Changes in mate recognition through alterations of pheromones and receptors in the multisexual mushroom fungus Schizophyllum commune

Schizophyllum commune has thousands of mating types defined in part by numerous lipopeptide pheromones and their G-protein-coupled receptors. These molecules are encoded within multiple versions of two redundantly functioning B mating-type loci, B alpha and B beta. Compatible combinations of pheromones and receptors, produced by individuals of different B mating types, trigger a pathway of fertilization required for sexual development. Analysis of the B beta 2 mating-type locus revealed a large cluster of genes encoding a single pheromone receptor and eight different pheromones. Phenotypic effects of mutations within these genes indicated that small changes in both types of molecules could significantly alter their specificity of interaction. For example, a conservative amino acid substitution in a pheromone resulted in a gain of function toward one receptor and a loss of function with another. A two-amino-acid deletion from a receptor precluded the mutant pheromone from activating the mutant receptor, yet this receptor was activated by other pheromones. Sequence comparisons provided clues toward understanding how so many variants of these multigenic loci could have evolved through duplication and mutational divergence. A three-step model for the origin of new variants comparable to those found in nature is presented.     

Ligand Recognition in Multiallelic Pheromone Receptors from the Basidiomycete Schizophyllum commune Studied in Yeast

The homobasidiomycete Schizophyllum commune encodes a multiallelic pheromone receptor system that distinguishes more than 20 nonself from at least 2 self pheromones. The well-investigated pheromone response system of the yeast Saccharomyces cerevisiae was used to link the FUS1::lacZ reporter system to the heterologous pheromone receptors from S. commune. To investigate yeast G-protein binding, the unchanged heterologous receptor was compared to constructs carrying an exchange of the 3rd cytoplasmatic loop for the Ste2 sequence. A better coupling could be achieved with the altered constructs. In order to examine activation by single pheromones, an artificial peptide based on the sequence of a new putative pheromone gene, bap2(1), in the Balpha2 mating-type locus encoding the shortest pheromone found so far in fungal mating types was used. Thus, we have reassembled the pheromone recognition of the basidiomycete S. commune and constructed a system ideal for specificity analysis in the yeast S. cerevisiae.     

Multiple Genes Encoding Pheromones and a Pheromone Receptor Define the Bβ1 Mating-Type Specificity in Schizophyllum Commune

The genes defining multiple B mating types in the wood-rotting mushroom Schizophyllum commune are predicted to encode multiple pheromones and pheromone receptors. These genes are clustered in each of two recombinable and independently functioning loci, Bα and Bβ. A difference in specificity at either locus between a mated pair of individuals initiates an identical series of events in sexual morphogenesis. The Bα1 locus was recently found to contain genes predicted to encode three lipopeptide pheromones and a pheromone receptor with a seven-transmembrane domain. These gene products interact in hetero-specific pairs, the pheromone of one Bα specificity with the receptor of any one of the other eight Bα specificities, and are likely to activate a signaling cascade similar to that known for mating in Saccharomyces cerevisiae. We report here that the Bβ1 locus also contains at least three pheromone genes and one pheromone receptor gene, which function similarly to the genes in the Bα1 locus, but only within the series of Bβ specificities. A comparison of the DNA sequences of the Bα1 and Bβ1 loci suggest that each arose from a common ancestral sequence, allowing us to speculate about the evolution of this unique series of regulatory genes.     

The mating-type locus B alpha 1 of Schizophyllum commune contains a pheromone receptor gene and putative pheromone genes.

Analysis of the multispecific B alpha mating-type locus of Schizophyllum commune provided evidence that pheromones and pheromone receptors govern recognition of self versus non-self and sexual development in this homobasidiomycetous fungus. Four subclones of an 8.2 kb genomic fragment carrying B alpha 1 specificity induced B-regulated sexual morphogenesis when introduced into a strain with one of the eight compatible B alpha specificities that are known to exist in nature. One of these clones, which activated all other B alpha specificities, contains a gene termed bar1. The predicted protein product of bar1, as well as that of bar2, a homologous gene isolated from a B alpha 2 strain, has significant homology to known fungal pheromone receptor proteins in the rhodopsin-like superfamily of G protein-linked receptors. The other three active B alpha 1 clones were subcloned further to identify the minimal active element in each clone. Every active subclone contains a putative pheromone gene ending in a signal for possible isoprenylation. A message of approximately 600 bp was observed for one of these genes, bap1(1). This paper presents the first evidence for a system of multiple pheromones and pheromone receptors as a basis for multispecific mating types in a fungus.     

Crossing the boundary between the Balpha and Bbeta mating-type loci in Schizophyllum commune

In this study, genes of the Schizophyllum commune Balpha and Bbeta mating-type loci are shown to be within a few kilobases of each other. The region between the nearest Balpha and Bbeta genes contains many short direct repeats. Predicted amino acid sequences and activity spectra of three pheromones encoded in the Balpha3 mating-type specificity are presented along with a re-evaluation of pheromone activity of many previously reported S. commune lipopeptide pheromones. This analysis showed that S. commune pheromones belong to five subtypes. Several pheromones activate both a Bbeta receptor and a Balpha receptor, a phenomenon previously unrecognized. Clues from mating tests and DNA hybridization led to the cloning of bar8, the gene encoding the Balpha8 pheromone receptor, Bar8. Bar8 is similar in sequence to Bbr1, the Bbeta1 pheromone receptor, and functionally identical to it. These data begin to elucidate the enigmatic recombination patterns previously encountered at the B mating-type complex.     

Self-compatible B mutants in coprinus with altered pheromone-receptor specificities

A successful mating in the mushroom Coprinus cinereus brings together a compatible complement of pheromones and G-protein-coupled receptors encoded by multiallelic genes at the B mating-type locus. Rare B gene mutations lead to constitutive activation of B-regulated development without the need for mating. Here we characterize a mutation that arose in the B6 locus and show that it generates a mutant receptor with a single amino acid substitution (R96H) at the intracellular end of transmembrane domain III. Using a heterologous yeast assay and synthetic pheromones we show that the mutation does not make the receptor constitutively active but permits it to respond inappropriately to a normally incompatible pheromone encoded within the same B6 locus. Parallel experiments carried out in Coprinus showed that a F67W substitution in this same pheromone enabled it to activate the normally incompatible wild-type receptor. Together, our experiments show that a single amino acid replacement in either pheromone or receptor can deregulate the specificity of ligand-receptor recognition and confer a self-compatible B phenotype. In addition, we use the yeast assay to demonstrate that different receptors and pheromones found at a single B locus belong to discrete subfamilies within which receptor activation cannot normally occur.     

Mating types and pheromone recognition in the Homobasidiomycete schizophyllum commune.

In the homobasidiomycete Schizophyllum commune the mating type genes of the B locus encode pheromones and pheromone receptors in multiple allelic specificities. Interaction of non-self pheromones and receptors leads to induction of B-regulated development easily scored in S. commune by the "flat" phenotype which lacks aerial mycelium formation and shows aberrant hyphal morphology. In contrast, self pheromones are not recognized and B-regulated development is not induced. Natural and mutant alleles of receptors have been analyzed for their specificity in transformation assays, and parts of the receptor involved in ligand discrimination can be described. The biological role of pheromone response in S. commune is assumed to be connected to nuclear migration based on the observation that wild-type cells with a receptor gene of different specificity lead to cells capable of nuclear uptake. Other possible roles for pheromone function are discussed.     

Expression and function of sex pheromones and receptors in the homothallic ascomycete Gibberella zeae

In heterothallic ascomycete fungi, idiomorphic alleles at the MAT locus control two sex pheromone-receptor pairs that function in the recognition and chemoattraction of strains with opposite mating types. In the ascomycete Gibberella zeae, the MAT locus is rearranged such that both alleles are adjacent on the same chromosome. Strains of G. zeae are self-fertile but can outcross facultatively. Our objective was to determine if pheromones retain a role in sexual reproduction in this homothallic fungus. Putative pheromone precursor genes (ppg1 and ppg2) and their corresponding pheromone receptor genes (pre2 and pre1) were identified in the genomic sequence of G. zeae by sequence similarity and microsynteny with other ascomycetes. ppg1, a homolog of the Saccharomyces alpha-factor pheromone precursor gene, was expressed in germinating conidia and mature ascospores. Expression of ppg2, a homolog of the a-factor pheromone precursor gene, was not detected in any cells. pre2 was expressed in all cells, but pre1 was expressed weakly and only in mature ascospores. ppg1 or pre2 deletion mutations reduced fertility in self-fertilization tests by approximately 50%. Deltappg1 reduced male fertility and Deltapre2 reduced female fertility in outcrossing tests. In contrast, Deltappg2 and Deltapre1 had no discernible effects on sexual function. Deltappg1/Deltappg2 and Deltapre1/Deltapre2 double mutants had the same phenotype as the Deltappg1 and Deltapre2 single mutants. Thus, one of the putative pheromone-receptor pairs (ppg1/pre2) enhances, but is not essential for, selfing and outcrossing in G. zeae whereas no functional role was found for the other pair (ppg2/pre1).     

Chimeric pheromone receptors in the basidiomycete Schizophyllum commune

The pheromone receptor system of the basidiomycete Schizophyllum commune is capable of ligand discrimination to confer mating specificity. The pheromone receptors of the B alpha locus were investigated for ligand discrimination in a strategy of domain swapping experiments. Several altered phenotypes of chimeric receptors have been found. These include constitutive pheromone receptors which need no ligand for activation of the downstream cascade of events. In addition, receptors still dependent on ligand were identified that had altered pheromone activation profiles, including promiscuous receptors that are activated by pheromones of all nine specificities, including the former self. In addition, highly discriminative receptors were created which are activated by only two of the eight non-self-specificities. The chimeric receptors identify the last third of the receptor as the determinant for B alpha 1 specificity, whereas B alpha 2 specificity resides in noncontiguous domains covering the first and middle parts of the receptor molecule.     

Defining pheromone-receptor signaling in Candida albicans and related asexual Candida species

Candida albicans is an important human fungal pathogen in which sexual reproduction is under the control of the novel white-opaque switch. Opaque cells are the mating-competent form, whereas white cells do not mate but can still respond to pheromones, resulting in biofilm formation. In this study, we first define the domains of the α-pheromone receptor Ste2 that are necessary for signaling in both white and opaque forms. Both cell states require the IC loop 3 (IC3) and the C-terminal tail of Ste2 for the cellular response, whereas the first IC loop (IC1) of Ste2 is dispensable for signaling. To also address pheromone-receptor interactions in related species, including apparently asexual Candida species, Ste2 orthologues were heterologously expressed in Candida albicans. Ste2 receptors from multiple Candida clade species were functional when expressed in C. albicans, whereas the Ste2 receptor of Candida lusitaniae was nonfunctional. Significantly, however, expression of a chimeric C. lusitaniae Ste2 receptor containing the C-terminal tail of Ste2 from C. albicans generated a productive response to C. lusitaniae pheromone. This system has allowed us to characterize pheromones from multiple Candida species and indicates that functional pheromone-receptor couples exist in fungal species that have yet to be shown to undergo sexual mating.     

A constitutively active G-protein-coupled receptor causes mating self-compatibility in the mushroom Coprinus.

In the mushroom Coprinus cinereus, the multiallelic B mating type genes are predicted to encode a large family of seven-transmembrane domain receptors and CaaX-modified pheromones. We have shown that a single amino acid change Q229P in transmembrane domain VI of one receptor confers a self-compatible mating phenotype. Using a heterologous yeast assay, we have demonstrated that this C.cinereus pheromone receptor is a G-protein-coupled receptor and that the Q229P mutation is constitutively activating. A C.cinereus pheromone precursor was processed to an active species specifically in yeast MATa cells and activated the co-expressed wild-type receptor. Yeast cells expressing the wild-type receptor were used to test the activity of synthetic peptides, enabling us to predict the structure of the mature C.cinereus pheromone and to show that the Q229P mutation does not compromise normal receptor function.     

The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein

The STE4 and STE18 genes are required for haploid yeast cell mating. Sequencing of the cloned genes revealed that the STE4 polypeptide shows extensive homology to the beta subunits of mammalian G proteins, while the STE18 polypeptide shows weak similarity to the gamma subunit of transducin. Null mutations in either gene can suppress the haploid-specific cell-cycle arrest caused by mutations in the SCG1 gene (previously shown to encode a protein with similarity to the alpha subunit of G proteins). We propose that the products of the STE4 and STE18 genes comprise the beta and gamma subunits of a G protein complex coupled to the mating pheromone receptors. The genetic data suggest pheromone-receptor binding leads to the dissociation of the alpha subunit from beta gamma (as shown for mammalian G proteins), and the free beta gamma element initiates the pheromone response.     

Pheromones and Pheromone Receptors Are the Primary Determinants of Mating Specificity in the Yeast Saccharomyces Cerevisiae

Saccharomyces cerevisiae has two haploid cell types, a and alpha, each of which produces a unique set of proteins that participate in the mating process. We sought to determine the minimum set of proteins that must be expressed to allow mating and to confer specificity. We show that the capacity to synthesize alpha-factor pheromone and a-factor receptor is sufficient to allow mating by mat alpha 1 mutants, mutants that normally do not express any alpha- or a-specific products. Likewise, the capacity to synthesize a-factor receptor and alpha-factor pheromone is sufficient to allow a ste2 ste6 mutants, which do not produce the normal a cell pheromone and receptor, to mate with wild-type a cells. Thus, the a-factor receptor and alpha-factor pheromone constitute the minimum set of alpha-specific proteins that must be produced to allow mating as an alpha cell. Further evidence that the pheromones and pheromone receptors are important determinants of mating specificity comes from studies with mat alpha 2 mutants, cells that simultaneously express both pheromones and both receptors. We created a series of strains that express different combinations of pheromones and receptors in a mat alpha 2 background. These constructions reveal that mat alpha 2 mutants can be made to mate as either a cells or as alpha cells by causing them to express only the pheromone and receptor set appropriate for a particular cell type. Moreover, these studies show that the inability of mat alpha 2 mutants to respond to either pheromone is a consequence of two phenomena: adaptation to an autocrine response to the pheromones they secrete and interference with response to alpha factor by the a-factor receptor.     

Expression analysis of PCSTE3, a putative pheromone receptor from the lung pathogenic fungus Pneumocystis carinii

The fungal pathogen Pneumocystis carinii remains the most prevalent opportunistic infection in patients infected with HIV. Fungal pheromone receptors are seven transmembrane domain G-protein-coupled receptors which are expressed on specific mating types, and have ligand-binding extracellular domains for specific pheromones from cells of the opposite mating type. We have cloned and characterized PCSTE3 from P. carinii, which encodes a seven transmembrane domain protein orthologous to the Saccharomyces cerevisiae pheromone receptor Ste3. We detect PCSTE3 by indirect immunofluorescence using antibodies designed to extracellular domains of the receptor in yeast expressing the protein. Using a downstream Fus1-lacZ reporter gene, we determined that PCSTE3 does not recognize a- or alpha-factor pheromones as ligands for the receptor. We isolated P. carinii life cycle stages and examined PCSTE3 expression by immunofluorescence microscopy and flow cytometry, and found PCSTE3 expression exclusively on a population of trophic forms. PCSTE3 receptor expression was not found on cysts.     

G protein signaling governing cell fate decisions involves opposing Galpha subunits in Cryptococcus neoformans

Communication between cells and their environments is often mediated by G protein-coupled receptors and cognate G proteins. In fungi, one such signaling cascade is the mating pathway triggered by pheromone/pheromone receptor recognition. Unlike Saccharomyces cerevisiae, which expresses two Galpha subunits, most filamentous ascomycetes and basidiomycetes have three Galpha subunits. Previous studies have defined the Galpha subunit acting upstream of the cAMP-protein kinase A pathway, but it has been unclear which Galpha subunit is coupled to the pheromone receptor and response pathway. Here we report that in the pathogenic basidiomycetous yeast Cryptococcus neoformans, two Galpha subunits (Gpa2, Gpa3) sense pheromone and govern mating. gpa2 gpa3 double mutants, but neither gpa2 nor gpa3 single mutants, are sterile in bilateral crosses. By contrast, deletion of GPA3 (but not GPA2) constitutively activates pheromone response and filamentation. Expression of GPA2 and GPA3 is differentially regulated: GPA3 expression is induced by nutrient-limitation, whereas GPA2 is induced during mating. Based on the phenotype of dominant active alleles, Gpa2 and Gpa3 signal in opposition: Gpa2 promotes mating, whereas Gpa3 inhibits. The incorporation of an additional Galpha into the regulatory circuit enabled increased signaling complexity and facilitated cell fate decisions involving choice between yeast growth and filamentous asexual/sexual development.     

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.