Comparative genetic and physiological studies of the MAP kinase Mpk1p from Kluyveromyces lactis and Saccharomyces cerevisiae.
MAP kinases are essential components of signal transduction pathways in yeasts and higher eukaryotes. Here, we report on the isolation of the gene encoding the MAP kinase KlMpk1p by complementation of the respective Saccharomyces cerevisiae deletion mutant with a genomic library from Kluyveromyces lactis. Sequencing revealed the presence of an open reading frame capable of encoding a protein of 520 amino acid residues with a deduced molecular mass of 59.726 Da. The deduced protein sequence displayed a high degree of similarity to known MAP kinases from yeast to man, with an overall identity of 70 % to ScMpk1p. One-hybrid analysis demonstrated the presence of a cryptic transcriptional activation domain in the C-terminal part of the protein. Deletion of this sequence in ScMpk1p resulted in a reduced MAP kinase activity (measured by an indirect assay), an increased sensitivity towards caffeine and an increased resistance against Calcofluor white. Complete deletion mutants of Klmpk1 display an osmo-remedial phenotype on rich medium, but are capable of growth in the absence of osmotic stabilization on synthetic medium. As Scmpk1 deletion mutants, they are sensitive to cell surface destabilizing agents such as Calcofluor white and SDS, and growth is inhibited in the presence of 5 mM caffeine. Overexpression of KlMPK1 did not produce a growth defect in S. cerevisiae or in K. lactis.
Kirchrath
L
L
Amersham Pharmacia Biotech, Munzinger-Str. 9, Freiburg, 79021, FRG.
Lorberg
A
A
Schmitz
H P
HP
Gengenbacher
U
U
Heinisch
J J
JJ
D000595:Amino Acid Sequence; D001557:Benzenesulfonates / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005816:Genetic Complementation Test; D007716:Kluyveromyces / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008826:Microbial Sensitivity Tests; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D020798:Two-Hybrid System Techniques
D001557:Benzenesulfonates; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D012967:Sodium Dodecyl Sulfate; D002110:Caffeine; C007061:C.I. Fluorescent Brightening Agent 28; D020928:Mitogen-Activated Protein Kinases; C072724:SLT2 protein, S cerevisiae