Pim1, a MAP kinase involved in cell wall integrity in Pichia pastoris
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O R I GI N A L P A P E R
I. C. Cosano á H. MartõÂ n á M. FlaÂndez á C. Nombela M. Molina
Pim1, a MAP kinase involved in cell wall integrity in Pichia pastoris
Received: 8 August 2000 / Accepted: 10 December 2000 / Published online: 3 March 2001 Ó Springer-Verlag 2001
Abstract 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 ®rst MAPK to be identi®ed 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 aected in the MAPK Slt2/ Mpk1 of Saccharomyces cerevisiae, which is involved in ensuring cell wall integrity. Furthermore, pim1 mutants are hypersensitive to caeine and cell wall-destabilising compounds. Pim1 is phosphorylated at two sites, and thereby activated, in response to heat stress, caeine 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 alsodoubly 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 onehybrid experiments, as does its S. cerevisiae counterpart, Slt2. Interestingly, however, Pim1 does not rescue the mutant phenotype of an slt2D 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.
Communicated by C. P. Hollenberg I. C. Cosano á H. MartõÂ n á M. FlaÂndez á C. Nombela M. Molina (&) Departamento de MicrobiologõÂ a II, Facultad de Farmacia, Universidad Complutense, Plaza de RamoÂn y Cajal s/n, 28040 Madrid, Spain E-mail: [email protected] Fax: 34 91 3941745
Keywords Cell wall integrity á Slt2 á MAP kinase á Pichia pastoris
Introduction Signal transduction pathways that lead to activation of MAP kinases (MAPKs) regulate many essential functions in eukaryotic cells. The activity of MAPKs is stimulated by a broad variety of signals, including mitogens, growth factors, cytokines, various stress conditions and other factors. MAPK activation requires dual phosphorylation on conserved threonine and tyrosine residues. This is mediated by a MAPK kinase (MAPKK) which is in turn activated by phosphorylation by a MAPK kinase kinase (MAPKKK) (Cobb and Goldsmith 1995). Such signal transduction modules, known as MAPK cascades, operate as intermediate switches between other signalling elements and are evolutionarily conserved; mammalians, plants, worms, ¯ies, and unicellular organisms such as yeasts, use these central transducers to respond to changes in the environment. The budding yeast Saccharomyces cerev
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