) Quantification of Map4-GFP and Mam3-mCherry fluorescence. The intensity was quantified by image analysis using Image-J software. At least 8 different areas were analyzed. Means of arbitrary units (A.U.) with standard deviations are presented. (C) Induced agglutination by overexpression of Map4. Strain SS1290 ectopically overexpressing <i>map4</i>⁺ driven by the <i>nmt1</i> promoter was precultured in MM+N without thiamine for 12 hr and then cultured in MM−N for 4 hr (Map4OP). The same strain was transformed by the pSLF173 empty vector [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069491#B43" target="_blank">43</a>] as a control.</p

Kinetics of sexual agglutination and polar cell growth in liquid culture.

<p>(A) Mating kinetics of a homothallic wild-type strain (L968) cultured in SSL−N liquid medium. At hourly intervals, the intensity of agglutination (AI) was determined. Portions of the culture were subjected to brief sonication and microphotographs were taken. The long (L) and short (S) axes of cells were measured to calculate the L/S ratio for triplicate samples (100 cells each). The frequencies of prezygotes and cells with pointy projections were determined. (B) Microphotographs showing typical cell morphology. Arrows, prezygotes with a pointy mating projection (shown by asterisks). Scale bar, 10 µm. (C, D) Mating kinetics of M-factor-less mutant cells incubated with (C) or without (D) 200 nM M-factor. The experimental procedures are as described in (A). (E) Left, polar cell growth and prezygote formation in M-factor-induced aggregated cells and floating free cells. An M-factor-less strain (FS55) and an M-factor-producing <i>fus1Δ</i> strain (FS123) were incubated with 200 nM M-factor. After 4 hr, aggregates and free cells were isolated. After brief sonication, the L/S ratio was determined (n=1,000 for both strains). Right, frequency of prezygotes formed with <i>fus1Δ</i> (Aggregates <i>vs</i> Free: each <i>p</i>-value was shown, <i>t</i>-test)..</p