Pim1, a MAP kinase involved in cell wall integrity in Pichia pastoris

Mitogen-activated protein kinases (MAPKs) are key enzymes in the signal tra nsduction pathways of eukaryotes. We report the isolation of a Pichia pasto ris gene, PIM1, which encodes the first MAPK to be identified in this yeast . Pim1 shows the greatest similarity to fungal MAPKs involved in the mainte nance of cell integrity. Disruption of the PIM1 gene results in an osmoreme diable thermosensitive phenotype reminiscent of that observed in mutants af fected in the MAPK Slt2/ Mpk1 of Saccharomyces cerevisiae, which is involve d in ensuring cell wall integrity. Furthermore, pim1 mutants are hypersensi tive to caffeine and cell wall-destabilising compounds. Pim1 is phosphoryla ted at two sites, and thereby activated, in response to heat stress, caffei ne and agents that alter the fungal cell wall, which is consistent with a r ole in adaptation to these conditions. These results support the idea that the MAPK-based mechanisms which regulate cell wall integrity are conserved in yeast species. Pim1 is alsodoubly phosphorylated in S. cerevisiae in res ponse to stimuli that activate the cell integrity pathway in this yeast. In addition, Pim1 is able to activate the transcription of a reporter gene in one-hybrid experiments, as does its S. cerevisiae counterpart, Slt2. Inter estingly, however, Pim1 does not rescue the mutant phenotype of an slt2 Del ta strain. This indicates some functional divergence in MAPK modulation and signal transmission by cell integrity pathways and provides a tool that ma y contribute to a better understanding of MAPK signalling.