Pim1, a MAP kinase involved in cell wall integrity in Pichia pastoris.
Mitogen-activated protein kinases (MAPKs) are key enzymes in the signal transduction pathways of eukaryotes. We report the isolation of a Pichia pastoris gene, PIM1, which encodes the first MAPK to be identified in this yeast. Pim1 shows the greatest similarity to fungal MAPKs involved in the maintenance of cell integrity. Disruption of the PIM1 gene results in an osmoremediable thermosensitive phenotype reminiscent of that observed in mutants affected in the MAPK Slt2/ Mpk1 of Saccharomyces cerevisiae, which is involved in ensuring cell wall integrity. Furthermore, pim1 mutants are hypersensitive to caffeine and cell wall-destabilising compounds. Pim1 is phosphorylated at two sites, and thereby activated, in response to heat stress, caffeine and agents that alter the fungal cell wall, which is consistent with a role 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 also doubly phosphorylated in S. cerevisiae in response 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. Interestingly, however, Pim1 does not rescue the mutant phenotype of an slt2delta strain. This indicates some functional divergence in MAPK modulation and signal transmission by cell integrity pathways and provides a tool that may contribute to a better understanding of MAPK signalling.
Cosano
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IC
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Martín
H
H
Flández
M
M
Nombela
C
C
Molina
M
M
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002110:Caffeine / Q000494:pharmacology; D002473:Cell Wall / Q000187:drug effects / Q000648:ultrastructure*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D005800:Genes, Fungal; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010843:Pichia / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D002110:Caffeine; D020928:Mitogen-Activated Protein Kinases; C432694:Pim1 protein, Pichia pastoris; C072724:SLT2 protein, S cerevisiae