Functional Characterization of an α-Factor-Like Sordaria macrospora Peptide Pheromone and Analysis of Its Interaction with Its Cognate Receptor in Saccharomyces cerevisiae

Abstract

The homothallic filamentous ascomycete Sordaria macrospora possesses genes which are thought to encode two pheromone precursors and two seven-transmembrane pheromone receptors. The pheromone precursor genes are termed ppg1 and ppg2. The putative products derived from the gene sequence show structural similarity to the ␣-factor precursors and a-factor precursors of the yeast Saccharomyces cerevisiae. Likewise, sequence similarity has been found between the putative products of the pheromone receptor genes pre2 and pre1 and the S. cerevisiae Ste2p ␣-factor receptor and Ste3p a-factor receptor, respectively. To investigate whether the ␣-factor-like pheromone-receptor pair of S. macrospora is functional, a heterologous yeast assay was used. Our results show that the S. macrospora ␣-factor-like pheromone precursor PPG1 is processed into an active pheromone by yeast MAT␣ cells. The S. macrospora PRE2 protein was demonstrated to be a peptide pheromone receptor. In yeast MATa cells lacking the endogenous Ste2p receptor, the S. macrospora PRE2 receptor facilitated all aspects of the pheromone response. Using a synthetic peptide, we can now predict the sequence of one active form of the S. macrospora peptide pheromone. We proved that S. macrospora wild-type strains secrete an active pheromone into the culture medium and that disruption of the ppg1 gene in S. macrospora prevents pheromone production. However, loss of the ppg1 gene does not affect vegetative growth or fertility. Finally, we established the yeast assay as an easy and useful system for analyzing pheromone production in developmental mutants of S. macrospora.The life cycle of ascomycetes can be either homothallic or heterothallic. Homothallic species are self-fertile and are able to complete the sexual cycle without a mating partner. In heterothallic ascomycetes, mating occurs only between cells of opposite mating type, which attract each other by secreting pheromones (for reviews see reference 9, 46, and 47). Each mating type produces its own specific pheromone. These can be divided into two groups depending on the pathway of synthesis and secretion.The pheromone response system of the heterothallic ascomycetous yeast Saccharomyces cerevisiae is a well-studied model of pheromone and pheromone receptor interaction. Binding of pheromones to their specific receptors triggers a G-proteinlinked signal transduction pathway that induces the expression of several genes, facilitating the fusion of MATa and MAT␣ cells to form diploid MATa/␣ cells (44). The peptide synthesized by MATa cells is derived from a precursor with a C-terminal CaaX (where C is cysteine, a is an aliphatic residue, and X is any amino acid residue) motif, a signal for carboxymethylation and farnesylation. The mature lipopeptide pheromone, called a-factor, is secreted via an ATP-binding cassette transporter (7). MAT␣ cells produce ␣-factor, which is secreted by the classical yeast secretory pathway (9,35,73). The ␣-factor is a 13-amino-acid peptide derived from a precursor containing several copies...

Pheromones Are Essential for Male Fertility and Sufficient To Direct Chemotropic Polarized Growth of Trichogynes during Mating in Neurospora crassa

“…The filamentous fungus Sordaria macrospora is closely related to N. crassa yet does not require a mating partner in order to complete the sexual cycle. The homothallic S. macrospora coexpresses two pheromone precursor and two pheromone receptor genes (33,34), a compatible pair of which were demonstrated to be functional in a recent study (27). In light of these results, we examined the N. crassa m1a70 strains for self-fertility traits, since they coexpress ccg-4 and pre-2, a compatible pair of pheromone and pheromone receptor genes (Fig.…”

Sexual reproduction and mating-type–mediated strain development in the penicillin-producing fungus Penicillium chrysogenum

“…The interaction of pheromones and pheromone receptors from P. chrysogenum was studied using the heterologous yeast system that was described previously (32) and is detailed further in SI Materials and Methods.…”

“…We therefore examined their functionality using yeast bioassays. Successful pheromone binding and signaling would be expected to result in cell cycle arrest and change in cell morphology and lead to formation of a halo in lawns of Saccharomyces cerevisiae (32)(33)(34). On the basis of similarity to the S. cerevisiae MFα proteins, the P. chrysogenum Pcppg1 gene was predicted to produce a decapeptide pheromone of sequence KWCGHIGQGC, expected to bind to the cognate PcPRE2 receptor protein.…”

Three α-Subunits of Heterotrimeric G Proteins and an Adenylyl Cyclase Have Distinct Roles in Fruiting Body Development in the Homothallic Fungus Sordaria macrospora

“…To date, several S. macrospora pro mutants that develop protoperithecia but no perithecia have been generated and characterized, thereby revealing essential components of fruiting body development Nowrousian et al 1999Nowrousian et al , 2007aPö ggeler and Kü ck 2004;Kü ck 2005;Engh et al 2007). In addition, despite the fact that S. macrospora completes the sexual cycle without a mating partner, two pheromone-precursor (ppg1 and ppg2) and two pheromonereceptor genes (pre1 and pre2) were shown to be involved in sexual development (Pö ggeler and Kü ck 2000; Mayrhofer and Pö ggeler 2005;Mayrhofer et al 2006;Pö ggeler et al 2006b). The two receptors show significant amino acid similarities to the pheromone receptors in S. cerevisiae (Pö ggeler and Kü ck 2001).…”