Yeast osmosensor Sln1 and plant cytokinin receptor Cre1 respond to changes in turgor pressure

Very little is known about how cellular osmosensors monitor changes in osmolarity of the environment. Here, we report that in yeast, Sln1 osmosensor histidine kinase monitors changes in turgor pressures. Reductions in turgor caused by either hyperosmotic stress, nystatin, or removal of cell wall activate MAPK Hog1 specifically through the SLN1 branch, but not through the SHO1 branch of the high osmolarity glycerol pathway. The integrity of the periplasmic region of Sln1 was essential for its sensor function. We found that activity of the plant histidine kinase cytokinin response 1 (Cre1) is also regulated by changes in turgor pressure, in a manner identical to that of Sln1, in the presence of cytokinin. We propose that Sln1 and Cre1 are turgor sensors, and that similar turgor-sensing mechanisms might regulate hyperosmotic stress responses both in yeast and plants.     

Identification of a novel gene family involved in osmotic stress response in Caenorhabditis elegans

Organisms exposed to the damaging effects of high osmolarity accumulate solutes to increase cytoplasmic osmolarity. Yeast accumulates glycerol in response to osmotic stress, activated primarily by MAP kinase Hog1 signaling. A pathway regulated by protein kinase C (PKC1) also responds to changes in osmolarity and cell wall integrity. C. elegans accumulates glycerol when exposed to high osmolarity, but the molecular pathways responsible for this are not well understood. We report the identification of two genes, osm-7 and osm-11, which are related members of a novel gene family. Mutations in either gene lead to high internal levels of glycerol and cause an osmotic resistance phenotype (Osr). These mutants also have an altered defecation rhythm (Dec). Mutations in cuticle collagen genes dpy-2, dpy-7, and dpy-10 cause a similar Osr Dec phenotype. osm-7 is expressed in the hypodermis and may be secreted. We hypothesize that osm-7 and osm-11 interact with the cuticle, and disruption of the cuticle causes activation of signaling pathways that increase glycerol production. The phenotypes of osm-7 are not suppressed by mutations in MAP kinase or PKC pathways, suggesting that C. elegans uses signaling pathways different from yeast to mount a response to osmotic stress.     

The Rgd1p Rho GTPase-activating protein and the Mid2p cell wall sensor are required at low pH for protein kinase C pathway activation and cell survival in Saccharomyces cerevisiae

The protein kinase C (PKC) pathway is involved in the maintenance of cell shape and cell integrity in Saccharomyces cerevisiae. Here, we show that this pathway mediates tolerance to low pH and that the Bck1 and Slt2 proteins belonging to the mitogen-activated protein kinase cascade are essential for cell survival at low pH. The PKC pathway is activated during acidification of the extracellular environment, and this activation depends mainly on the Mid2p cell wall sensor. Rgd1p, which encodes a Rho GTPase-activating protein for the small G proteins Rho3p and Rho4p, also plays a role in low-pH response. The rgd1Delta strain is sensitive to low pH, and Rgd1p activates the PKC pathway in an acidic environment. Inactivation of both genes in the double mutant rgd1Delta mid2Delta strain renders yeast cells unable to survive at low pH as in bck1Delta and slt2Delta strains. Our data provide evidence for the existence of two distinct ways, one involving Mid2p and the other involving Rgd1p, with both converging to the cell integrity pathway to mediate low-pH tolerance in Saccharomyces cerevisiae. Nevertheless, even if Rgd1p acts on the PKC pathway, it seems that its mediating action on low-pH tolerance is not limited to this pathway. As the Mid2p amount plays a role in rgd1Delta sensitivity to low pH, Mid2p seems to act more like a molecular rheostat, controlling the level of PKC pathway activity and thus allowing phenotypical expression of RGD1 inactivation.     

Modulation of yeast Sln1 kinase activity by the CCW12 cell wall protein

The yeast Sln1p sensor kinase is best known as an osmosensor involved in the regulation of the hyperosmolarity glycerol mitogen-activated protein kinase cascade. Down-regulation of Sln1 kinase activity occurs under hypertonic conditions and leads to phosphorylation of the Hog1p mitogen-activated protein kinase and increased osmotic stress-response gene expression. Conditions leading to kinase up-regulation include osmotic imbalance caused by glycerol retention in the glycerol channel mutant, fps1 (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367). The hypothesis that Sln1p kinase activity is responsive to turgor was first suggested by the increased Sln1p kinase activity in mutants lacking Fps1p in which glycerol accumulation leads to water uptake. Also consistent with the turgor hypothesis is the observation that reduced turgor caused by treatment of cells with nystatin, a drug that increases membrane permeability and causes cell shrinkage, reduced Sln1p kinase activity (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367; Reiser, V., Raitt, D. C., and Saito, H. (2003) J. Cell Biol. 161, 1035-1040). The turgor hypothesis is revisited here in the context of the identification and characterization of the cell wall gene, CCW12, as a determinant of Sln1p activity. Results of this analysis suggest that the activity of the plasma membrane localized Sln1p is affected by the presence or absence of specific outer cell wall proteins and that this effect is independent of turgor.     

A simple mathematical model of adaptation to high osmolarity in yeast

We present a simple ordinary differential equation (ODE) model of the adaptive response to an osmotic shock in the yeast Saccharomyces cerevisiae. The model consists of two main components. First, a biophysical model describing how the cell volume and the turgor pressure are affected by varying extra-cellular osmolarity. The second component describes how the cell controls the biophysical system in order to keep turgor pressure, or equivalently volume, constant. This is done by adjusting the glycerol production and the glycerol outflow from the cell. The complete model consists of 4 ODEs, 3 algebraic equations and 10 parameters. The parameters are constrained from various literature sources and estimated from new and previously published absolute time series data on intra-cellular and total glycerol. The qualitative behaviour of the model has been successfully tested on data from other genetically modified strains as well as data for different input signals. Compared to a previous detailed model of osmoregulation, the main strength of our model is its lower complexity, contributing to a better understanding of osmoregulation by focusing on relationships which are obscured in the more detailed model. Besides, the low complexity makes it possible to obtain more reliable parameter estimates.     

Biophysical properties of Saccharomyces cerevisiae and their relationship with HOG pathway activation

Parameterized models of biophysical and mechanical cell properties are important for predictive mathematical modeling of cellular processes. The concepts of turgor, cell wall elasticity, osmotically active volume, and intracellular osmolarity have been investigated for decades, but a consistent rigorous parameterization of these concepts is lacking. Here, we subjected several data sets of minimum volume measurements in yeast obtained after hyper-osmotic shock to a thermodynamic modeling framework. We estimated parameters for several relevant biophysical cell properties and tested alternative hypotheses about these concepts using a model discrimination approach. In accordance with previous reports, we estimated an average initial turgor of 0.6 ± 0.2 MPa and found that turgor becomes negligible at a relative volume of 93.3 ± 6.3% corresponding to an osmotic shock of 0.4 ± 0.2 Osm/l. At high stress levels (4 Osm/l), plasmolysis may occur. We found that the volumetric elastic modulus, a measure of cell wall elasticity, is 14.3 ± 10.4 MPa. Our model discrimination analysis suggests that other thermodynamic quantities affecting the intracellular water potential, for example the matrix potential, can be neglected under physiological conditions. The parameterized turgor models showed that activation of the osmosensing high osmolarity glycerol (HOG) signaling pathway correlates with turgor loss in a 1:1 relationship. This finding suggests that mechanical properties of the membrane trigger HOG pathway activation, which can be represented and quantitatively modeled by turgor.     

Aberrant processing of the WSC family and Mid2p cell surface sensors results in cell death of Saccharomyces cerevisiae O-mannosylation mutants

Protein O mannosylation is a crucial protein modification in uni- and multicellular eukaryotes. In humans, a lack of O-mannosyl glycans causes congenital muscular dystrophies that are associated with brain abnormalities. In yeast, protein O mannosylation is vital; however, it is not known why impaired O mannosylation results in cell death. To address this question, we analyzed the conditionally lethal Saccharomyces cerevisiae protein O-mannosyltransferase pmt2 pmt4Delta mutant. We found that pmt2 pmt4Delta cells lyse as small-budded cells in the absence of osmotic stabilization and that treatment with mating pheromone causes pheromone-induced cell death. These phenotypes are partially suppressed by overexpression of upstream elements of the protein kinase C (PKC1) cell integrity pathway, suggesting that the PKC1 pathway is defective in pmt2 pmt4Delta mutants. Congruently, induction of Mpk1p/Slt2p tyrosine phosphorylation does not occur in pmt2 pmt4Delta mutants during exposure to mating pheromone or elevated temperature. Detailed analyses of the plasma membrane sensors of the PKC1 pathway revealed that Wsc1p, Wsc2p, and Mid2p are aberrantly processed in pmt mutants. Our data suggest that in yeast, O mannosylation increases the activity of Wsc1p, Wsc2p, and Mid2p by enhancing their stability. Reduced O mannosylation leads to incorrect proteolytic processing of these proteins, which in turn results in impaired activation of the PKC1 pathway and finally causes cell death in the absence of osmotic stabilization.     

Stimulation of the yeast high osmolarity glycerol (HOG) pathway: evidence for a signal generated by a change in turgor rather than by water stress

The Saccharomyces cerevisiae HOG pathway controls responses to osmotic shock such as production of the osmolyte glycerol. Here we show that the HOG pathway can be stimulated by addition of glycerol. This stimulation was strongly diminished in cells expressing an unregulated Fps1p glycerol channel, presumably because glycerol rapidly equilibrated across the plasma membrane. Ethanol, which passes the plasma membrane readily and causes water stress by disturbing the hydration of biomolecules, did not activate the HOG pathway. These observations suggest that stimulation of the HOG pathway is mediated by a turgor change and not by water stress per se.     

Ptk2 contributes to osmoadaptation in the filamentous fungus Neurospora crassa

Hyphal tip-growing organisms often rely upon an internal hydrostatic pressure (turgor) to drive localized expansion of the cell. Regulation of the turgor in response to osmotic shock is mediated primarily by an osmotic MAP kinase cascade which activates osmolyte synthesis and ion uptake to effect turgor recovery. We characterized a Neurospora crassa homolog (PTK2) of ser/thr kinase regulators of ion transport in yeast to determine its role in turgor regulation in a filamentous fungi. The ptk2 mutant is osmosensitive, and has lower turgor poise than wildtype. The cause appears to be lower activity of the plasma membrane H⁺-ATPase. Its role in osmoadaptation is unrelated to the activity of the osmotic MAP kinase cascade. Instead, it acts in an alternative pathway that, like the osmotic MAP kinase cascade, also involves ion transport mediated osmoadaptation.     

Cell surface expression of bacterial esterase A by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg(-1) protein for Kre1/EstA/Cwp2p and 72 mU mg(-1) protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg(-1) protein for Kre1/EstA/Cwp2p and 1.27 U mg(-1) protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.     

Functional and genetic interactions of TOR in the budding yeast Saccharomyces cerevisiae with myosin type II-deficiency (myo1Delta)

ABSTRACT: BACKGROUND: Yeast has numerous mechanisms to survive stress. Deletion of myosin type II (myo1Delta) in Saccharomyces cerevisiae results in a cell that has defective cytokinesis. To survive this genetically induced stress, this budding yeast up regulates the PKC1 cell wall integrity pathway (CWIP). More recently, our work indicated that TOR, another stress signaling pathway, was down regulated in myo1Delta strains. Since negative signaling by TOR is known to regulate PKC1, our objectives in this study were to understand the cross-talk between the TOR and PKC1 signaling pathways and to determine if they share upstream regulators for mounting the stress response in myo1Delta strains RESULTS: Here we proved that TORC1 signaling was down regulated in the myo1Delta strain. While a tor1Delta mutant strain had increased viability relative to myo1Delta, a combined myo1Deltator1Delta mutant strain showed significantly reduced cell viability. Synthetic rescue of the tor2-21ts lethal phenotype was observed in the myo1Delta strain in contrast to the chs2Delta strain, a chitin synthase II null mutant that also activates the PKC1 CWIP and exhibits cytokinesis defects very similar to myo1Delta, where the rescue effect was not observed. We observed two pools of Slt2p, the final Mitogen Activated Protein Kinase (MAPK) of the PKC1 CWIP; one pool that is up regulated by heat shock and one that is up regulated by the myo1Delta stress. The cell wall stress sensor WSC1 that activates PKC1 CWIP under other stress conditions was shown to act as a negative regulator of TORC1 in the myo1Delta mutant. Finally, the repression of TORC1 was inversely correlated with the activation of PKC1 in the myo1Delta strain. CONCLUSIONS: Regulated expression of TOR1 was important in the activation of the PKC1 CWIP in a myo1Delta strain and hence its survival. We found evidence that the PKC1 and TORC1 pathways share a common upstream regulator associated with the cell wall stress sensor WSC1. Surprisingly, essential TORC2 functions were not required in the myo1Delta strain. By understanding how yeast mounts a concerted stress response, one can further design pharmacological cocktails to undermine their ability to adapt and to survive.     

Aspergillus nidulans HOG pathway is activated only by two-component signalling pathway in response to osmotic stress

Genome sequencing analyses revealed that Aspergillus nidulans has orthologous genes to all those of the high-osmolarity glycerol (HOG) response mitogen-activated protein kinase (MAPK) pathway of Saccharomyces cerevisiae. A. nidulans mutant strains lacking sskA, sskB, pbsB, or hogA, encoding proteins orthologous to the yeast Ssk1p response regulator, Ssk2p/Ssk22p MAPKKKs, Pbs2p MAPKK and Hog1p MAPK, respectively, showed growth inhibition under high osmolarity, and HogA MAPK in these mutants was not phosphorylated under osmotic or oxidative stress. Thus, activation of the A. nidulans HOG (AnHOG) pathway depends solely on the two-component signalling system, and MAPKK activation mechanisms in the AnHOG pathway differ from those in the yeast HOG pathway, where Pbs2p is activated by two branches, Sln1p and Sho1p. Expression of pbsB complemented the high-osmolarity sensitivity of yeast pbs2Delta, and the complementation depended on Ssk2p/Ssk22p, but not on Sho1p. Pbs2p requires its Pro-rich motif for binding to the Src-homology3 (SH3) domain of Sho1p, but PbsB lacks a typical Pro-rich motif. However, a PbsB mutant (PbsB(Pro)) with the yeast Pro-rich motif was activated by the Sho1p branch in yeast. In contrast, HogA in sskADelta expressing PbsB(Pro) was not phosphorylated under osmotic stress, suggesting that A. nidulans ShoA, orthologous to yeast Sho1p, is not involved in osmoresponsive activation of the AnHOG pathway. We also found that besides HogA, PbsB can activate another Hog1p MAPK orthologue, MpkC, in A. nidulans, although mpkC is dispensable in osmoadaptation. In this study, we discuss the differences between the AnHOG and the yeast HOG pathways.     

Independent signaling pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection by Magnaporthe grisea.

The phytopathogenic fungus Magnaporthe grisea elaborates a specialized infection cell called an appressorium with which it mechanically ruptures the plant cuticle. To generate mechanical force, appressoria produce enormous hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control of cellular turgor, we analyzed the response of M. grisea to hyperosmotic stress. During acute and chronic hyperosmotic stress adaptation, M. grisea accumulates arabitol as its major compatible solute in addition to smaller quantities of glycerol. A mitogen-activated protein kinase-encoding gene OSM1 was isolated from M. grisea and shown to encode a functional homolog of HIGH-OSMOLARITY GLYCEROL1 (HOG1), which encodes a mitogen-activated protein kinase that regulates cellular turgor in yeast. A null mutation of OSM1 was generated in M. grisea by targeted gene replacement, and the resulting mutants were sensitive to osmotic stress and showed morphological defects when grown under hyperosmotic conditions. M. grisea deltaosm1 mutants showed a dramatically reduced ability to accumulate arabitol in the mycelium. Surprisingly, glycerol accumulation and turgor generation in appressoria were unaltered by the Deltaosm1 null mutation, and the mutants were fully pathogenic. This result indicates that independent signal transduction pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection. Consistent with this, exposure of M. grisea appressoria to external hyperosmotic stress induced OSM1-dependent production of arabitol.     

Functional interaction between the PKC1 pathway and CDC31 network of SPB duplication genes

The earliest known step in yeast spindle pole body (SPB) duplication requires Cdc31p and Kar1p, two physically interacting SPB components, and Dsk2p and Rad23p, a pair of ubiquitin-like proteins. Components of the PKC1 pathway were found to interact with these SPB duplication genes in two independent genetic screens. Initially, SLG1 and PKC1 were obtained as high-copy suppressors of dsk2Delta rad23Delta and a mutation in MPK1 was synthetically lethal with kar1-Delta17. Subsequently, we demonstrated extensive genetic interactions between the PKC1 pathway and the SPB duplication mutants that affect Cdc31p function. The genetic interactions are unlikely to be related to the cell-wall integrity function of the PKC1 pathway because the SPB mutants did not exhibit cell-wall defects. Overexpression of multiple PKC1 pathway components suppressed the G2/M arrest of the SPB duplication mutants and mutations in MPK1 exacerbated the cell cycle arrest of kar1-Delta17, suggesting a role for the PKC1 pathway in SPB duplication. We also found that mutations in SPC110, which encodes a major SPB component, showed genetic interactions with both CDC31 and the PKC1 pathway. In support of the model that the PKC1 pathway regulates SPB duplication, one of the phosphorylated forms of Spc110p was absent in pkc1 and mpk1Delta mutants.     

Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation

The accumulation of compatible solutes, such as glycerol, in the yeast Saccharomyces cerevisiae, is a ubiquitous mechanism in cellular osmoregulation. Here, we demonstrate that yeast cells control glycerol accumulation in part via a regulated, Fps1p-mediated export of glycerol. Fps1p is a member of the MIP family of channel proteins most closely related to the bacterial glycerol facilitators. The protein is localized in the plasma membrane. The physiological role of Fps1p appears to be glycerol export rather than uptake. Fps1 delta mutants are sensitive to hypo-osmotic shock, demonstrating that osmolyte export is required for recovery from a sudden drop in external osmolarity. In wild-type cells, the glycerol transport rate is decreased by hyperosmotic shock and increased by hypo-osmotic shock on a subminute time scale. This regulation seems to be independent of the known yeast osmosensing HOG and PKC signalling pathways. Mutants lacking the unique hydrophilic N-terminal domain of Fps1p, or certain parts thereof, fail to reduce the glycerol transport rate after a hyperosmotic shock. Yeast cells carrying these constructs constitutively release glycerol and show a dominant hyperosmosensitivity, but compensate for glycerol loss after prolonged incubation by glycerol overproduction. Fps1p may be an example of a more widespread class of regulators of osmoadaptation, which control the cellular content and release of compatible solutes.