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

Mating pheromone signaling is essential for conjugation between haploid cells of P-type (P-cells) and haploid cells of M-type (M-cells) in Schizosaccharomyces pombe. 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”.     

Mating pheromones of the fission yeast Schizosaccharomyces pombe: purification and structural characterization of M-factor and isolation and analysis of two genes encoding the pheromone.

Conjugation in the fission yeast Schizosaccharomyces pombe is controlled by the action of mating pheromones. Here I describe the isolation and characterization of M-factor, the pheromone released by M-type cells. M-factor is a nanopeptide in which the carboxy-terminal cysteine residue is carboxy-methylated and S-alkylated, probably with a farnesyl residue: Tyr-Thr-Pro-Lys-Val-Pro-Tyr-Met-Cys(S-farnesyl)-OCH3. Evidence for this structure was obtained by amino acid analysis, mass spectrometry and tandem mass spectrometry of the native M-factor. Two genes encoding the M-factor were also identified and characterized. It appears that M-factor is synthesized as a larger precursor which is post-translationally cleaved and modified to yield the active pheromone. The proposed modifications are consistent with mechanisms known to exist in other yeast and higher eukaryotes.     

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 a-factor pheromone of Saccharomyces cerevisiae is essential for mating.

The Saccharomyces cerevisiae pheromone a-factor is produced by a cells and interacts with alpha cells to cause cell cycle arrest and other physiological responses associated with mating. Two a-factor structural genes, MFA1 and MFA2, have been previously cloned with synthetic probes based on the a-factor amino acid sequence (A. Brake, C. Brenner, R. Najarian, P. Laybourn, and J. Merryweather, cited in M.-J. Gething [ed.], Protein transport and secretion, 1985). We have examined the function of these genes in a-factor production and mating by construction and analysis of chromosomal null mutations. mfa1 and mfa2 single mutants each exhibited approximately half the wild-type level of a-factor activity and were proficient in mating, whereas the mfa1 mfa2 double mutant produced no a-factor and was unable to mate. These results demonstrate that both genes are functional, that each gene makes an equivalent contribution to the a-factor activity and mating capacity of a cells, and that a-factor plays an essential role in mating. Strikingly, exogenous a-factor did not alleviate the mating defect of the double mutant, suggesting that an a cell must be producing a-factor to be an effective mating partner.     

Mutations in a gene encoding the alpha subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling.

Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identical to the gene GPA1 (also known as SCG1), recently shown to be highly homologous to genes encoding the alpha subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpa1 mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones. Mutational disruption of G-protein activity causes cell-cycle arrest in G1, deposition of mating-specific cell surface agglutinins, and induction of pheromone-specific mRNAs, all of which are responses to pheromone in wild-type cells. In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor. A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.     

Identification of Differentially Expressed Genes in the Pheromone Glands of Mated and Virgin Bombyx mori by Digital Gene Expression Profiling

Background

Mating decreases female receptivity and terminates sex pheromone production in moths. Although significant progress has been made in elucidating the mating-regulated inactivation of pheromone biosynthesis-activating neuropeptide (PBAN) secretion, little is known about the mating induced gene expression profiles in pheromone glands (PGs). In this study, the associated genes involved in Bombyx mori mating were identified through digital gene expression (DGE) profiling and subsequent RNA interference (RNAi) to elucidate the molecular mechanisms underlying the mating-regulated gene expression in PGs.

Results

Eight DGE libraries were constructed from the PGs of mated and virgin females: 1 h mating (M1)/virgin (V1) PGs, 3 h mating (M3)/virgin (V3) PGs, 24 h mating (M24)/virgin (V24) PGs and 48 h mating (M48)/virgin (V48) PGs (M48 and V48). These libraries were used to investigate the gene expression profiles affected by mating. DGE profiling revealed a series of genes showing differential expression in each set of mated and virgin female samples, including immune-associated genes, sex pheromone synthesis-associated genes, juvenile hormone (JH) signal-associated genes, etc. Most interestingly, JH signal was found to be activated by mating. Application of the JH mimics, methoprene to the newly-emerged virgin females leaded to the significant reduction of sex pheromone production. RNAi-mediated knockdown of putative JH receptor gene, Methoprene tolerant 1 (Met1), in female pupa resulted in a significant decrease in sex pheromone production in mature females, suggesting the importance of JH in sex pheromone synthesis.

Conclusion

A series of differentially expressed genes in PGs in response to mating was identified. This study improves our understanding of the role of JH signaling on the mating-elicited termination of sex pheromone production.     

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.     

Remarkably simple sequence requirement of the M-factor pheromone of Schizosaccharomyces pombe

The mating reaction is triggered by specific pheromones in a wide variety of organisms. Small peptides are used as mating pheromones in yeasts and fungi. In the fission yeast Schizosaccharomyces pombe, M-factor is a C terminally farnesylated nonapeptide secreted from M-cells, and its counterpart, P-factor, is a simple peptide composed of 23 amino acids. The primary structure requirements for the biological activity of pheromone peptides remain to be elucidated. Here, we conducted comprehensive substitution of each of the amino acids in M-factor peptide and inspected the mating ability of these missense mutants. Thirty-five sterile mutants were found among an array of 152 mutants with single amino acid substitutions. Mapping of the mutation sites clearly indicated that the sterile mutants were associated exclusively with four amino acid residues (VPYM) in the carboxyl-terminal half. In contrast, the substitution of four amino-terminal residues (YTPK) with any amino acid had no or only a slightly deleterious effect on mating. Furthermore, deletion of the three N-terminal residues caused no sterility, although truncation of a fourth residue had a marked effect. We conclude that a farnesylated hexapeptide (KVPYMC(Far)-OCH(3)) is the minimal M-factor that retains pheromone activity. At least 15 nonfunctional peptides were found to be secreted, suggesting that these mutant M-factor peptides are no longer recognized by the cognate receptor.     

Far3 and five interacting proteins prevent premature recovery from pheromone arrest in the budding yeast Saccharomyces cerevisiae

In budding yeast, diffusible mating pheromones initiate a signaling pathway that culminates in several responses, including cell cycle arrest. Only a handful of genes required for the interface between pheromone response and the cell cycle have been identified, among them FAR1 and FAR3; of these, only FAR1 has been extensively characterized. In an effort to learn about the mechanism by which Far3 acts, we used the two-hybrid method to identify interacting proteins. We identified five previously uncharacterized open reading frames, dubbed FAR7, FAR8, FAR9, FAR10, and FAR11, that cause a far3-like pheromone arrest defect when disrupted. Using two-hybrid and coimmunoprecipitation analysis, we found that all six Far proteins interact with each other. Moreover, velocity sedimentation experiments suggest that Far3 and Far7 to Far11 form a complex. The phenotype of a sextuple far3far7-far11 mutant is no more severe than any single mutant. Thus, FAR3 and FAR7 to FAR11 all participate in the same pathway leading to G1 arrest. These mutants initially arrest in response to pheromone but resume budding after 10 h. Under these conditions, wild-type cells fail to resume budding even after several days whereas far1 mutant cells resume budding within 1 h. We conclude that the FAR3-dependent arrest pathway is functionally distinct from that which employs FAR1.