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

Abstract

When treated with a hyperosmotic stimulus, Kluyveromyces lactis cells respond by activating the mitogen-activated protein kinase (MAPK) K. lactis Hog1 (KlHog1) protein via two conserved branches, SLN1 and SHO1. Mutants affected in only one branch can cope with external hyperosmolarity by activating KlHog1p by phosphorylation, except for single ⌬Klste11 and ⌬Klste50 mutants, which showed high sensitivity to osmotic stress, even though the other branch (SLN1) was intact. Inactivation of both branches by deletion of KlSHO1 and KlSSK2 also produced sensitivity to high salt. Interestingly, we have observed that in ⌬Klste11 and ⌬Klsho1 ⌬Klssk2 mutants, which exhibit sensitivity to hyperosmotic stress, and contrary to what would be expected, KlHog1p becomes phosphorylated. Additionally, in mutants lacking both MAPK kinase kinases (MAPKKKs) present in K. lactis (KlSte11p and KlSsk2p), the hyperosmotic stress induced the phosphorylation and nuclear internalization of KlHog1p, but it failed to induce the transcriptional expression of KlSTL1 and the cell was unable to grow in high-osmolarity medium. KlHog1p phosphorylation via the canonical HOG pathway or in mutants where the SHO1 and SLN1 branches have been inactivated requires not only the presence of KlPbs2p but also its kinase activity. This indicates that when the SHO1 and SLN1 branches are inactivated, high-osmotic-stress conditions activate an independent input that yields active KlPbs2p, which, in turn, renders KlHog1p phosphorylation ineffective. Finally, we found that KlSte11p can alleviate the sensitivity to hyperosmotic stress displayed by a ⌬Klsho1 ⌬Klssk2 mutant when it is anchored to the plasma membrane by adding the KlSho1p transmembrane segments, indicating that this chimeric protein can substitute for KlSho1p and KlSsk2p.

Role of the Sln1‐phosphorelay pathway in the response to hyperosmotic stress in the yeast Kluyveromyces lactis

“…B), even though this mutant was highly sensitive to hyperosmotic stress. This observation agreed with the amount of KlHog1 phosphorylation that was detected in the double ΔKlste11ΔKlssk2 mutant (Velázquez‐Zavala et al ., ).…”

“…However, this phosphorylation failed to protect the cells against hyperosmotic stress, which was contrary to the outcome we expected. These results correlate with the phenotype displayed by a ΔKlssk2ΔKlste11 mutant (Velázquez‐Zavala et al ., ) in which KlHog1 was phosphorylated under hyperosmotic stress. This could mean that phosphorylation of KlHog1, under these conditions, was performed in a different manner that made the reaction inefficient for an osmostress response or that KlHog1 activity has not reached a threshold to trigger an osmoresponse.…”

“…This would mean that in K. lactis , the phosphorelay system either does not participate in the response to hyperosmotic stress or that the K. lactis phosphorelay pathway acts in a different manner in this yeast species. The first hypothesis was discarded since a double ΔKlsln1 ΔKlsho1 mutant was osmosensitive, which contrasted the observed phenotype of the single ΔKlsho1 mutant, which was osmoresistant (Velázquez‐Zavala et al ., ). This indicated that both K. lactis branches were active and worked in parallel for hyperosmotic adaptation.…”

Activation of the Hog1 MAPK by the Ssk2/Ssk22 MAP3Ks, in the absence of the osmosensors, is not sufficient to trigger osmostress adaptation in Saccharomyces cerevisiae

“…An aliquot was used to determine protein concentrations by the Bradford assay. Cells were centrifuged and the pellets were lysed with SB‐DTT buffer as previously described . Protein samples were resolved by SDS/PAGE and transferred to Immobilon‐Polyvinylidene difluoride membrane (Millipore, Edo.…”

“…de México, México). Inmunoblotting was performed using anti‐Hog1 (1 : 5000 yC‐20; Santa Cruz Biotechnology, Dallas, TX, USA) for total Hog1 detection and anti‐phospho‐p38 (1 : 10 000; Cell Signaling Technology, Danvers, MA, USA) for Phospho‐Hog1 detection as previously described . All westerns were repeated three times and representative blots are shown in figures.…”