Molecular and functional characterization of a mutant allele of the mitogen-activated protein-kinase gene SLT2(MPK1) rescued from yeast autolytic mutants.
We have further characterized the functionality of the Saccharomyces cerevisiae gene SLT2(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 of lyt2 mutants. Both slt2delta and lyt2 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, the SSD1 allele being very significant in this regard. The SLT2 allele of several lyt2 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 (slt2delta) and lyt2 strains. After nucleotide sequencing of the recovered allele, we found that the defect of lyt2 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. Keywords Yeast middle dot SLT2 middle dot MAP-kinase middle dot Caffeine
Martín
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H
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, E-28040-Madrid, Spain.
Castellanos
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MC
Cenamor
R
R
Sánchez
M
M
Molina
M
M
Nombela
C
C
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002110:Caffeine / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D009154:Mutation; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D017931:DNA Primers; D004271:DNA, Fungal; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D002110:Caffeine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C072724:SLT2 protein, S cerevisiae