The Eukaryotic Two-Component Histidine Kinase Sln1p RegulatesOCH1via the Transcription Factor, Skn7p

Abstract

The yeast "two-component" osmotic stress phosphorelay consists of the histidine kinase, Sln1p, the phosphorelay intermediate, Ypd1p and two response regulators, Ssk1p and Skn7p, whose activities are regulated by phosphorylation of a conserved aspartyl residue in the receiver domain. Dephospho-Ssk1p leads to activation of the hyper-osmotic response (HOG) pathway, whereas phospho-Skn7p presumably leads to activation of hypo-osmotic response genes. The multifunctional Skn7 protein is important in oxidative as well as osmotic stress; however, the Skn7p receiver domain aspartate that is the phosphoacceptor in the SLN1 pathway is dispensable for oxidative stress. Like many well-characterized bacterial response regulators, Skn7p is a transcription factor. In this report we investigate the role of Skn7p in osmotic response gene activation. Our studies reveal that the Skn7p HSF-like DNA binding domain interacts with a cis-acting element identified upstream of OCH1 that is distinct from the previously defined HSE-like Skn7p binding site. Our data support a model in which Skn7p receiver domain phosphorylation affects transcriptional activation rather than DNA binding to this class of DNA binding site.

Modulation of Yeast Sln1 Kinase Activity by the Ccw12 Cell Wall Protein

“…Subclones containing portions of the left arm of chromosome XII were tested for complementation of the pathway activation phenotype of the ccw12 and ylr111w deletion mutants from the Research Genetics Collection. The effects of subclones on pathway activity was measured using the pathway-responsive P OCH1 -lacZ reporter, pJF1320 (10). ␤-Galactosidase (␤-gal) activity values are the average of three or more transformants and are represented as Miller units normalized to protein.…”

“…The same strain carrying a complementing CCW12 plasmid (pSS1559) or empty vector (pRS313) is shown for comparison. SLN1-SKN7 pathway activation was measured using the P OCH1 -lacZ reporter, pZL1320 (10). ␤-Galactosidase (␤-gal) activity values are the average of three of more transformants and are represented in Miller units normalized to protein.…”

“…An increase in kinase activity leads to shutdown of the HOG pathway and activation of the Skn7p transcription factor (9). Sln1p kinase activation is apparent by the increase in expression of SLN1-SKN7 response genes such as OCH1 (10) and NCA3.…”

“…A role for the SLN1-SKN7 pathway in cell wall integrity was suggested by the original isolation of skn7 mutants on the basis of their resistance to killer factor (kre, for killer resistance) (12). A large body of data link Skn7p to the PKC cell wall integrity pathway (10,(13)(14)(15) and suggest that the SLN1-SKN7 and the PKC pathways may function in parallel to protect cells from lysis because of the build up of turgor pressure.…”

“…Although genes involved in cell wall integrity had previously been identified as targets of the pathway (10), the role of specific wall components in osmotic stress sensing and signaling has not received much attention. Here we find that the relative abundance in the wall of the abundant GPI-anchored wall mannoprotein, Ccw12p, affects SLN1-SKN7 pathway activity.…”