Jan S. Fassler

The yeast histidine protein kinase, Sln1p, mediates phosphotransfer to two response regulators, Ssk1p and Skn7p

The Saccharomyces cerevisiae Sln1 protein is a ‘two‐component’ regulator involved in osmotolerance. Two‐component regulators are a family of signal‐transduction molecules with histidine kinase activity common in prokaryotes and recently identified in eukaryotes. Phosphorylation of Sln1p inhibits the HOG1 MAP kinase osmosensing pathway via a phosphorelay mechanism including Ypd1p and the response regulator, Ssk1p. SLN1 also activates an MCM1‐dependent reporter gene, P‐lacZ, but this function is independent of Ssk1p. We present genetic and biochemical evidence that Skn7p is the response regulator for this alternative Sln1p signaling pathway. Thus, the yeast Sln1 phosphorelay is actually more complex than appreciated previously; the Sln1 kinase and Ypd1 phosphorelay intermediate regulate the activity of two distinct response regulators, Ssk1p and Skn7p. The established role of Skn7p in oxidative stress is independent of the conserved receiver domain aspartate, D427. In contrast, we show that Sln1p activation of Skn7p requires phosphorylation of D427. The expression of TRX2, previously shown to exhibit Skn7p‐dependent oxidative‐stress activation, is also regulated by the SLN1 phosphorelay functions of Skn7p. The identification of genes responsive to both classes of Skn7p function suggests a central role for Skn7p and the SLN1‐SKN7 pathway in integrating and coordinating cellular response to various types of environmental stress.

Intracellular glycerol levels modulate the activity of Sln1p, a Saccharomyces cerevisiae two-component regulator.

The HOG mitogen-activated protein kinase pathway mediates the osmotic stress response in Saccharomyces cerevisiae, activating genes like GPD1 (glycerol phosphate dehydrogenase), required for survival under hyperosmotic conditions. Activity of this pathway is regulated by Sln1p, a homolog of the “two-component” histidine kinase family of signal transduction molecules prominent in bacteria. Sln1p also regulates the activity of a Hog1p-independent pathway whose transcriptional output can be monitored using an Mcm1p-dependent lacZreporter gene. The relationship between the two Sln1p branches is unclear, however, the requirement for unphosphorylated pathway intermediates in Hog1p pathway activation and for phosphorylated intermediates in the activation of the Mcm1p reporter suggests that the two Sln1p branches are reciprocally regulated. To further investigate the signals and molecules involved in modulating Sln1p activity, we have screened for new mutations that elevate the activity of the Mcm1p-dependent lacZ reporter gene. We find that loss of function mutations in FPS1, a gene encoding the major glycerol transporter in yeast activates the reporter in a SLN1-dependent fashion. We propose that elevated intracellular glycerol levels in the fps1mutant shift Sln1p to the phosphorylated state and trigger the Sln1-dependent activity of the Mcm1 reporter. These observations are consistent with a model in which Sln1p autophosphorylation is triggered by a hypo-osmotic stimulus and indicate that the Sln1p osmosensor is tied generally to osmotic balance, and may not specifically sense an external osmolyte.

Identification of novel Yap1p and Skn7p binding sites involved in the oxidative stress response of Saccharomyces cerevisiae

The Saccharomyces cerevisiae Yap1p and Skn7p transcription factors collaborate in the activation of oxidative stress response (OSR) genes. Although Yap1p and Skn7p oxidative stress response elements (YRE, OSRE) have been characterized and identified in some OSR genes, many OSR genes lack such elements. In this study, the complex, oxidative responsive, CCP1 promoter was used as a model to investigate the cis‐acting elements responsible for activation by oxidative stress. In addition to consensus YRE and OSRE sequences, novel Yap1p and Skn7p binding sites were identified in the CCP1 promoter. These new sites were found to mediate Yap1p‐ and Skn7p‐dependent activation of OSR genes including TSA1 and CTT1 previously thought to lack Yap1p and Skn7p binding sites. The novel YREs and OSREs were found to be enriched in the promoter regions of a set of 179 OSR genes. The widespread existence of novel Yap1p and Skn7p binding sites strongly suggest that direct binding of Yap1p and Skn7p is responsible for activation of many more OSR genes than previously believed.

Saccharomyces cerevisiae Histidine Phosphotransferase Ypd1p Shuttles between the Nucleus and Cytoplasm for SLN1-Dependent Phosphorylation of Ssk1p and Skn7p

ABSTRACT Sln1p is a plasma membrane-localized two-component histidine kinase that functions as an osmotic stress sensor in Saccharomyces cerevisiae. Changes in osmotic pressure modulate Sln1p kinase activity, which, together with Ypd1p, a phosphorelay intermediate, changes the phosphorylation status of two response regulators, Ssk1p and Skn7p. Ssk1p controls the activity of the HOG1 mitogen-activated protein kinase pathway. Skn7p is a nuclearly localized transcription factor that regulates genes involved in cell wall integrity and other processes. Subcellular compartmentalization may therefore play an important role in eukaryotic two-component pathway regulation. We have studied the subcellular localization of SLN1 pathway components and find that Ypd1p is a dynamic protein with a role in shuttling the osmotic stress signal from Sln1p to Ssk1p in the cytosol and to Skn7p in the nucleus. The need to translocate the signal into different intracellular compartments contributes a spatial dimension to eukaryotic two-component pathways compared to the prototypical two-component pathways of prokaryotes.

The Saccharomyces cerevisiae SPT13/GAL11 gene has both positive and negative regulatory roles in transcription.

To understand the function of SPT13, a gene encoding a trans-acting factor involved in regulation of Ty-mediated gene expression, we have cloned and sequenced the gene. Our analysis revealed that SPT13 is the same gene as GAL11, a gene in which mutations cause reduced expression of some GAL4-regulated genes. Further analysis of spt13/gal11 mutants suggested that the SPT13 gene also affects transcription of genes involved in mating-type specialization. We show here that SPT13 has both positive and negative regulatory roles in transcription.

A cytoplasmic coiled-coil domain is required for histidine kinase activity of the yeast osmosensor, SLN1

The yeast histidine kinase, Sln1p, is a plasma membrane‐associated osmosensor that regulates the activity of the osmotic stress MAP kinase pathway. Changes in the osmotic environment of the cell influence the autokinase activity of the cytoplasmic kinase domain of Sln1p. Neither the nature of the stimulus, the mechanism by which the osmotic signal is transduced nor the manner in which the kinase is regulated is currently clear. We have identified several mutations located in the linker region of the Sln1 kinase (just upstream of the kinase domain) that cause hyperactivity of the Sln1 kinase. This region of histidine kinases is largely uncharacterized, but its location between the transmembrane domains and the cytoplasmic kinase domain suggests that it may have a potential role in signal transduction. In this study, we have investigated the Sln1 linker region in order to understand its function in signal transduction and regulation of Sln1 kinase activity. Our results indicate that the linker region forms a coiled‐coil structure and suggest a mechanism by which alterations induced by osmotic stress influence kinase activity by altering the alignment of the phospho‐accepting histidine with respect to the catalytic domain of the kinase.

Oxidative Stress Function of the Saccharomyces cerevisiae Skn7 Receiver Domain

ABSTRACT The bifunctional Saccharomyces cerevisiae Skn7 transcription factor regulates osmotic stress response genes as well as oxidative stress response genes; however, the mechanisms involved in these two types of regulation differ. Skn7 osmotic stress activity depends on the phosphorylation of the receiver domain aspartate, D427, by the Sln1 histidine kinase. In contrast, D427 and the SLN1-SKN7 phosphorelay are dispensable for the oxidative stress response, although the receiver domain is required. The majority of oxidative stress response genes regulated by Skn7 also are regulated by the redox-responsive transcription factor Yap1. It is therefore possible that the nuclearly localized Skn7 does not itself respond to the oxidant but simply cooperates with Yap1 when it translocates to the nucleus. We report here that oxidative stress leads to a phosphatase-sensitive, slow-mobility Skn7 variant. This suggests that Skn7 undergoes a posttranslational modification by phosphorylation following exposure to oxidant. Oxidant-dependent Skn7 phosphorylation was eliminated in strains lacking the Yap1 transcription factor. This suggests that the phosphorylation of Skn7 is regulated by Yap1. Mutations in the receiver domain of Skn7 were identified that affect its oxidative stress function. These mutations were found to compromise the association of Yap1 and Skn7 at oxidative stress response gene promoters. A working model is proposed in which the association of Yap1 with Skn7 in the nucleus is a prerequisite for Skn7 phosphorylation and the activation of oxidative stress response genes.

Fungal Skn7 Stress Responses and Their Relationship to Virulence

ABSTRACT The histidine kinase-based phosphorelay has emerged as a common strategy among bacteria, fungi, protozoa, and plants for triggering important stress responses and interpreting developmental cues in response to environmental as well as chemical, nutritional, and hormone signals. The absence of this type of signaling mechanism in animals makes the so-called “two-component” pathway an attractive target for development of antimicrobial agents. The best-studied eukaryotic example of a two-component pathway is the SLN1 pathway in Saccharomyces cerevisiae, which responds to turgor and other physical properties associated with the fungal cell wall. One of the two phosphoreceiver proteins known as response regulators in this pathway is Skn7, a highly conserved stress-responsive transcription factor with a subset of activities that are dependent on SLN1 pathway phosphorylation and another subset that are independent. Interest in Skn7as a determinant in fungal virulence stems primarily from its well-established role in the oxidative stress response; however, the involvement of Skn7 in maintenance of cell wall integrity may also be relevant. Since the cell wall is crucial for fungal survival, structural and biosynthetic proteins affecting wall composition and signaling pathways that respond to wall stress are likely to play key roles in virulence. Here we review the molecular and phenotypic characteristics of different fungal Skn7 proteins and consider how each of these properties may contribute to fungal virulence.

Activated alleles of yeast SLN1 increase Mcm1-dependent reporter gene expression and diminish signaling through the Hog1 osmosensing pathway.

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 cerevisiaeis 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 (designatednrp2) that regulate the essential transcription factor Mcm1. nrp2 alleles cause a 5-fold increase in the activity of an Mcm1-dependent reporter, whereas deletion ofSLN1 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.

SPT13 (GAL11) of Saccharomyces cerevisiae negatively regulates activity of the MCM1 transcription factor in Ty1 elements.

The Ty transposable elements of Saccharomyces cerevisiae consist of a single large transcription unit whose expression is controlled by a combination of upstream and downstream regulatory sequences. Errede (B. Errede, Mol. Cell. Biol. 13:57-62, 1993) has shown that among the downstream control sequences is a binding site for the transcription factor, MCM1. A small restriction fragment containing the Ty1 MCM1-binding site exhibits very weak activation of heterologous gene expression. The absence of SPT13 (GAL11) causes a dramatic increase in activity directed by these sequences. This effect is mediated through the MCM1-binding site itself. MCM1 mRNA and protein levels, as well as its affinity for its binding site, are unchanged in the absence of SPT13. Our results suggest that SPT13 has a role in the negative control of MCM1 activity that is likely to be posttranslational. A role for SPT13 in the negative regulation of the activity of the Ty1 MCM1-binding site is consistent with our previous proposal that spt13-mediated suppression of Ty insertion mutations could be attributed to the loss of negative regulation of genes adjacent to Ty elements.