Activated Alleles of Yeast SLN1 Increase Mcm1-dependent Reporter Gene Expression and Diminish Signaling through the Hog1 Osmosensing Pathway

Abstract

Two-component signal transduction systems involving histidine autophosphorylation and phosphotransfer to an aspartate residue on a receiver molecule have only recently been discovered in eukaryotes, although they are well studied in prokaryotes. The Sln1 protein of Saccharomyces cerevisiae is a two-component regulator involved in osmotolerance. Phosphorylation of Sln1p leads to inhibition of the Hog1 mitogen-activated protein kinase osmosensing pathway. We have discovered a second function of Sln1p by identifying recessive activated alleles (designated nrp2) that regulate the essential transcription factor Mcm1. nrp2 alleles cause a 5-fold increase in the activity of an Mcm1-dependent reporter, whereas deletion of SLN1 causes a 10-fold decrease in reporter activity and a corresponding decrease in expression of Mcm1-dependent genes. In addition to activating Mcm1p, nrp2 mutants exhibit reduced phosphorylation of Hog1p and increased osmosensitivity suggesting that nrp2 mutations shift the Sln1p equilibrium toward the phosphorylated state. Two nrp2 mutations map to conserved residues in the receiver domain (P1148S and P1196L) and correspond to residues implicated in bacterial receivers to control receiver phosphorylation state. Thus, it appears that increased Sln1p phosphorylation both stimulates Mcm1p activity and diminishes signaling through the Hog1 osmosensing pathway.

Intracellular Glycerol Levels Modulate the Activity of Sln1p, aSaccharomyces cerevisiae Two-component Regulator

“…The fps1⌬-dependent Increase in P-lacZ Reporter Activity Requires Sln1 and Skn7-The elevation in P-lacZ activity in an fps1 mutant is similar to the effect of sln1* activating mutations (17,18). To investigate the relationship between SLN1 and FPS1, we measured P-lacZ activity in the double mutant.…”

“…Null mutations in SLN1 decrease P-lacZ reporter gene expression whereas activator mutations in the gene (sln1*) increase P-lacZ reporter gene expression. As is the case for the HOG pathway, Sln1p control of the P-lacZ reporter appears to require the kinase and phosphotransfer functions of Sln1p (17). The sln1* mutations that result in increased P-lacZ reporter gene activity map to positions likely to affect phosphorylation.…”

“…Recently, we have shown that Sln1p also regulates the activity of a Hog1p-independent pathway whose transcriptional output can be monitored using a lacZ reporter gene we call P-lacZ, which consists of a CYC1-lacZ fusion in which the UAS of CYC1 has been replaced by a palindromic-binding site (P site) for the Mcm1p transcription factor (17,18). Potential and known target genes of Mcm1p encode proteins involved in cell type determination, cell wall and membrane integrity, cellular metabolism, and cell cycle progression (19).…”

“…The sln1* mutations that result in increased P-lacZ reporter gene activity map to positions likely to affect phosphorylation. More compelling evidence for the role of phosphorylation and phosphotransfer in sln1* activation of the P-lacZ reporter comes from the osmosensitive phenotype of these mutants which is a reflection of reduced HOG pathway activity (17). The demonstration of a second pathway controlled by Sln1p led us to propose that the Sln1p osmosensor sends two signals in response to changes in osmolarity.…”

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

“…In addition to inactivation by hyper-osmotic conditions, the Sln1p kinase can also be stimulated. 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.…”