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Microbiology and Molecular Biology Reviews, June 2002, p. 300-372, Vol. 66, No. 2
1092-2172/02/$04.00+0     DOI: 10.1128/MMBR.66.2.300-372.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Osmotic Stress Signaling and Osmoadaptation in Yeasts

Department of Cell and Molecular Biology/Microbiology, Göteborg University, S-405 30 Göteborg, Sweden

SUMMARY

INTRODUCTION

Yeasts Live in a Variable Environment

Yeasts as Model Systems

Striving for an Integrative View

SENSING OSMOTIC CHANGES

Osmosensors

Sho1p.

Sln1p.

What Are Osmosensors Sensing?

SIGNALING OSMOTIC CHANGES

Overview of Signaling Pathways Involved in Osmoadaptation

HOG MAP Kinase Pathway in S. cerevisiae

MAP kinase pathways.

S. cerevisiae MAP kinase pathways.

HOG pathway architecture.

Activation of HOG pathway function: subcellular localization and dynamics.

(i) Role of the two branches of the HOG pathway.

(ii) Activation via the Sln1 branch.

(iii) Activation via the Sho1 branch.

(iv) Events downstream of the MAPKKKs.

Modulation and feedback control of the HOG pathway.

Cross talk between HOG pathway and other MAP kinase pathways.

Transcriptional Regulators of the HOG Pathway

Sko1/Acr1p.

Hot1p.

Msn1p.

Sgd1p.

General Stress Response: Msn2p, Msn4p, and Protein Kinase A

STREs and the transcription factors Msn2p and Msn4p.

Control of Msn2p and Msn4p localization.

What mechanisms control protein kinase A under stress?

Control of Msn2p/Msn4p-dependent genes by the HOG pathway.

Nutrient-Controlled Signaling and Stress Responses

Cell Integrity Pathway

Pathway architecture: MAP kinase cascade.

Pathway control: the Rho1p G-protein.

Cell surface sensors of the cell integrity pathway.

Other systems controlling the cell integrity pathway.

Pathways controlled by Rho1p and Pkc1p.

Transcriptional targets of PKC signaling.

(i) Rlm1p.

(ii) SBF (Swi4p and Swi6p).

Calcium-Dependent Signaling: Calcium Pulse and Calcineurin

Skn7p: Integrating Input from Different Osmosensing Pathways?

Skn7p is controlled by the Sln1p-Ypd1p osmosensing phosphorelay system and osmotic signals.

Skn7p interacts with the cell integrity pathway.

Could Skn7p be a master regulator of responses to hypo-osmolarity?

Could coordination of cell wall biogenesis be the common denominator of Skn7p function?

Is there a common mechanism by which Skn7p mediates its effects?

Stress-Inducible Sty1 MAP Kinase Pathway in S. pombe

Known components and architecture of the Sty1 pathway.

Signaling to the Sty1 pathway.

Transcriptional responses mediated by the Sty1 pathway.

Osmosensing Signaling Pathways in Other Yeasts

CELLULAR SYSTEMS INVOLVED IN RESPONSE TO HYPEROSMOTIC SHOCK

Genome-Wide Expression Analyses Reveal Genes and Proteins Responsive to Osmotic Shock

Hallmarks of a General Response to Environmental Challenges

Why have a general stress response?

Adjustments of protein production.

Enhanced gene expression under different stress conditions.

Systems potentially upregulated by different stress conditions.

Redox metabolism and overlap with oxidative stress responses.

Metabolism and Transport of Glycerol

Glycerol metabolic pathway.

Control of glycerol production under osmotic stress.

Transmembrane flux of glycerol.

Glycerol transport via Fps1p.

Metabolism of Trehalose and Glycogen

Other osmolytes.

Genes Whose Expression Responds Specifically to Hyperosmotic Shock

Genes Whose Expression Responds to Hypo-Osmotic Shock

TRANSPORT SYSTEMS INVOLVED IN OSMOADAPTATION

MIP Channels

Fps1p.

Yeast aquaporins.

Ion Transport

Osmolyte Uptake

Possible Roles of the Vacuole in Osmoadaptation

STRIVING FOR AN INTEGRATED VIEW OF OSMOADAPTATION

Time Course of Events in Adaptation to Hyperosmotic Shock

Time Course of Events in Adaptation to Hypo-Osmotic Shock

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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On substrates such as fruits and flowers, yeasts are exposed to a highly variable environment with respect to the availability and quality of nutrients, temperature, pH, radiation, access to oxygen, and especially water activity (230). Water activity is defined as the chemical potential of free water in solution. Low water activity limits yeast growth, a fact that has been used for centuries for the preservation of fruits in dry form or with very high sugar levels, such as in marmalades (486). In the yeast's natural environment, the water activity can range widely and rapidly, due to both external influences and the activity of the yeast itself. In order to maintain an appropriate cell volume and a ratio of free to bound water favorable for biochemical reactions, the water activity of the cytosol and its organelles has to be lower than that of the surrounding medium. In this way, a constant force is maintained, driving water into the cell along its concentration gradient. This force is counteracted by turgor pressure, which is established by the limited ability for expansion of the plasma membrane and especially the cell wall (48, 653).

From the yeast's point of view, (at least) two different aspects need to be considered: survival of sudden changes in the water activity and the acquisition of tolerance to low water activity, i.e., to high external osmolarity. For instance, yeast cells in a water droplet on a grape berry may suddenly be exposed to high sugar levels when the berry breaks open due to animal or fungal activity. Then yeast cells experience a hyperosmotic shock (or osmotic upshift), accompanied by rapid water outflow and cell shrinking. On the other hand, cells adapted to high sugar levels on drying fruits or flowers may be washed away in a rain shower into essentially distilled water. Such a hypo-osmotic shock (or osmotic downshift) increases the water concentration gradient and leads to rapid influx of water, cell swelling, and hence increased turgor pressure. Within wide limits, the yeast cell wall prevents cell bursting (565).

The ability to survive a sudden change in water activity must be an intrinsic property of the cell, which means that the appropriate survival systems are in place under all conditions. Survival mechanisms need to operate within the first seconds after a sudden osmotic shift because passive water loss or uptake occurs very fast (reviewed in references 48, 65, and 66). While relatively little is known about the mechanisms ensuring survival of a hyperosmotic shock, we have insight into the cell's strategy to survive a hypo-osmotic shock, and those will be discussed in this review.

Yeast cells may also be exposed to slowly decreasing water activity, for instance, when their substrate is drying in the sun. Cellular water follows its concentration gradient by passive diffusion, so that the cells lose water and the concentration of biomolecules and ions in the cell increases, eventually resulting in an arrest of cellular activity: the cell suffers high osmolarity or hyperosmotic stress (in the literature, often synonymous with osmotic stress).

Yeast cells have developed mechanisms to adjust, within certain limits, to high external osmolarity and maintain or reestablish an inside-directed driving force for water (although accurate measurements quantifying this force do not exist). Adaptation to altered osmolarity is an active process based on sensing of osmotic changes and appropriate cellular responses aimed at maintaining cellular activity. Adaptation after a hyperosmotic shock may well take several hours (reviewed in references 48, 228, and 360). As will be discussed in detail, the accumulation of chemically inert osmolytes, such as glycerol, plays a central role in osmoadaptation (63, 568, 661). Therefore, yeast cells can be metabolically active and proliferate over a range of external water activities. This range is species specific (48, 64). Beyond those limits of cellular activity, yeasts have the ability to survive almost complete dehydration, a property that is used for the production of dry yeast known to every home baker (112). Dry yeast contains less than 10% residual water.

The underlying molecular mechanisms for survival of a hyperosmotic shock and adaptation to high osmolarity are probably distinct but overlapping: cells adapted to moderately high osmolarity survive a severe osmotic shock better than nonadapted cells (49, 533, 612, 627). The main body of this article will address mechanisms involved in the adaptation to high osmolarity. In the laboratory, yeast cells are usually exposed to a hyperosmotic shock and then their responses are studied. Alternatively, yeast cells growing in media of low and high osmolarity, i.e., fully adapted cells, are compared.

Initial interest in the molecular mechanisms of yeast osmoadaptation originated from the need to improve the performance of yeast strains under industrial conditions, which are often associated with rapid alterations in water activity and especially with high osmolarity (112, 478, 491). Additional practical aims are the improvement of food preservation methods, which require a better understanding of the impact of low water activity on yeast cells, especially in combination with other stress factors such as heat, cold, acidity, and chemical food preservatives (168). These aspects have motivated early research into the control of the cellular content of glycerol and trehalose, low-molecular-weight compounds serving as compatible solutes to adjust intracellular water activity and to protect biomolecules from denaturation (48, 63, 67, 173, 415, 429, 593).

The field of yeast osmoadaptation has received much wider scientific interest with the discovery in 1993 of the involvement in osmoadaptation of a mitogen-activated protein kinase (MAP kinase) cascade, a conserved eukaryotic signal transduction module (61, 205). Present knowledge confirms that many principles of osmoadaptation are conserved across eukaryotes, and therefore yeasts are ideal model systems with which to study the underlying mechanisms. Osmoadaptation is part of cellular osmoregulation, which plays an important yet not fully appreciated role in cell growth and morphogenesis.

This review deals with the responses and mechanisms of adaptation to changes in the relative water concentration between the inside and the outside of cells which affect cell volume and turgor pressure. The water activity in the cytosol can also change due to the presence of substances that cross the plasma membrane readily. The most prominent of such substances is ethanol, the product of sugar fermentation by yeasts. In certain wine fermentations, the ethanol concentration can reach 15 to 20% per volume. Ethanol causes water stress because it affects hydration of biomolecules (reviewed in references 208 and 465). The underlying response mechanisms are different from, though overlapping with, those for volume or turgor changes. I will also not discuss in any detail the mechanisms with which yeast cells manage to survive two extreme forms of osmotic stress, desiccation and freezing; relatively little is known about the underlying molecular mechanisms. This review also does not discuss the interesting question of why certain yeasts can tolerate or even prefer much lower water activities than others. The molecular bases for the species-specific differences in osmotolerance are not well understood (48). Finally, this review will discuss to only a very limited extent yeast responses to salt stress. NaCl is very commonly used in the laboratory to increase medium osmolarity. NaCl stimulates osmotic responses in essentially the same way as sugars or sugar alcohols at concentrations causing similar water activity (86, 500, 501). Na⁺, however, is toxic, because it replaces K⁺ in biomolecules (537, 538). Therefore, Na⁺ stimulates additional detoxification responses.

SENSING OSMOTIC CHANGES

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Numerous proteins have been classified as osmosensors, often based on "guilt by association." The mere fact that a protein is needed for mediating responses to an osmotic shock plus its predicted location in the plasma membrane is used as an argument for a role in osmosensing. However, the molecular mechanism(s) by which osmosensors detect osmotic changes remains a matter of intensive research. Ultimate proof that a protein functions as an osmosensor requires defined in vitro studies (518, 653). In light of the difficulties associated with expression, purification, and reconstitution of transmembrane proteins (38), such in vitro data are still rare. Moreover, in vitro studies on osmosensors that do not at the same time transport substances require suitable monitoring and reporter systems.

A genuine osmosensor does not, per definitionem, function as a receptor for a certain (range of) compound, distinguishing it from chemosensors. Rather, an osmosensor might detect changes in the physicochemical properties of the solvent due to altered water concentration or water structure. Alternatively, it may sense mechanical stimuli that may occur as a consequence of the changes in water activity (205, 653). It is usually anticipated that osmosensors operate at the cell surface as integral membrane proteins. However, an osmosensor could also be a soluble protein. I will return to the possible mechanisms of sensing after discussing the known yeast proteins that are (probably) involved in this process. An excellent recent review discusses the physicochemical bases and possible mechanisms of osmosensing in detail, with an emphasis on bacterial systems, but the principles apply to any cell (653).

Two different types of proteins have been most intensely studied with regard to their control by osmotic changes. On the one hand, there are transmembrane transport proteins whose function is controlled by mechanical stimulation or changes in medium osmolarity. It is generally assumed that these mechanosensitive channels sense osmotic changes solely to control their own transport activity, but they could of course also connect to signaling pathways. I will discuss the mechanisms controlling them in more detail together with the osmoregulated yeast osmolyte exporter Fps1p. The second category of proteins are bona fide sensors that control signaling pathways leading to osmoadaptive responses.

Sho1p. Sho1p is a protein of 367 amino acids consisting of four predicted transmembrane domains within the N-terminal part, a linker domain, and an SH3 domain for protein-protein interaction (Fig. 1) (473, 488). Functional homologs of Sho1p have been isolated from the yeasts Candida utilis and Kluyveromyces lactis by complementation of the S. cerevisiae sho1 mutant (554). As expected, sequence conservation is highest in the transmembrane region (the C. utilis and K. lactis homologs show 56 and 54% identity to the S. cerevisiae protein, respectively) and the SH3 domain (62 and 73% identity). The sequence of the linker domain is only poorly conserved except for the 20 to 25 amino acids immediately flanking the transmembrane and SH3 domains (in the case of the K. lactis homolog) as well as a 10-amino-acid peptide in the center of the linker, whose function is not known. Homologs from higher eukaryotes have not been reported.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Topology of Sho1p and Sln1p. Numbers indicate amino acid positions.

The role of Sho1p as an anchor protein rather than a genuine osmosensor makes previous observations suggesting a role of Sho1p in the pseudohyphal development pathway more comprehensible (439). As outlined below, the Sho1p branch of the osmosensing HOG pathway and the pseudohyphal development pathway share several components, apparently including Sho1p. Sho1p is located at places on the cell surface where growth and cell expansion occur, such as the bud neck, the growing bud, and mating projections, as well as in internal structures, possibly the vacuole (488, 498). It is plausible that the cell has to monitor osmotic changes very closely as well as plasma membrane and cell wall remodeling in expanding areas of its surface to ensure coordinated cell morphogenesis. Sho1p could function as a protein that directs signal transduction complexes to such areas.

It is not known how Sho1p itself achieves its specific localization at areas of cell growth. It has, however, been reported that latrunculin A disturbs the location of Sho1p within the bud but not at the bud neck or growing bud (498). Latrunculin A disrupts the actin cytoskeleton (394). This observation could hint at the involvement of components of the actin skeleton in locating Sho1p to certain areas of the cell surface. Consistent with this idea, Sho1p-dependent signaling requires the G-protein Cdc42p (488, 498), which in turn is involved in actin nucleation and in localization of signaling components to places of active cell growth (reviewed in references 264 and 287). However, Cdc42p is not needed for the specific localization of Sho1p (488, 498).

It is known that an osmotic shock disrupts the actin cytoskeleton, which is reorganized during recovery and adaptation (62). If Sho1p interacts with the actin cytoskeleton, directly or via other proteins, osmotic changes may affect this interaction, thereby enabling Sho1p to recruit the Pbs2p kinase and other pathway components to the cell surface. This scenario, though speculative at this point, illustrates that osmosensing could well occur within the cell rather than within the plasma membrane. It should be noted that there is also evidence arguing against an involvement of the actin cytoskeleton in controlling the Sho1p branch: latrunculin A does not seem to affect osmosensing (498). However, the effects of such compounds are difficult to control and quantitate.

It should also be noted that although anchoring of Sho1p to the cell surface is necessary for signaling, the specific localization of Sho1p at the cell surface does not seem to be needed for sensing of an osmotic shock. As outlined above, derivatives of Sho1p lacking any of its three apparent structural domains can perform the function, as long as they recruit Pbs2p to the surface (488). It is unlikely that all these different constructs attain the same specific cell surface localization, although this has not been tested. In any case, the osmotic shock treatments performed in laboratory experiments may not reflect the true physiological role for Sho1p and associated proteins, which might have specific functions in monitoring subtle osmotic changes during cell growth and surface remodeling. Certainly more work is needed to understand the precise role of Sho1p in osmoregulated signaling.

Sln1p. Sln1p is a protein of 1,220 amino acids (Fig. 1) (442). As discussed in detail later, Sln1p is a negative regulator of the HOG signaling pathway, and deletion of SLN1 is lethal because of pathway overactivation (357). The protein is organized into four distinct regions: (i) an N-terminal section containing two predicted transmembrane domains separated by a loop, probably facing the periplasmic space; (ii) a linker region; (iii) a histidine kinase domain; and (iv) a receiver domain (442). Histidine kinases and receiver domains form so-called two-component systems, which are the prototype sensing and signaling units of prokaryotes (521, 579). Up to 80 such systems have been predicted from the genome sequences of certain species (387). Eukaryotic organisms employ histidine kinase signaling systems much less frequently.

Sln1p is the only sensor histidine kinase in the S. cerevisiae proteome, while other fungi may in fact have several: C. albicans has at least three (12, 79, 403, 573, 657), S. pombe also has at least three (72), and one each have so far been reported for Neurospora crassa (11), Aspergillus nidulans (634), and Aspergillus fumigatus (477). The slime mold Dictyostelium discoideum has at least 11 (579). At least eight histidine kinase systems have been found in plants (240, 521, 620), but so far none has been reported from animals (579).

The Sln1p histidine kinase domain, which is highly homologous to that of histidine kinases from bacteria and other eukaryotes, contains a phosphorylated histidine in position 576. The receiver domain of typical prokaryotic two-component systems is usually located within the second protein component, the response regulator, often a transcription factor (579). Sln1p, like other known eukaryotic systems, is a hybrid histidine kinase containing a receiver domain within the same polypeptide. This receiver domain is also well conserved; the phosphate-receiving aspartate residue is located at position 1144.

Only three of the known eukaryotic histidine kinases may be true Sln1p homologs, based on functional data or sequence comparison. The closest homolog is C. albicans Sln1 (CaSln1). The protein has a similar size (1,377 amino acids versus 1,220) and structural organization (403). It also shows significant sequence similarity with Sln1p not only in its histidine kinase (69% identity over 162 amino acids) and receiver domains (61% over 122 amino acids) but also in the putative sensor domain (31% over 297 amino acids). Within this sensor domain, the two transmembrane domains and the sequences immediately surrounding them are especially well conserved (49 and 45% identity), while the external loop shows only 28% identity spread over the entire domain. These data are in line with deletion analysis of Sln1p, which indicates that the first transmembrane domain is needed for the sensitivity of the histidine kinase activity to osmotic upshift (440). CaSln1 can suppress the lethality caused by an sln1 mutation in S. cerevisiae, but the receiver domain is not needed for suppression, suggesting that phosphotransfer bypasses two steps in the phosphorelay system (see below), although this has not been tested experimentally (403). The sln1 deletion mutant of C. albicans is viable and shows only some slight growth retardation in high-osmolarity medium (403). Hence, the precise role of CaSln1 in signaling or that of the downstream pathway may be different.

A protein from the filamentous fungus Aspergillus nidulans (1,070 amino acids; NCBI accession number BAB07814) has a similar architecture. Its external loop is somewhat longer (350 amino acids, versus about 300 in the two yeasts), but it exhibits weak homology to S. cerevisiae Sln1p even in the sensor domain (27% identity over the first 167 amino acids). The histidine kinase domain (52% over 91 amino acids) and the receiver domain (52% over 128 amino acids) are well conserved. Functional data for the A. nidulans protein have not been reported.

The Arabidopsis thaliana histidine kinase ATHK1 (1,207 amino acids) also has a domain structure similar to that of Sln1p, although the first transmembrane domain is preceded by an approximately 60-amino-acid extension. ATHK1 has been shown to complement the lethality of the S. cerevisiae sln1 mutation (618-620). When comparing the structurally related putative sensor domains of S. cerevisiae and C. albicans Sln1p with that of the A. nidulans protein and further with the A. thaliana homolog, significant sequence similarity disappears. The ATHK1 histidine kinase and receiver domains are significantly similar to those of Sln1p (49% identity over 76 and 78 amino acids, respectively).

All available data suggest that the Sho1p and Sln1p osmosensing systems function independently and upstream of two different branches of the HOG pathway (see below). However, a possible interaction, in physical or regulatory terms, between the two systems has so far not been studied directly. Also, the precise localization of Sln1p on the cell surface has not been reported so far.

Significant work on the mechanisms of osmosensing has been done especially on bacterial sensors such as the KdpD-KpdE system, which controls expression of the components of the high-affinity K⁺ transporter Kpd and the EnvZ-OmpR system, which controls expression of outer membrane porins. The histidine kinase KdpD has been proposed to be a turgor sensor, but the actual mechanism of stimulation is not understood (653). The protein has been purified and reconstituted in proteoliposomes (268). In this in vitro system, KdpD is activated by increased ionic strength and K⁺ ions attenuate the activity, suggesting that osmolarity sensing by KdpD is overlapped by some solute specificity (269). Interestingly, KdpD function in vitro seems to require negatively charged phospholipids, indicating that interaction with the surrounding lipids might be important for regulation and signal transmission (575). Although KdpD-KdpE is a two-component system, the domain organization of the sensor histidine kinase is different from that of Sln1p: KdpD has four transmembrane domains located in the central part of the protein.

EnvZ, however, has a domain structure that is very similar to that of Sln1p, with two transmembrane domains in the N-terminal part separated by an external loop; however, apart from the histidine kinase domain, there is no apparent sequence similarity to Sln1p. EnvZ has been studied intensively, with a focus on the function of the histidine kinase domain and its intrinsic phosphatase activity (147). Distinct domains have been purified and their structures have been analyzed (590, 607), but EnvZ has not been purified as a complete protein, nor has it been reported to be functionally reconstituted in proteoliposomes. Mutational analysis suggests that the transmembrane regions are required for sensing (603-606). This observation again suggests that sensing occurs in the membrane, for instance, in response to membrane stretching, but it does not exclude the possibility that the transmembrane domains play their role by positioning, for instance, the external loop in an osmoresponsive way. In such a scenario, the transmembrane domains would be required for intramolecular signaling through the membrane without being directly involved in sensing.

Since a range of different substances that alter water activity can stimulate the HOG pathway, it seems unlikely that binding of a ligand is the primary event. As for KdpD and EnvZ, it is rather anticipated that Sln1p is affected by a physical stimulus. The role of the sensor domain of Sln1p has been studied by a low-density deletion analysis (440). The data suggest that the first transmembrane domain is required for the control of histidine kinase activity by osmotic changes, since deletion of this domain renders the kinase constitutively active and unresponsive to an osmotic shock. This observation, however, does not reveal if the first transmembrane domain has any particular features important for osmosensing. Replacing it with a different transmembrane domain, as was done with Sho1p, could possibly provide such information. The external loop region seems to contain a dimerization domain, which can be replaced by an unrelated leucine zipper element. Dimerization is necessary for hybrid kinases because histidine autophosphorylation occurs in trans (579).

Truncation of Sln1p so that it lacks both transmembrane segments and the loop strongly diminishes its activity, resulting in hyperactivation of the downstream pathway (440). Clearly, more detailed functional analyses of Sln1p are needed to better understand its mechanism in osmosensing. In particular, it would be most revealing if Sln1p histidine kinase activity could be monitored in yeast secretory vesicles (406) or even reconstituted in proteoliposomes; such systems could allow more detailed studies on the biophysical mechanisms controlling Sln1p.

As indicated earlier, it is intuitively anticipated that osmosensing occurs at the cell surface, although this is by no means mechanistically necessary (653). In fact, the osmosensing histidine kinase of the cell wall-less slime mold Dictyostelium discoideum appears to be a cytosolic protein, since it is lacking any apparent transmembrane domains (535). The following scenarios, although all speculative, illustrate some possibilities for osmosensing beyond physical impact on the plasma membrane.

As mentioned above, an osmotic upshock causes the actin cytoskeleton to dissociate, and it is subsequently reorganized during adaptation. Possible sites of osmosensing are cortical actin patches, which consist of helical bundles of actin wrapped around plasma membrane invaginations (58, 400). Although this has not been studied in detail, it is likely that the size and shape of the invaginations change upon osmotic shock, affecting the conformation of actin bundles (205). In fact, the dynamic reestablishment of the cytoskeleton during adaptation (62, 97) illustrates that the cell senses that its actin network is disturbed. Therefore, the actin cytoskeleton may serve as a genuine osmosensor, at least for its own reorganization. This dynamic process would also make an excellent system for termination of the response. Although it has been known for many years that certain actin alleles (428, 644) as well as mutations in different components of the cytoskeleton cause osmosensitivity, there is no direct experimental evidence for an involvement of actin or its associated proteins in the control of signaling pathways or any other osmoadaptive processes in the cell. The notable and important exception is the G-protein Cdc42p, which plays an important role in the control of assembly of the actin cytoskeleton (198, 264) and is involved in the Sho1 branch of the HOG pathway (488, 498), as well as in other signaling pathways (264, 287).

An osmotic upshock causes water to flow out of the cell. One consequence of this is an increase in the concentration of all cellular components. In particular, the higher concentration of certain ions could serve as a signal. Recently, a higher internal concentration of K⁺ has been proposed to control the osmoregulated BetP channel in Corynebacterium glutamicum as a kind of concentration-dependent second messenger (518). A sensor protein could undergo a conformational change on binding of such an ion and thereby initiate a response. Since many proteins and especially enzymes are well known to respond to low-molecular-weight effectors, the cell is rich in putative osmosensors in this sense. Posttranslational effects, such as changes in metabolism (see below), could be controlled via altered concentration of low-molecular-weight compounds in the shrunken cell.

SIGNALING OSMOTIC CHANGES

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In addition to the HOG pathway, protein kinase A (cyclic AMP [cAMP]-dependent protein kinase) has been shown to affect expression of genes upon an osmotic upshift (423). Protein kinase A mediates a general stress response that is observed under essentially all stress conditions, such as heat shock, nutrient starvation, high ethanol levels, oxidative stress, and osmotic stress (362, 519, 555). Hence, protein kinase A most probably does not respond directly to osmotic changes. It is not well understood how the activity of protein kinase A is controlled by stress.

Other signaling pathways have recently been associated with responses to an osmotic upshift. It has been observed that an osmotic shock stimulates production of phosphatidylinositol-3,5-bisphosphate, which could serve as a second messenger in an osmotic signaling system (145). In addition, evidence has been provided that the Snf1p AMP-dependent kinase, which controls the general glucose repression system in S. cerevisiae, is involved in transcriptional responses to osmotic shock (613; M. Krantz and S. Hohmann, unpublished data).

Less is known about responses to osmotic downshifts. Rapid, posttranslational mechanisms seem to play an important role in survival, such as glycerol export through the Fps1p channel. In addition, it has been observed that a hypo-osmotic shock stimulates influx of calcium into yeast cells (30), but the physiological significance is unclear. It has also been shown that a hypo-osmotic shock rapidly stimulated the cell integrity pathway (125), but again the physiological significance is unclear. Finally, recent data indicate that Sln1p, Ypd1p, and Skn7p form a phosphorelay system that could activate gene expression upon cell swelling (338, 591), although this has not been tested directly and the overall impact on cellular physiology also remains to be elucidated. Global gene expression analysis has not yet revealed a characteristic gene expression pattern caused by an osmotic downshift but rather a reversal of the expression pattern observed in cells growing at high osmolarity (191), although more thorough analyses of hypo-osmotic shock responses may be performed in the near future.

MAP kinase pathways. MAP kinase pathways (for nomenclature of proteins, see Fig. 2) are highly conserved signaling units apparently occurring in all eukaryotes, where they play essential roles in the response to environmental signals or hormones, growth factors, and cytokines. MAP kinase pathways control cell growth, morphogenesis, proliferation, and stress responses, and they are involved in many disease processes. Excellent reviews on MAP kinase pathways are published frequently (for instance, see references 28, 88, 205, 304, 308, and 339), and therefore I summarize only essential principles relevant to the further discussion.

View larger version (25K): [in this window] [in a new window]   FIG. 2. Nomenclature of proteins in MAP kinase pathways. Arrows only indicate the flow of information; pathway-specific protein complexes are common and are required for signal transmission.

MAPKKKs consist of an N-terminal regulatory and a C-terminal catalytic kinase domain. The regulatory domain locks the C-terminal kinase domain in the inactive state. Activation may occur by phosphorylation through an upstream protein kinase or through interaction with other proteins, a process that often involves small G-proteins. The activation mechanisms and sensor systems upstream of MAP kinase pathways are diverse and include receptor-tyrosine kinase (in animal systems), G-protein-coupled receptors, phosphorelay systems, and others.

Different MAP kinase pathways form interacting signaling systems. For instance, one MAPKK may control several different MAP kinases, as is observed even in the relatively simple yeast system. Different pathways within the same organism often share kinases. Especially in higher eukaryotes but even in S. cerevisiae, this situation results in highly complex network systems of signaling pathways. Pathway specificity is commonly but probably not exclusively achieved by scaffold proteins. Scaffolds may be proteins apparently dedicated to this purpose, such as S. cerevisiae Ste5p, or may be part of active components of the MAP kinase cascade, such as the yeast MAPKK Pbs2p. Hence, presentation of MAP kinase systems as linear pathways, as shown in Fig. 2 to explain nomenclature, is actually an oversimplification.

MAP kinase pathways are negatively controlled by protein phosphatases acting on both the MAPKK and the MAP kinase (serine-threonine phosphatases) or only on the MAP kinase (tyrosine phosphatases) (283).

S. cerevisiae MAP kinase pathways. Since components of the MAP kinase cascade can be recognized by sequence similarity, the set of relevant kinases in the yeast proteome is known. S. cerevisiae has five MAP kinases. Based on genetic analyses as well as studies on the transcriptional readout upon physiological, pharmacological and genetic stimulation, the five MAP kinases are allocated to six distinct MAP kinase pathways (Fig. 3): (i) the mating pheromone response pathway (MAP kinase Fus3p), (ii) the pseudohyphal development pathway (Kss1p), (iii) the HOG pathway (Hog1p), (iv) the protein kinase C (PKC) or cell integrity pathway (Slt2/Mpk1p), and (v) the spore wall assembly pathway (Smk1p) (149, 205, 222, 474).

View larger version (46K): [in this window] [in a new window]   FIG. 3. Outline of the yeast MAP kinase pathways, illustrating similarities and difference in architecture. Gpa1p, Ste4p, and Ste18p form a heterotrimeric G-protein; Ras2p is one of two yeast Ras proteins. Other proteins are explained in the text.

The specificity of signal transmission is ensured by scaffold proteins (457), which are specific to their respective pathway: Ste5p is needed for the mating pheromone response pathway (74), and Pbs2p also functions as the MAPKK of the HOG pathway (473). Additional mechanisms ensuring pathway specificity and involving the MAP kinases Fus3p and Hog1p have been discussed; deletion of these two kinases results in inappropriate cross talk (354, 439). For the spore wall assembly pathway, only a MAP kinase and an upstream protein kinase have been found; the MAPKK and MAPKKK are missing. Hence, Smk1p is activated either by an unusual mechanism or by any of the known MAPKKs and MAPKKKs.

The sensing input systems differ between pathways, and it appears that yeast MAP kinase pathways represent a range of possible input devices. The mating pheromone response pathway is controlled by the pheromone receptors Ste2p and Ste3p (for the mating pheromones and a, respectively), which are G-protein-coupled receptors. For signal transmission, Ste50p, whose molecular function is not well understood, and the upstream kinase Ste20p, a member of the PAK (p21-activated protein kinase) family (122), are required. Ste20p serves as upstream kinase not only in the mating pheromone response pathway but apparently in all Ste11p-dependent pathways (321, 439, 508); at least in some instances, Cla4p (35) and perhaps Sps1p (368), yeast kinases related to Ste20p, may perform related or redundant functions. Localization of Ste20p to sites of cell growth and activation of Ste20p as well as of Cla4p require the G-protein Cdc42p and its exchange factor, Cdc24p. As mentioned above, Cdc42p also interacts with the actin cytoskeleton (reviewed in references 89, 149, 154, and 287).

The sensor of the pseudohyphal development pathway has not been unambiguously identified, although evidence is accumulating that Mep2p is needed for pathway activation through nitrogen-dependent signals (348). Mep2, which has strong similarity to ammonium transporters, is a member of a new class of sensor proteins derived from transporters (171). The two branches of the HOG pathway are controlled by apparently completely different input systems. As outlined above, the sensor for the Sho1 branch is presently not known; the formation upon stimulation of a protein complex between Sho1p and Pbs2p, probably also involving Ste50p, the upstream kinase Ste20p, the G-protein Cdc42p, and the MAPKKK Ste11, is needed for the activation of this system (488, 498). The Sln1 branch of the HOG pathway is controlled by a phosphorelay system. The upstream sensing system of the STE vegetative growth pathway seems to involve proteins from the Sho1 branch of the HOG pathway, including Sho1p itself (149). Multiple putative sensors have been described for the cell integrity pathway, as discussed below (216). The MAP kinase cascade is controlled by the upstream kinase Pkc1p, the single yeast homolog of protein kinase C, which also has additional functions. For the spore wall formation pathway, the upstream kinase Sps1p has been identified, but the mechanisms that control this protein are not known.

Upon activation, a portion of the MAP kinase moves to the nucleus, where it controls transcriptional regulatory proteins. Transcription factors have been associated with most of the yeast pathways, and the control of these factors by the MAP kinase is known to different degrees of molecular detail. The type of transcription factor and the way it is controlled are apparently not conserved between MAP kinase pathways. Fus3p of the mating pheromone response pathway activates the zinc finger protein Ste12p, which binds to pheromone response elements and activates a set of genes whose products play roles in mating and cell fusion (205, 499, 507). Ste12p activation requires inactivation of two negative regulators, Dig1p and Dig2p. Ste12p cooperates on target promoters with Mcm1p, which is a factor binding to numerous promoters. Ste12p is also the transcription factor needed for the transcriptional output of the pseudohyphal development pathway, where it cooperates with Tec1p on target promoters or controls expression of TEC1 (329). The Hog1p kinase mediates its effects through at least five different transcription factors: the possibly redundant zinc finger proteins Msn2p and Msn4p (533), Hot1p (which does not belong to a known family of factors) (503), the bZIP protein Sko1/Acr1p (480), and the MADS box protein Smp1p (F. Posas, 2001, personal communication). The cell integrity pathway mediates transcriptional responses through Rlm1p, which controls genes encoding proteins required for cell wall assembly, as well as SBF (Swi4/Swi6 cell cycle box-binding factor) (216). The transcription factor(s) controlled by Smk1p has not been identified.

Present knowledge of the architecture, sensors, and outputs of the yeast MAP kinase leads to an emerging picture where these pathways, in concert, orchestrate morphogenesis, directed cell growth, and remodeling of the cell surface. While the primary decisions for cell polarity seem to be controlled by other mechanisms (89), the MAP kinase pathways are required for directed cell growth (bud formation, mating projections, and pseudohyphal growth), the necessary remodeling of the cell surface associated with growth (cell wall integrity, cell integrity, and HOG pathways), and maintenance of the appropriate turgor pressure (HOG and cell integrity pathways). Spore formation is also accompanied by remodeling of the surface. While cellular morphogenesis in response to developmental and external stimuli seems to be the primary role of the yeast MAP kinase pathways, several of those (the mating pheromone response, cell integrity, and HOG pathways) have demonstrated roles in also coordinating cell proliferation (cell cycle control) with morphogenesis.

HOG pathway architecture. The architecture of the HOG pathway (Fig. 4) has been elucidated through several clever genetic screens and epistasis analysis, the latter partly based on expected analogy to other pathways. In the following I summarize the genetic evidence for the architecture of the HOG pathway, while in the next section I discuss in more detail the dynamic operation of the pathway as elucidated by tools of biochemistry and cell biology. Phenotypes of key HOG pathway mutants are summarized in Table 1.

View larger version (39K): [in this window] [in a new window]   FIG. 4. Outline of the HOG pathway. Pbs2p functions both as a scaffold and as a MAPKKK; it is not known exactly with which components of the Sho1 branch it interacts.

hog1

pbs2

hog1

pbs2

hog1 pbs2

This pioneering work of Gustin and colleagues marked the realization that osmotic responses are mediated by specific and potentially conserved signaling pathways (61). Saito and colleagues discovered further pathway components. They were searching for mutations that caused synthetic lethality in combination with deletion of PTP2, which encodes a protein tyrosine phosphatase. This screen identified the genes SLN1 and YPD1 (357, 476). It subsequently turned out that deletion of SLN1 and YPD1 by themselves causes lethality. This lethality, however, was suppressed by overexpression of PTP2, confirming a genetic link (357, 476). The SLN1 gene had initially been identified in a screen for mutations causing synthetic lethality with a mutation affecting protein degradation by the ubiquitin-dependent pathway (441, 442). In light of the presently known role of Sln1p, this early finding remains mysterious. Perhaps presently unknown ubiquitin-dependent proteolytic mechanisms are part of osmotic responses. The sln1 lethality seems to depend on the genetic background: sln1 mutants of some strains grow poorly, especially on defined (SC) medium (442).

Mutations that suppressed the lethality of the sln1 mutation identified the already known genes HOG1 and PBS2 (357), linking Sln1p to the HOG pathway. Since cellular depletion of Sln1p, as achieved by expression of SLN1 from the repressible GAL1 promoter, caused heavy tyrosine phosphorylation of Hog1p, and because of the similarity of Sln1p to sensor histidine kinases, it was concluded that Sln1p is an upstream sensor of the HOG pathway. The same suppressor search identified further components of this pathway, the MAPKKs Ssk2p and Ssk1p, which contain a response regulator domain (357). A search for genes homologous to SSK2 revealed SSK22 (355). These two proteins are 69% identical in the C-terminal kinase domain and 47% identical in the N-terminal regulatory domain. At least within the HOG pathway, both proteins seem to perform the same function (355).

Epistasis analysis confirmed the pathway organization indicated by the similarity of the protein kinases to components of MAP kinase pathways. For instance, inappropriate activation of the HOG pathway by N-terminal truncation of the Ssk2p and Ssk22p kinases causes lethality, as does deletion of Sln1p. This lethality is suppressed by deletion of PBS2 and HOG1, but not by deletion of SSK1 (355), placing Ssk2p and Ssk22p upstream of Pbs2p and Hog1p but downstream of Ssk1p. Multicopy suppression of the sln1 lethality also generated a rich harvest: the genes encoding the protein phosphatases Ptp2p and Ptp3p, which encode phosphotyrosine phosphatases, and Ptc1p and Ptc3p, which encode serine/threonine protein phosphatases (356, 357, 441). Interestingly, none of the suppressor screens revealed any candidate downstream targets of the pathway, such as transcription factors or genes whose expression is controlled by the pathway. This is due to the fact that the HOG pathway controls the expression of many genes via different transcriptional regulators, and individual deletion of such genes causes only moderate suppression, if any (503).

Several observations suggested that the pathway had not yet been completely uncovered. Even the double ssk2 ssk22 mutant still showed Hog1p phosphorylation upon osmotic shock, and the mutant did not display an osmosensitive phenotype, indicating alternative routes to activate the downstream MAPKK Pbs2p (355). Several components of this route were identified in an exhaustive search for mutations that caused osmosensitivity in an ssk2 ssk22 background. This screen identified an allele of Pbs2p as well as Sho1p (355), and direct interaction between Pbs2p and Sho1p via the SH3 domain of Sho1p was demonstrated. The same screen also identified the Ste11p MAPKKK. Triple ssk2 ssk22 sho1 and ssk2 ssk22 ste11 mutants are as osmosensitive as pbs2 and hog1 mutants and fail to phosphorylate Hog1p upon osmotic shock (355, 473), indicating that the three MAPKKKs Ssk2p, Ssk22, and Ste11p represent the activators of Pbs2p. Only when more than 1.4 M NaCl is applied do such triple mutants show Hog1p phosphorylation; it is not known if this is a truly physiological response (622), and it may be caused by Hog1p autophosphorylation (33). Finally, the same screen also identified another component of the pheromone response pathway, Ste50p (475). Ste50p is needed for activation of Hog1p via the Sho1 branch, and an ssk2 ssk22 ste50 triple mutant is osmosensitive (439, 475).

The fact that Ste11p is involved in both the pheromone response pathway and the HOG pathway prompted a screen for mutations that allowed cross talk, i.e., activation of the pheromone response pathway by osmotic shock (439). It was found that mutations in the gene for Hog1p caused such cross talk and that stimulation of the pheromone response pathway by osmotic shock in a hog1 mutant required Sho1p, Ste50p, and also Ste20p. In fact, a triple ssk2 ssk22 ste20 mutant is osmosensitive and defective in Hog1p phosphorylation, identifying Ste20p as a pathway component. However, the osmosensitivity is not as strong as, e.g., in an ssk2 ssk22 ste50 mutant (439, 488). This is probably due to the Ste20p homolog, Cla4p (117); overexpression of CLA4 rescues the osmosensitivity of the ssk2 ssk22 ste20 triple mutant (488), indicating that the proteins have overlapping functions. Finally, the essential G-protein Cdc42p, which binds Ste20p as well as Cla4p (35, 117), is involved in signal transduction through the Sho1p branch according to several lines of evidence: the Ste20p domain for interaction with Cdc42p is required for signaling through the HOG pathway, a dominant negative allele of Cdc42p reduces Hog1p phosphorylation in an ssk2 ssk22 background, a partially defective allele of CDC42 reduces Hog1p phosphorylation in an ssk1 background, and Cdc42p is required for the osmoshock-induced localization of Ste20p to the cell surface (see below) (488, 498).

At this point, genetic analyses leave a couple of open questions. Which protein functions as the osmosensor in the Sho1 branch of the pathway? Recent analysis indicates that Sho1p itself probably only functions as a recruiting factor (488). The Saito group has done an exhaustive search for mutations conferring osmosensitivity on an ssk2 ssk22 double mutant (475), but since no candidate sensor was found, it (they) may be encoded by redundant genes or one of the known components might actually function as the sensor. Integrin receptor-like type I transmembrane proteins, such as the Wsc proteins (629), are possible sensor candidates. Alternatively, the actin cytoskeleton may be involved in the sensing process, as discussed above.

Which proteins are involved in a possible third input system into the HOG pathway? The existence of a third sensing system is based on the observations that even in sho1 ssk1 and sho1 ssk2 ssk22 mutants, deletion of HOG1 leads to osmo-induced activation of the pheromone response pathway. In addition, it was observed that a sho1 ssk1 strain is less osmosensitive than an ste11 ssk1 or ste50 ssk1 strain, suggesting that Ste11p can also be activated by osmotic shock independently of Sho1p (439).

What is the mechanism that stimulates Hog1p phosphorylation by severe osmotic stress in an sho1 ssk2 ssk22 mutant (622)? Principally, the second and third questions might lead to the same answer, although Hog1p autophosphorylation may also be a simple possibility. More work is needed to clarify the possible existence of alternative input systems into the HOG pathway.

Activation of HOG pathway function: subcellular localization and dynamics. (i) Role of the two branches of the HOG pathway. The genetic evidence outlined above suggests that the upstream branches of the HOG pathway operate independently of each other; blocking one branch of the pathway still allows rapid Hog1p phosphorylation upon an osmotic shock, and such cells are apparently fully resistant to high osmolarity. Although these observations suggest redundant functions of the two branches, it is unlikely that the cell maintains two different complex pathways to activate Pbs2p.

It has been proposed that different sensitivities of the two branches may allow the cell to respond over a wide range of osmolarity changes (355). In an ssk2 ssk22 double mutant, which completely relies on the Sho1 branch, stimulation of Hog1p tyrosine phosphorylation requires at least 300 mM NaCl, becomes visible after about 2 min, and reaches a maximum at 5 min. In contrast, in an sho1 ssk22 mutant, which relies on the Sln1 branch only, Hog1p phosphorylation is already apparent with 100 mM NaCl and is maximal after 1 min with 300 mM NaCl. These data suggest that Sln1p is more sensitive than the sensor of the Sho1 branch. It also appears from the same set of experiments that the Sho1 branch operates in an on-off fashion, while the Sln1 branch shows an approximately linear dose response up to about 600 mM NaCl. Different sensitivities seem to be a plausible though not fully satisfactory explanation for the existence of the two branches.

At an osmolarity where only one of the two branches is activated, i.e., below 300 mM NaCl, hog1 mutants still grow, indicating that the pathway is not absolutely necessary under these conditions. In addition, in nature yeast cells are commonly exposed to far more dramatic osmolarity changes. At high osmolarity, when Hog1p phosphorylation is apparently saturated, the period of Hog1p phosphorylation becomes progressively longer the more osmoticum is used to shock the cells. In other words, the cell seems to modulate the response by the amplitude only at lower osmolarity and by the period of activation at higher osmolarity (622; B. Nordlander, M. Rep, M. J. Tamás, and S. Hohmann, unpublished observations). Taken together, the observed different sensitivities and responsiveness of the two branches may rather reflect different mechanisms of stimulation, i.e., the two branches may interpret osmotic changes via different physical stimuli, such as membrane stretch, cell wall stress, or impact on the cytoskeleton. If so, it should be possible to find conditions under which only one of the two branches is activated.

The recent finding that components of the Sho1 branch are localized or recruited to places of active cell growth (488, 498) indicates that it fulfils a specific localized role in osmosensing. The precise subcellular localization of components of the Sln1 branch has not been reported, but on the basis of our present understanding, one might speculate that the Sho1 branch monitors (mainly) osmotic changes during cell growth and expansion, while the Sln1p branch (mainly) senses osmotic changes in the environment.

Such an idea is further supported by several observations. If we assume that (one branch of) the HOG pathway monitors osmotic changes during cell growth, we would expect the pathway to perform functions even at normal, constant external osmolarity. Simultaneous deletion of the genes PTC1 and PTP2, which encode protein phosphatases, is lethal under normal growth conditions, and this lethality is suppressed by additional deletion of HOG1 (254, 356). This suggests that Hog1p is activated even at low osmolarity but that this activation is counteracted by the phosphatases; the upstream branch involved in this homeostatic activation has not been determined.

Furthermore, evidence has been reported for a role of the HOG pathway in controlling cell surface composition under ambient osmolarity: Hog1p is required for the proper localization of Mnn1p, a Golgi glycosyltransferase (505). In addition, mutations in the HOG pathway, in fact in both branches, increase tolerance to the antifungal drug calcofluor, which targets chitin-containing fungal cell walls (188). Moreover, a shift to low pH was shown to cause Hog1p-dependent changes in the expression of genes encoding cell wall proteins and a concomitant change in cell wall composition (276). Low pH is not known to stimulate the HOG pathway, and hence these effects may be secondary. Which upstream branches of the HOG pathway were required for the effect was not studied. Taking the results together, it is apparent that the HOG pathway confers activity even without osmotic stress.

This function could be a reflection of its role in monitoring turgor during cell growth, and this in turn could be a specific role of the Sho1 branch. Sho1p together with Ste20p, Ste11p, Ste7p, Kss1p, and Ste12p appears to form an independent MAP kinase pathway, the STE vegetative growth pathway, which seems to be part of the systems that control cell wall integrity (114, 324). That pathway is required for growth at normal osmolarity. In addition, a genetic screen for multicopy suppressors of the growth defect of an ste11 ssk2 ssk22 triple mutant at moderately high osmolarity and elevated temperature, which is probably due to simultaneous inactivation of the HOG and STE vegetative growth pathways, revealed the genes LRE1 and HLR1 (16). The products of these two genes are thought to affect cell wall composition. Overexpression of LRE1 and HLR1 also partially suppressed the osmosensitivity of a hog1 and a pbs2 mutant, suggesting that the HOG pathway, directly or via interaction with other pathways, affects cell wall composition (16).

Together, both branches of the HOG pathway may then orchestrate osmotic responses and integrate the need for cell expansion in response to osmotic signals generated by growth and by the environment.

(ii) Activation via the Sln1 branch. While the physical signal