Protein Kinases Involved in Mating and Osmotic Stress in the Yeast Kluyveromyces lactis

Abstract

Systematic disruption of genes encoding kinases and mitogen-activated protein kinases (MAPKs) was performed in Kluyveromyces lactis haploid cells. The mutated strains were assayed by their capacity to mate and to respond to hyperosmotic stress. The K. lactis Ste11p (KlSte11p) MAPK kinase kinase (MAPKKK) was found to act in both mating and osmoresponse pathways while the scaffold KlSte5p and the MAPK KlFus3p appeared to be specific for mating. The p21-activated kinase KlSte20p and the kinase KlSte50p participated in both pathways. Protein association experiments showed interaction of KlSte50p and KlSte20p with G␣ and G␤, respectively, the G protein subunits involved in the mating pathway. Both KlSte50p and KlSte20p also showed interaction with KlSte11p. Disruption mutants of the K. lactis PBS2 (KlPBS2) and KlHOG1 genes of the canonical osmotic response pathway resulted in mutations sensitive to high salt and high sorbitol but dispensable for mating. Mutations that eliminate the MAPKK KlSte7p activity had a strong effect on mating and also showed sensitivity to osmotic stress. Finally, we found evidence of physical interaction between KlSte7p and KlHog1p, in addition to diminished Hog1p phosphorylation after a hyperosmotic shock in cells lacking KlSte7p. This study reveals novel roles for components of transduction systems in yeast.

Ineffective Phosphorylation of Mitogen-Activated Protein Kinase Hog1p in Response to High Osmotic Stress in the Yeast Kluyveromyces lactis

“…However, we observed that when KlSte11p or its adaptor protein, KlSte50p, was inactivated, growth was severely affected. All the observed phenotypes are in agreement with those described previously (25), except for that of the ⌬Klste20 mutant. We attribute this discrepancy to the different genetic background in which mutants in this study were constructed.…”

“…We included in this analysis the genes that code for the transmembrane protein KlSho1p, the PAK KlSte20p, the MAPKKK KlSte11p, and its adaptor protein, KlSte50p. For comparison, we also included ⌬Klpbs2 and ⌬Klhog1 mutants, which have been shown to be sensitive to high osmotic stress (25). As can be seen in Fig.…”

“…pGEM-KlSTE11, which was previously described (25), was digested with XhoI, generating a 2,193-bp fragment, which was incubated with the Klenow enzyme and then ligated into YEpKDGalURA (28), which had previously been digested with EcoRI and filled in with the Klenow enzyme, yielding YEpKDGal-KlSTE11. A protein consisting of a fusion between full-length KlSte11p and the transmembrane segments of KlSHO1 was constructed as follows: a 360-bp fragment of KlSHO1 tagged with 6 histidine residues was obtained by PCR using pGEM-KlSHO1 as the template.…”

“…The pGEM-T Easy vector (Promega) was used for cloning and sequencing of PCR products. The integrative vector YIp352-URA (25) was used for yeast gene disruptions. YEpKDGal (29) was used for galactose induction of gene expression.…”

“…Comparative genomic analyses have shown that K. lactis contains orthologs of most proteins of the S. cerevisiae HOG pathway (24). However, besides one genetic study (25) and the report that a mutant with a deletion of the K. lactis HSL1 (KlHSL1) gene, encoding a serine-threonine protein kinase involved in the cell cycle, was sensitive to hyperosmotic stress and showed constitutive KlHog1p phosphorylation (26), very little is known about the architecture and regulation of the K. lactis hyperosmotic signaling system (reviewed in reference 23). Here we describe some aspects of K. lactis Hog1p regulation, focusing on the role of the SHO1 branch.…”