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.
Project description:Yeast Sln1p is an osmotic stress sensor with histidine kinase activity. Modulation of Sln1 kinase activity in response to changes in the osmotic environment regulates the activity of the osmotic response mitogen-activated protein kinase pathway and the activity of the Skn7p transcription factor, both important for adaptation to changing osmotic stress conditions. Many aspects of Sln1 function, such as how kinase activity is regulated to allow a rapid response to the continually changing osmotic environment, are not understood. To gain insight into Sln1p function, we conducted a two-hybrid screen to identify interactors. Mog1p, a protein that interacts with the yeast Ran1 homolog, Gsp1p, was identified in this screen. The interaction with Mog1p was characterized in vitro, and its importance was assessed in vivo. mog1 mutants exhibit defects in SLN1-SKN7 signal transduction and mislocalization of the Skn7p transcription factor. The requirement for Mog1p in normal localization of Skn7p to the nucleus does not fully account for the mog1-related defects in SLN1-SKN7 signal transduction, raising the possibility that Mog1p may play a role in Skn7 binding and activation of osmotic response genes.
Project description:Ypd1p, a histidine-containing phosphotransfer protein, plays an important role in a branched His-Asp phosphorelay signal transduction pathway that regulates cellular responses to hyperosmotic stress in Saccharomyces cerevisiae. Ypd1p is required for phosphoryl group transfer from the membrane-bound Sln1p sensor histidine kinase to two downstream response regulator proteins, Ssk1p and Skn7p. To investigate the molecular basis for interaction of Ypd1p with these response regulator domains, we used an approach that coupled alanine-scanning mutagenesis of surface-exposed residues in Ypd1p with a yeast two-hybrid interaction screen. Mutated residues that adversely affected the interaction of Ypd1p with the C-terminal response regulator domain of Ssk1p were identified and found to cluster on or near the alphaA helix in Ypd1p. Our results, supported by analysis of a modeled complex, identify a binding site on Ypd1p for response regulators that is composed of a cluster of conserved hydrophobic residues surrounded by less conserved polar residues. We propose that molecular interactions involving Ypd1p are mediated primarily through hydrophobic contacts, whereas binding specificity and strength of interaction may be influenced by select polar side chain interactions.
Project description:We recently characterized the histidine kinase receptor genes of Candida lusitaniae. For the present study, we have further investigated the role of SSK1 and SKN7, encoding response regulators. The results of functional analysis of mutants indicated that Ssk1p is involved in osmotolerance and pseudohyphal development, whereas Skn7p appears crucial for oxidative stress adaptation.
Project description:Filamentous ascomycetous fungi possess many histidine kinases and two conserved response regulators, Ssk1p and Skn7p, in their two-component signaling systems. We previously reported that the fungus unique group III histidine kinase regulates high-osmolarity adaptation and iprodione/fludioxonil fungicide sensitivity by controlling the phosphorylation of Hog1-type mitogen-activated protein kinase (MAPK) in filamentous ascomycetes. Here, we have characterized the response regulator genes ChSsk1 and ChSkn7 in the southern corn leaf blight fungus Cochliobolus heterostrophus. Both ChSsk1- and ChSkn7-disrupted mutants showed little sensitivity to high-osmolarity stress and moderate resistance to the iprodione/fludioxonil fungicides. The phosphorylation of Hog1-type MAPK BmHog1p induced by high-osmolarity stress and fungicide treatments was only regulated by ChSsk1p, indicating that ChSkn7p has roles in high-osmolarity adaptation and fungicide sensitivity that are independent from the activation of BmHog1p. The Chssk1 Chskn7 double mutants clearly showed higher sensitivity to osmolar stress and higher resistance to fungicides than the single mutants. The dose responses of the double mutants fit well with those of the group III histidine kinase-deficient strain. These results suggest that in filamentous ascomycetes, the Ssk1- and Skn7-type response regulators control high-osmolarity adaptation and fungicide sensitivity additively with differential mechanisms under the regulation of the group III histidine kinase. This study provides evidence that filamentous fungi have a unique two-component signaling system that is different from that of yeast and is responsible for high-osmolarity adaptation and fungicide sensitivity.
Project description:Saccharomyces cerevisiae strains of the ?1278b background generate biofilms, referred to as mats, on low-density agar (0.3%) plates made with rich media (YPD). Mat formation involves adhesion of yeast cells to the surface of the agar substrate and each other as the biofilm matures, resulting in elaborate water channels that create filigreed patterns of cells. The cell wall adhesion protein Flo11p is required for mat formation; however, genetic data indicate that other unknown effectors are also required. For example, mutations in vacuolar protein sorting genes that affect the multivesicular body pathway, such as vps27?, cause mat formation defects independently of Flo11p, presumably by affecting an unidentified signaling pathway. A cell wall signaling protein, Wsc1p, found at the plasma membrane is affected for localization and function by vps27?. We found that a wsc1 mutation disrupted mat formation in a Flo11p-independent manner. Wsc1p appears to impact mat formation through the Rom2p-Rho1p signaling module, by which Wsc1p also regulates the cell wall. The Bck1p, Mkk1/Mkk2, Mpk1p MAP kinase signaling cascade is known to regulate the cell wall downstream of Wsc1p-Rom2p-Rho1p but, surprisingly, these kinases do not affect mat formation. In contrast, Wsc1p may impact mat formation by affecting Skn7p instead. Skn7p can also receive signaling inputs from the Sln1p histidine kinase; however, mutational analysis of specific histidine kinase receiver residues in Skn7p indicate that Sln1p does not play an important role in mat formation, suggesting that Skn7p primarily acts downstream of Wsc1p to regulate mat formation.
Project description:Histidine kinase (HK) phosphorelay signaling is a major mechanism by which fungi sense their environment. The maize pathogen Cochliobolus heterostrophus has 21 HK genes, 4 candidate response regulator (RR) genes (SSK1, SKN7, RIM15, REC1), and 1 gene (HPT1) encoding a histidine phosphotransfer domain protein. Because most HKs are expected to signal through RRs, these were chosen for deletion. Except for pigment and slight growth alterations for rim15 mutants, no measurable altered phenotypes were detected in rim15 or rec1 mutants. Ssk1p is required for virulence and affects fertility and proper timing of sexual development of heterothallic C. heterostrophus. Pseudothecia from crosses involving ssk1 mutants ooze masses of single ascospores, and tetrads cannot be found. Wild-type pseudothecia do not ooze. Ssk1p represses asexual spore proliferation during the sexual phase, and lack of it dampens asexual spore proliferation during vegetative growth, compared to that of the wild type. ssk1 mutants are heavily pigmented. Mutants lacking Skn7p do not display any of the above phenotypes; however, both ssk1 and skn7 mutants are hypersensitive to oxidative and osmotic stresses and ssk1 skn7 mutants are more exaggerated in their spore-type balance phenotype and more sensitive to stress than single mutants. ssk1 mutant phenotypes largely overlap hog1 mutant phenotypes, and in both types of mutant, the Hog1 target gene, MST1, is not induced. ssk1 and hog1 mutants were examined in the homothallic cereal pathogen Gibberella zeae, and pathogenic and reproductive phases of development regulated by Ssk1 and Hog1 were found to mirror, but also vary from, those of C. heterostrophus.
Project description: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.
Project description:The Ssk1p response regulator of Candida albicans is required for oxidant adaptation, survival in human neutrophils, and virulence in a disseminated murine model of candidiasis. We have previously shown that the amino acid residues D556 and D513 of the Ssk1p receiver domain are critical to the Ssk1p in oxidant stress adaptation and morphogenesis. Herein, transcriptional profiling is used to explain the oxidant sensitivity and morphogenesis defect of two point mutants (D556N and D513K, respectively) compared with a WT strain. In the D556N mutant, during oxidative stress (5 mM H(2)O(2)), a downregulation of genes associated with redox homeostasis and oxidative stress occurred, which accounted for about 5% of all gene changes, including among others, SOD1 (superoxide dismutase), CAP1 (required for some types of oxidant stress), and three genes encoding glutathione biosynthesis proteins (GLR1, GSH1, and GSH2). Mutant D513K was not sensitive to peroxide but was impaired in its yeast $/to hyphal transition. We noted downregulation of genes associated with morphogenesis and cell elongation. Virulence of each mutant was also evaluated in a rat vaginitis model of candidiasis. Clearance of an SSK1 null and the D556N mutants from the vaginal canal was significantly greater than wild type or the D513K mutant, indicating that a change in a single amino acid of the Ssk1p alters the ability of this strain to colonize the rat vaginal mucosa.
Project description:The whole genome analysis has demonstrated that wine yeasts undergo changes in promoter regions and variations in gene copy number, which make them different to lab strains and help them better adapt to stressful conditions during winemaking, where oxidative stress plays a critical role. Since cytoplasmic thioredoxin II, a small protein with thiol-disulphide oxidoreductase activity, has been seen to perform important functions under biomass propagation conditions of wine yeasts, we studied the involvement of Trx2p in the molecular regulation of the oxidative stress transcriptional response on these strains. In this study, we analyzed the expression levels of several oxidative stress-related genes regulated by either Yap1p or the co-operation between Yap1p and Skn7p. The results revealed a lowered expression for all the tested Skn7p dependent genes in a Trx2p-deficient strain and that Trx2p is essential for the oxidative stress response during respiratory metabolism in wine yeast. Additionally, activity of Yap1p and Skn7p dependent promoters by ?-galactosidase assays clearly demonstrated that Skn7p-dependent promoter activation is affected by TRX2 gene deficiency. Finally we showed that deleting the TRX2 gene causes Skn7p hyperphosphorylation under oxidative stress conditions. We propose Trx2p to be a new positive efector in the regulation of the Skn7p transcription factor that controls phosphorylation events and, therefore, modulates the oxidative stress response in yeast.
Project description:The histidine kinase-based signal transduction pathway was first uncovered in bacteria and is a prominent form of regulation in prokaryotes. However, this type of signal transduction is not unique to prokaryotes; over the last decade two-component signal transduction pathways have been identified and characterized in diverse eukaryotes, from unicellular yeasts to multicellular land plants. A number of small but important differences have been noted in the architecture and function of eukaryotic pathways. Because of the powerful genetic approaches and facile molecular analysis associated with the yeast system, the SLN1 osmotic response pathway in Saccharomyces cerevisiae is particularly useful as a eukaryotic pathway model. This chapter provides an overview of genetic and biochemical methods that have been important in elucidating the stimulus-response events that underlie this pathway and in understanding the details of a eukaryotic His-Asp phosphorelay.