Functional characterization of an alpha-factor-like Sordaria macrospora peptide pheromone and analysis of its interaction with its cognate receptor in Saccharomyces cerevisiae.
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 alpha-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 alpha-factor receptor and Ste3p a-factor receptor, respectively. To investigate whether the alpha-factor-like pheromone-receptor pair of S. macrospora is functional, a heterologous yeast assay was used. Our results show that the S. macrospora alpha-factor-like pheromone precursor PPG1 is processed into an active pheromone by yeast MATalpha 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.
Mayrhofer
Severine
S
Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität, Bochum, Germany.
Pöggeler
Stefanie
S
D000595:Amino Acid Sequence; D005298:Fertility; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010675:Pheromones / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism*; D043563:Receptors, Pheromone / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D020037:Sordariales / Q000737:chemistry / Q000235:genetics*
D005656:Fungal Proteins; D010446:Peptide Fragments; D010675:Pheromones; C415591:Ppg1 protein, Sordaria macrospora; C415592:Ppg2 protein, Sordaria macrospora; D011498:Protein Precursors; D043562:Receptors, G-Protein-Coupled; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D043563:Receptors, Pheromone; C081863:STE3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins