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The asymmetric chemical structures of two mating pheromones reflect their differential roles in mating of fission yeast
Taisuke Seike, Hiromi Maekawa, Taro Nakamura, Chikashi Shimoda
Journal of Cell Science 2019 132: jcs230722 doi: 10.1242/jcs.230722 Published 25 June 2019

ABSTRACT

In the fission yeast Schizosaccharomyces pombe, the mating reaction is controlled by two mating pheromones, M-factor and P-factor, secreted by M- and P-type cells, respectively. M-factor is a C-terminally farnesylated lipid peptide, whereas P-factor is a simple peptide. To examine whether this chemical asymmetry in the two pheromones is essential for conjugation, we constructed a mating system in which either pheromone can stimulate both M- and P-cells, and examined whether the resulting autocrine strains can mate. Autocrine M-cells responding to M-factor successfully mated with P-factor-lacking P-cells, indicating that P-factor is not essential for conjugation; by contrast, autocrine P-cells responding to P-factor were unable to mate with M-factor-lacking M-cells. The sterility of the autocrine P-cells was completely restored by expressing the M-factor receptor. These observations indicate that the different chemical characteristics of the two types of pheromone, a lipid and a simple peptide, are not essential; however, a lipid peptide might be required for successful mating. Our findings allow us to propose a model of the differential roles of M-factor and P-factor in conjugation of S. pombe.

This article has an associated First Person interview with the first author of the paper.

INTRODUCTION

Sexual reproduction accelerates evolution by increasing the diversity of the gene pool. The fission yeast Schizosaccharomyces pombe has two mating types (sexes): h+ (Plus) and h− (Minus) (Gutz et al., 1974; Egel, 1989, 2004). Under nitrogen starvation, two haploid cells of opposite mating-type mate to produce a diploid zygote (Egel, 1971). The mating reaction is controlled by pheromonal communication, as illustrated in Fig. 1A. M-factor, secreted by M-cells, is a C-terminally farnesylated and o-methylated nonapeptide (Davey, 1991, 1992), whereas P-factor, secreted by P-cells, is a simple peptide of 23 amino acids (Imai and Yamamoto, 1994). These pheromone peptides are accepted by a specific G-protein-coupled receptor (GPCR); Mam2 for P-factor (Kitamura and Shimoda, 1991) and Map3 for M-factor (Tanaka et al., 1993). Activation of the G-protein associated with the receptor, Gpa1, transmits signals through a MAPK cascade comprising Byr2 (MAPKKK), Byr1 (MAPKK) and Spk1 (MAPK), resulting in the transcription of pheromone-induced genes essential for mating (Obara et al., 1991; Xu et al., 1994; Barr et al., 1996). Thus, the pheromone signaling pathway downstream of the activated GPCRs is shared in both cell types.

A key function of mating pheromones in yeasts is to guide a mating projection called a ‘shmoo’ (Moore et al., 2008). During mating, a cell senses a gradient of pheromones secreted by an opposite mating-type cell (Jackson and Hartwell, 1990; Segall, 1993), and forms a shmoo that is directed toward the center of the pheromone source. This polarized growth of S. pombe is regulated by the central regulator of cell polarity Cdc42, in complex with the guanine nucleotide exchange factor Scd1 and the scaffold protein Scd2 (Bendezú and Martin, 2013). Cells first adhere to opposite mating-type cells to form aggregates, which may help to stabilize the pheromone gradient, especially in liquid environments (Seike et al., 2013). Two mating-type-specific glycoproteins, Mam3 of M-cells (Xue-Franzén et al., 2006) and Map4 of P-cells (Sharifmoghadam et al., 2006; Xue-Franzén et al., 2006), are responsible for this sexual agglutination. Because cell fusion does not occur among cells lacking these proteins, even on solid medium (Sharifmoghadam et al., 2006; Seike et al., 2013), the close cell-to-cell contact mediated by Mam3 and Map4 is essential for cell fusion in S. pombe.

Pheromones in yeasts also play a role in the choice of a favorable mating partner. For example, cells of the budding yeast Saccharomyces cerevisiae choose a mating partner that produces the strongest pheromone signal (Jackson and Hartwell, 1990; Rogers and Greig, 2009). This is probably because the Cdc42 polarization complex forms at the highest concentration of pheromones from which the polarized growth starts (Bendezú and Martin, 2013). In fact, addition of exogenous pheromone to cells unable to produce their own pheromone does not rescue their ability to mate (Kjaerulff et al., 1994; Seike et al., 2013). Our previous study showed that the mating pheromones of S. pombe have a distal and proximal mode of action (Seike et al., 2013); that is, the general secretion of pheromones first induces sexual agglutination to increase cell density (‘distal’ action), and then locally secreted pheromones establish the polarity to influence mating partner choice (‘proximal’ action). Thus, mating steps regulated by these two different modes of pheromone action lead to successful conjugation.

In S. pombe, the pheromones for two mating types differ with respect to several properties. M-factor is a lipid-modified peptide (hydrophobic), and is specifically secreted by the ATP-binding cassette (ABC) transporter Mam1 (Christensen et al., 1997; Davey et al., 1997; Kjaerulff et al., 2005), whereas P-factor is an unmodified peptide (hydrophilic) that is secreted by exocytosis (Imai and Yamamoto, 1994) (see Fig. 1A). This chemical asymmetry between mating pheromone peptides is widely conserved across ascomycetes (Table S1) (Martin et al., 2011), although previous studies have suggested that pheromone asymmetry may not be required for mating in S. cerevisiae (Gonçalves-Sá and Murray, 2011). Furthermore, the biological significance of such asymmetric modifications of mating pheromones is not fully understood.

In this study, we have investigated the necessity of the chemical asymmetry of pheromone peptides in S. pombe by constructing autocrine cells that respond to their own secreted pheromone. We found that autocrine M-cells can mate with P-factor-lacking P-cells, whereas autocrine P-cells cannot mate with M-factor-lacking M-cells. Our findings clearly indicate that the chemical asymmetry of pheromones is not necessarily required for cell fusion, whereas a lipid peptide may be essential for successful mating in S. pombe. We propose a model in which lipid peptide pheromones (i.e. M-factor) secreted locally from one cell might become concentrated near a cell of the opposite mating type, resulting in successful conjugation.

RESULTS AND DISCUSSION

Construction of haploid autocrine M- and P-strains

To investigate the necessity of the chemical asymmetry of the S. pombe mating pheromones, first, we attempted to construct a self-activated M-strain that responds to its own pheromone (i.e. M-factor). The 5′-upstream sequence of the mam2+ gene, which is likely to contain the promoter, was cloned into the plasmid pFA6a-hphMX6, which is used for chromosome integration (Fig. 1B). The activity of the promoter in M-type cells was confirmed by a lacZ fusion construct (Fig. S1). The pFA6a-hphMX6 (mam2PRO-map3) plasmid was integrated at the map3 region of chromosome I of the heterothallic mam2Δ M-strain FS324. Transcription of the map3+ gene in the resultant M-strain (FS600) was examined by semiquantitative RT-PCR. As shown in Fig. 1B, the map3 mRNA level markedly increased after 1 day of incubation of this M-strain in nitrogen-free medium (SSL−N), indicating that the M-factor receptor (map3+) was ectopically expressed. Therefore, we considered this self-activated M-strain to be an autocrine M-strain (Auto-M).

Fig. 1.
Fig. 1.

Strategy for creating autocrine haploid M- and P-strains. (A) Illustration of mating pheromone signaling in S. pombe. (B) Construction of an autocrine haploid M-strain (FS600), in which Map3 is expressed by the mam2 promoter (1027 bp) in M-cells instead of native Mam2. (C) Construction of an autocrine haploid P-strain (FS683), in which Mam2 is expressed by the map3 promoter (1501 bp) in P-cells instead of native Map3. Semiquantitative RT-PCR shows ectopic mating-type-specific expression of map3+ and mam2+ in the respective autocrine M- and P-strains. The act1+ gene was used as a control. Graphs in B,C show mean±s.d. (n=3).

Next, we constructed a self-activated P-strain that responds to its own pheromone P-factor. To express the P-factor receptor gene (mam2+) in P-cells, the 5′-upstream sequence of the map3+ gene, likely to contain the promoter function, was cloned into the plasmid pFA6a-hphMX6 (Fig. 1C). The map3PRO-mam2+ fusion gene was integrated in the authentic mam2+ region on chromosome I of the heterothallic map3Δ P-strain FS618. In the resultant P-strain (FS683), an increase in mam2 mRNA was confirmed by semiquantitative RT-PCR after 1 day of incubation in SSL−N medium (Fig. 1C). We thus conclude that an autocrine P-strain expressing only the P-factor receptor gene (mam2+) had been successfully constructed.

Pheromone-induced polarized growth and sexual agglutinability in autocrine M- and P-cells

Next, we evaluated self-activation of the autocrine cells by their own secreted M-factor or P-factor by observing shmoo formation (marked elongation of polarized cells). On solid medium (MEA), the two autocrine haploid strains (FS600 and FS683) showed distinct shmoo formations (Fig. 2A), which were quantitatively assayed by measuring the ratio of cell length (L) to cell width (W) of individual cells (see Materials and Methods). Whereas the L:W ratio in two wild-type haploid strains (FS324 and FS618) did not increase during 2 days of incubation, the L:W ratio of the autocrine haploid cells began to increase gradually after incubation started (Fig. 2A). Based on a definition of shmooing as an L:W ratio of more than 3.0 at 2 days, the autocrine M- and P-cells formed shmoos at a frequency of 26% and 44% (n=200 cells, each), respectively (Fig. 2B). In addition to shmooing, the autocrine M-cells were readily autolysed, as previously reported (Dudin et al., 2016), at a frequency of 39.3±3.7% (mean±s.d., n=1180), although lysis of the autocrine P-cells was not observed. This might be due to differences in the mechanisms of cellular pheromone signaling in M- and P-cells. Taken together, these data clearly demonstrate that the autocrine haploid cells are self-activated by their own pheromones.

Fig. 2.
Fig. 2.

Polarized growth and sexual agglutination in autocrine M- and P-strains. (A) Cell morphology after induction of mating. The arrow highlights an autolyzed cell. Scale bars: 10 µm. The ability for polarized growth was assessed by determining the length (L) to width (W) ratio of individual cells, and the frequency distribution of the L:W ratio is shown. Cells with a ratio of 3.0 or higher were defined as shmooing cells. For each strain, 200 cells were counted on each day of incubation. Data are given as the mean±s.d. (B) Measurement of the agglutination index (AI). Cultures in 3.5-cm petri dishes were gently shaken. The mean±s.d. of triplicate samples is presented.

Conjugation of yeast cells commences after sexual agglutination in response to mating pheromones (Miyata et al., 1997; Seike et al., 2013). We therefore examined sexual agglutinability in the autocrine M- and P-cells. We assayed agglutination intensity in two homothallic autocrine M- and P-strains (FS393 and TS167) cultured in SSL−N liquid medium. Two receptor-less homothallic strains (FS323 and TS160) were examined as a negative control. In both autocrine strains, strong agglutination was induced after 8 h of incubation (Fig. 2B), suggesting that Mam3 and Map4 proteins were fully induced by their own pheromones at the cell surface. Our previous study showed that expression of the P-type-specific adhesin Map4 is completely dependent on pheromone signaling, whereas that of the M-type-specific Mam3 is induced only by starvation and further enhanced by pheromone treatment (Xue-Franzén et al., 2006; Seike et al., 2013). As expected, the receptor-lacking cells showed no agglutination (no visible aggregates, Fig. 2B). Collectively, these data indicate that both autocrine strains are fully self-activated, as judged by shmooing and sexual agglutination.

Distinct differences in the mating ability of autocrine M- and P-cells

The mating competency of heterothallic autocrine M-cells (FS600) was examined by crossing with either wild-type heterothallic P-cells (FS127) or heterothallic P-factor-lacking P-cells (FY23424). As shown in Fig. 3A,B, the autocrine M-cells completed conjugation with both wild-type and P-factor-lacking P-cells, although the mating frequency was significantly lower than that observed between wild-type pairs (40.7±3.8% versus 14.7±2.6%; Fig. 3B; Table S2), consistent with previous observations (Kitamura et al., 1996; Dudin et al., 2016). This result indicates that the asymmetry in the chemical nature of the two different mating pheromones is not essential for mating because S. pombe cells mated by using only M-factor.

Fig. 3.
Fig. 3.

Mating ability of autocrine M- and P-strains. (A) Morphology of asci (arrows) in various combinations of two haploid strains. Scale bar: 5 µm. (B) Summary of the mating ability between two haploid strains seen in A. Mating (%) was determined by measuring the frequency of zygotes. Data are the mean±s.d. (n>300, each). ***P<0.001. The exact percentages of mating are reported in Table S2. (C) Recombinant frequency of wild-type and autocrine strains. Heterothallic haploid strains were differentially marked by kanMX6, hphMX6 or natMX6 drug-resistant markers. The mean±s.d. of triplicate samples is presented. (D) Effects on mating frequency of the co-expression of Map3 and Mam2 in autocrine P-cells (TS159). Expression of map3+ in the autocrine P-strain restored mating ability (43.9±2.3%) to a level comparable to that of the wild-type pair (45.7±4.9%). Data are given as the mean±s.d. (n>300, each); n.s., not significant. Scale bar: 5 µm.

To examine more quantitatively the lower mating ability of autocrine M-cells as compared with wild-type M-cells, wild-type P-cells (FS405), wild-type M-cells (FS424) and autocrine M-cells (FS600) were mixed in SSL−N at a cell number ratio of 2:1:1, and then followed by performing a quantitative assay of hybrid formation (see Materials and Methods). As shown in Fig. 3C, the recombinant frequency between autocrine M-cells and wild-type P-cells was significantly lower than that of the wild-type combination; that is, the frequency was about one-thirtieth of that of wild-type pairs. It is possible that the low selectability of autocrine M-cells as a mating partner might be due to the fixed (‘default’) location of the polarity complex at cell poles.

The mating ability of autocrine P-cells (FS683) was also tested. Unexpectedly, the autocrine P-cells did not mate with either wild-type or M-factor-lacking M-cells (Fig. 3A,B). Furthermore, the ability of the autocrine P-cells to mate with M-cells was not restored by exogenously added synthetic P-factor (5 µM), and no diploid zygotes were observed between autocrine M- and P-cells by microscopy (Table S2). The sterility of the autocrine P-cells was further confirmed by the lack of recombinant colonies in a mixed culture of autocrine P-cells (FS683), wild-type P-cells (FS504) and wild-type M-cells (FS703) (Fig. 3C). Finally, we verified that autocrine P-cells themselves generate a sufficiently strong pheromone signal to induce mating, by both inactivating the P-factor-degrading protease Sxa2 and adding exogenous synthetic P-factor to the autocrine P-cells (Imai and Yamamoto, 1992) (Fig. S3). Taken together, these experiments suggest that the ability of P-cells to undergo cell fusion is likely to require stimulation by M-factor, but not P-factor.

Establishment of cell polarity during mating

Our above findings suggest that cell fusion requires the correct localization of secreted pheromones. Previously, Bendezú and Martin (2013) reported that the Cdc42 forms dynamic zones of activity at distinct locations over time prior to cell fusion, and that these zones contain Mam1. We therefore observed the localization of GFP-tagged Mam1 and Map3 proteins during mating in the wild-type cells. As expected, the fluorescence signals were mostly localized at the conjugation tip [Mam1 at the contact site, 86% (n=44); Map3 at the contact site, 88% (n=24); Fig. S2], consistent with previous data (Dudin et al., 2016). To observe the localization of Mam1 in more detail, Mam1–3×GFP was expressed together with mCherry–Psy1, a marker of the forespore membrane (Nakamura et al., 2001). We found that the Mam1 signal was concentrated at the fusion site on the membrane [co-localization of GFP and mCherry signal, 100% (n=29); Fig. S2]. In addition, we noticed that the Mam2 receptor for P-factor was frequently localized at the conjugation tip [Mam2 at the contact site, 82% (n=27); Fig. S2]. These observations suggest that the local concentration of transporter and receptors at the fusion site leads to secure conjugation between mating partners.

We therefore considered that, for mating competency, P-cells might require local activation of the Map3 receptor. To examine this hypothesis, we tested the effect of expressing Map3 in autocrine P-cells (TS159), in addition to the P-factor receptor Mam2. Notably, this strain mated with wild-type M-cells at a level comparable to that of wild-type cell pairs (Fig. 3D). This result clearly indicates that the expression of Map3 in autocrine P-cells completely restores mating ability, suggesting that local signaling through Map3 is essential at the contact site between M- and P-cells. The cell polarity of P-cells is probably established by receiving M-factor secreted locally by M-cells. The haploid P-strain (TS159) co-expressing both Map3 and Mam2 produced a shorter shmoo as compared with autocrine haploid P-cells (FS683) (L:W ratio of 2.8±0.7 versus 1.9±0.4; Figs 2B and 3D), probably because the two receptors compete for the same G protein, Gpa1, within the cell.

Working hypothesis of conjugation in S. pombe

On the basis of the above experimental data, we propose the following model of the mating process in S. pombe (Fig. 4). In the first step, pheromone peptides are secreted in response to an environmental cue, such as nutritional starvation (Stage I). The hydrophilic simple peptide, P-factor, is easily diffused into the surrounding medium, and thus it will reach cells that are far away, enabling those cells to become rapidly aware of the existence of favorable mating partners. Next, the relatively low concentrations of pheromones secreted into the milieu enhance the production of agglutinin for cell-to-cell contact, resulting in cell agglutination (Stage II). This physical contact between cells raises local pheromone concentrations at the cell surface, which fixes the active cell polarity complex, containing Cdc42, at the contact site (Bendezú and Martin, 2013). The M-factor transporter Mam1 is localized at the polarization site in M-cells and, simultaneously, the M-factor receptor Map3 is located (or locally activated) near Mam1 in P-cells; thus, M-factor might be mainly secreted at the contact site. The hydrophobic lipid peptide M-factor is thought to diffuse for a shorter distance into the surrounding medium, and thus its concentration might be relatively high near P-cells. This local concentration of M-factor establishes the polarity of P-cells (Stage III), and polarized cell growth is induced. Cells of both mating types are firmly paired at the conjugation tip, and ultimately a pair of cells fuse to form a zygote.

Fig. 4.
Fig. 4.

Working hypothesis of conjugation in S. pombe. The chemical asymmetry in the two mating pheromones reflects their differential roles in conjugation of S. pombe. In Stage I, uniform secretion of pheromone peptides facilitates the expression of pheromone-inducible genes, such as the mating-type-specific agglutinins, Mam3 and Map4 (distal effect). In Stage II, pheromones induce sexual agglutinability in both M- and P-cells, which leads to stable cell-to-cell contact. In Stage III, polarized growth occurs at the contact site after agglutination. Hydrophobic M-factors are temporarily concentrated around P-cells, and these locally secreted M-factors establish the polarity of P-cells (proximal effect), leading to successful mating.

MATERIALS AND METHODS

Strains, media and culture conditions

The S. pombe strains used in this study are listed in Table S3. Standard methods were used for growth, transformation and genetic manipulation (Moreno et al., 1991). S. pombe cells were vegetatively grown in yeast extract (YE) medium supplemented with adenine sulfate (75 mg/l), uracil (50 mg/l), and leucine (50 mg/l). For solid medium, 15 g/l of Bacto Agar (BD Bioscience, Sparks, USA) was added to YE medium (YEA). Antibiotics [G418 (Nacalai Tesque, Kyoto, Japan), Hygromycin B (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and Nourseothricin (Cosmo Bio Co., Ltd., Tokyo, Japan)] were added to YEA at a final concentration of 100 µg/ml. The solid medium used for mating was malt extract agar (MEA). To induce mating in liquid medium, cells were shifted from SSL+N to nitrogen-free medium (SSL−N; minimal sporulation liquid medium without nitrogen) (Egel and Egel-Mitani, 1974; Gutz et al., 1974). Cells were incubated at 30°C for growth and at 28°C for mating, unless stated otherwise.

RNA extraction followed by semiquantitative real-time PCR

Cells [at an optical density at 600 nm (OD600)=0.1] were pre-cultured in YE liquid medium for 20 h at 30°C. Cells at a density of 4×107 cells/ml in 50-ml of SSL−N were continuously shaken at 28°C for 48 h after sampling at the start point (0 h). Next, 1 ml of culture was harvested for RNA extraction using an RNeasy® Mini Kit (Qiagen, Hilden, Germany). DNA digestion was performed to remove contaminating DNA in the solution before RNA clean up. For semiquantitative real-time PCR (RT-PCR), cDNA was synthesized from 500-ng samples of RNA using a SuperScript® VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, USA) according to the manufacturer's protocol. The program for RT-PCR was 94°C for 2 min, followed by 30 cycles of 98°C for 10 s, 58°C for 30 s, and 68°C for 30 s. DNA segments containing map3+, mam2+ and act1+ (control) were amplified by using the following sets of primers: 5′-CTCCATGCCTGTTGTCTTTTGGTG-3′/5′-GCTGCCAAACATAGCAAGCGTAAG-3′; 5′-CTCCACTTAGCAGCAAGACCATTG-3′/5′-GCATCGGACCAAATGGAAATTGC-3′; and 5′-TGCTATCATGCGTCTTGATCTCGC-3′/5′-AAGCGACGTAGCAAAGTTTCTCC-3′, respectively.

Observation of pheromone-induced cell growth

Cells were pre-cultured on a YEA plate at 30°C overnight, resuspended in sterilized water to a cell density of 1×108 cells/ml, and 30-µl aliquots were spotted onto MEA plates, which were incubated for 1–2 days at 28°C. In the experiment in Fig. S3B, cells were grown in SSL+N liquid medium overnight, washed with SSL−N liquid medium three times and then resuspended in SSL−N at a cell density of 4×107 cells/ml. The cells were then treated with synthetic P-factor and incubated for 1–2 days with gentle shaking. Cells were observed under a differential interference contrast (DIC) microscope, and photographs were taken. The length (L) and width (W) of individual cells were measured, and the L:W ratio was determined. In this study, cells with an L:W ratio of 3.0 or higher were defined as shmooing cells. In all experiments, at least 200 cells each were measured.

Quantitative assay of sexual agglutination

The intensity of cell agglutination was photometrically determined as described previously (Seike et al., 2013). In brief, sexual agglutination was induced by shifting cells from SSL+N to SSL−N liquid medium at a density of 4×107 cells/ml at 28°C. Cultures were incubated in 3.5-cm petri dishes (Thermo Fisher Scientific) with gentle shaking. The OD600 of an aliquot of the culture was measured, the cell suspension was then subjected to sonication (20 kHz) for 10 s to disperse cell aggregates completely, and the OD600 was immediately measured again. The agglutination index (AI) was calculated by:Embedded Image This value has been shown to be a function of the mean size of cell aggregates. In our experience, visible agglutination can be seen in samples with an AI of more than 1.1.

Mating frequency between mating partners

Mating frequency was determined as described previously (Seike et al., 2012). Two haploid strains were separately grown on YEA plates overnight and then resuspended in sterilized water to a cell density of 1×108 cells/ml. A 30-µl aliquot (15 µl of each strain) of cell suspension was then spotted onto an MEA plate, which was incubated for 2 days at 28°C. Cells were counted under a DIC microscope as described above. Cell types were classified into four groups: vegetative cells (V), zygotes (Z), asci (A), and free spores (S). The mating frequency was calculated according to the following equation:Embedded Image In general, triplicate samples (at least 1000 cells) were counted, and the mean±s.d. was calculated.

Quantitative assay of hybrid formation

The quantitative assay was performed as described previously (Seike et al., 2013). Heterothallic haploid strains carrying a different drug-resistance marker (kanMX6, hphMX6, or natMX6) on their chromosomes (Seike et al., 2015; Seike et al., 2019) were mixed and then incubated in SSL−N medium for 2 days to induce mating. The cell suspension was diluted and spread on plates containing the appropriate combinations of drugs. The number of colonies was counted after 3 days of incubation. Three separate tests were carried out, and the mean±s.d. was calculated as the recombinant frequency.

Fluorescence imaging

Cells carrying Mam1–3×GFP, Map3–GFP, Mam2–GFP or mCherry–Psy1 were immediately analyzed by fluorescence microscopy without washing or fixation. Z-series images in 0.4-μm steps were captured with a DeltaVision system (Applied Precision) equipped with GFP and TRITC filters (Chroma Technology Corp.), a 100× NA 1.4 UPlanSApo oil immersion objective (IX71; Olympus), and a camera (CoolSNAP HQ) and were quantified/processed with SoftWoRx 3.5.0 (Applied Precision).

Construction of a lacZ fusion gene and assay

A pDB248′-based multi-copy plasmid carrying the mat1-Pm/lacZ fusion gene (named pTA14) (Aono et al., 1994) was reconstructed to carry a mam2 promoter-lacZ fusion construct. In brief, the mat1-Pm promoter sequence was replaced by an ∼1-kb DNA fragment (Chr.I 4779661–4780687) containing the 5′-upstream sequence of the mam2+ gene. The resulting plasmid pTA(mam2PRO-lacZ) was transformed into each of the heterothallic M- and P-strains (FS493 and FS494).

Enzyme activity was calculated as previously described (Miller, 1972). Cells were suspended in Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4·7H2O and 50 mM β-mercaptoethanol) and stored at 80°C before the assay. Cells were permeabilized with chloroform and 0.1% sodium dodecyl sulfate. A solution of o-nitrophenyl-β-galactoside was added to the cell lysate and incubated at 28°C. The reaction mixture was centrifuged to remove cell debris (15,000 g for 5 min) and the optical density of the supernatant was determined at 420 nm (OD420). β-gal activity (Miller units) was calculated according to the following equation:Embedded Image where v is the sample volume and t is the reaction time. The mean of triplicate samples was calculated.

Statistical analysis

All experiments in this study were performed at least three times. The sample numbers used for statistical analysis are reported in the corresponding figure legends. A two-tailed unpaired Student's t-test was used to evaluate the differences between strains. P-values are indicated in the figures (***P<0.001).

Acknowledgements

We thank the National BioResource Project (NBRP), Japan, for providing yeast strains and plasmids.

Footnotes

  • Competing interests

    The authors declare no competing or financial interests.

  • Author contributions

    Conceptualization: T.S., C.S.; Methodology: T.S., H.M., C.S.; Validation: T.S., C.S.; Formal analysis: T.S., H.M., C.S.; Investigation: T.S., H.M., C.S.; Resources: T.S., H.M., T.N., C.S.; Data curation: T.S., C.S.; Writing - original draft: T.S.; Writing - review & editing: H.M., T.N., C.S.; Visualization: T.S., H.M., C.S.; Supervision: C.S.; Funding acquisition: T.S., T.N.

  • Funding

    This work was supported by the Japan Society for the Promotion of Science (JSPS) [KAKENHI Grant-in-Aid for JSPS Fellows JP15J03416 to T.S., Grant-in-Aid for Young Scientists (B) JP17K15181 to T.S., Scientific Research (C) JP15K07057 to T.N.] and in part by a grant for Basic Science Research Projects from The Sumitomo Foundation to T.S.

  • Supplementary information

    Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.230722.supplemental

  • Received February 13, 2019.
  • Accepted June 3, 2019.

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The asymmetric chemical structures of two mating pheromones reflect their differential roles in mating of fission yeast
Taisuke Seike, Hiromi Maekawa, Taro Nakamura, Chikashi Shimoda
Journal of Cell Science 2019 132: jcs230722 doi: 10.1242/jcs.230722 Published 25 June 2019
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