Molecular and functional characterization of a mutant allele of the mitogen-activated protein-kinase geneSLT2(MPK1) rescued from yeast autolytic mutants
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We have further characterized the functionality of theSaccharomyces cerevisiae geneSLT2(MPK1), coding for a MAP-kinase homolog essential for cell integrity, which is involved in the Pkc1p signalling pathway. This gene was isolated on the basis of its capacity to complement the thermosensitive-autolytic, osmotic-remediable phenotype oflyt2 mutants. Bothslt2A andlyt2 mutants displayed a caffeine-sensitive phenotype consisting of cell lysis that was not dependent on temperature. Caffeine concentrations affecting the growth of these mutant strains were dependent on the genetic background, theSSD1 allele being very significant in this regard. TheSLT2 allele of severallyt2 strains was both rescued and amplified by PCR. The recovered allele was shown to be non-functional as it could not complement the lytic phenotype of both deletion (slt2Δ) andlyt2 strains. After nucleotide sequencing of the recovered allele, we found that the defect oflyt2 mutants consists in a substitution of an aspartic acid for a glycine at position 35 of the amino-acid sequence of Slt2p. Gly35 is the third glycine of a glycine cluster (Gly-X-Gly-X-X-Gly), a conserved region in protein kinases and other nucleotide-binding proteins.
KeywordsYeast SLT2 MAP-kinase Caffeine
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