In our previous study, the specific interaction between P-factor, a peptide pheromone and its receptor, Mam2, on the cell surface of the fission yeast Schizosaccharomyces pombe was investigated by two methods, an atom...In our previous study, the specific interaction between P-factor, a peptide pheromone and its receptor, Mam2, on the cell surface of the fission yeast Schizosaccharomyces pombe was investigated by two methods, an atomic force microscope (AFM) and a GFP reporter assay. The removal of Leu at C-terminal of P-factor resulted in an inactivation of P-factor function to bind Mam2 and induce the signal transduction pathway. Here, we used truncated P-factor derivatives lacking N-terminal of P-factor (P12 ~ P22: 12 ~ 22 amino acid residues from C-terminal) as ligands for Mam2. From the dose-dependent analysis of the GFP reporter assay ranging from 1 nM to 100 μM of the peptide concentration, the peptides can be classified into three groups based on EC50 and maximal GFP production level, group1 (P-factor), group2 (P17 ~ 22), and group3 (P12 ~ P16). At 0.1 μM, only P-factor induced the signal transduction pathway. At 1 μM, peptides from group2 partially induced the pathway and peptides from group3 induced the pathway a little. At 10 μM, all peptides induced the pathway mostly depending on the length of peptides. We also performed AFM experiments using P-factor and peptides from group3 to investigate the interaction between the peptides and Mam2 for comparison between the two methods.展开更多
文摘In our previous study, the specific interaction between P-factor, a peptide pheromone and its receptor, Mam2, on the cell surface of the fission yeast Schizosaccharomyces pombe was investigated by two methods, an atomic force microscope (AFM) and a GFP reporter assay. The removal of Leu at C-terminal of P-factor resulted in an inactivation of P-factor function to bind Mam2 and induce the signal transduction pathway. Here, we used truncated P-factor derivatives lacking N-terminal of P-factor (P12 ~ P22: 12 ~ 22 amino acid residues from C-terminal) as ligands for Mam2. From the dose-dependent analysis of the GFP reporter assay ranging from 1 nM to 100 μM of the peptide concentration, the peptides can be classified into three groups based on EC50 and maximal GFP production level, group1 (P-factor), group2 (P17 ~ 22), and group3 (P12 ~ P16). At 0.1 μM, only P-factor induced the signal transduction pathway. At 1 μM, peptides from group2 partially induced the pathway and peptides from group3 induced the pathway a little. At 10 μM, all peptides induced the pathway mostly depending on the length of peptides. We also performed AFM experiments using P-factor and peptides from group3 to investigate the interaction between the peptides and Mam2 for comparison between the two methods.
