FEMS Yeast Research 2 (2002) 251^257
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MiniReview
Novel insights into the osmotic stress response of yeast
Willem H. Mager, Marco Siderius
Department of Biochemistry and Molecular Biology, Biocentrum Amsterdam, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam,
The Netherlands
Received 17 December 2001; received in revised form 29 April 2002; accepted 3 May 2002
First published online 3 June 2002
Abstract
Response to hyperosmolarity in the baker’s yeast Saccharomyces cerevisiae has attracted a great deal of attention of molecular and
cellular biologists in recent years, from both the fundamental scientific and applied viewpoint. Indeed the underlying molecular
mechanisms form a clear demonstration of the intricate interplay of (environmental) signalling events, regulation of gene expression and
control of metabolism that is pivotal to any living cell. In this article we briefly review the cellular response to conditions of
hyperosmolarity, with focus on the high-osmolarity glycerol mitogen-activated protein kinase pathway as the major signalling route
governing cellular adaptations. Special attention will be paid to the recent finding that in the yeast cell also major structural changes occur
in order to ensure maintenance of cell integrity. The intriguing role of glycerol in growth of yeast under (osmotic) stress conditions is
highlighted. 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Stress response; Hyperosmolarity ; Glycerol; Cell wall; Saccharomyces cerevisiae
1. General overview
Tentatively, in the response of Saccharomyces to hyperosmolarity, di¡erent stages can be distinguished: (i) immediate cellular changes that occur as the direct consequence
of the physico-mechanical forces operating under those
conditions, (ii) primary defense processes elicited in order
to set protection, repair and recovery in motion and (iii)
sustained adaptive events that permit restoration of cellular homeostasis under the new circumstances.
(i) As an immediate consequence of the exposure of
yeast to high osmolarity in the surrounding medium, cells
rapidly loose intracellular water which leads to loss of
turgor and hence shrinkage of cells [1]. Also intracellularly
the e¡ects of an osmotic challenge are manifest. Water
is recruited from the vacuole into the cytoplasm, thus
partially compensating for the sudden increase in (macro)molecular concentration. In addition, depending on the
severity of the osmotic stress, cytoskeleton collapses
* Corresponding author. Tel. : +31 (20) 4447570;
Fax : +31 (20) 4447553. E-mail address: [email protected]
(M. Siderius).
leading to depolarization of actin patches. These immediate e¡ects are caused by the physico-mechanical forces
operating under hyperosmotic conditions.
(ii) The primary response to hyperosmotic stress consists
of several molecular events that are triggered by the sudden cellular changes. First, cells temporarily arrest growth.
It is as yet unclear what determines the cell cycle point at
which arrest occurs. The block in the cell cycle has been
reported to occur at G1, probably by downregulation of
Cln3p-Cdc28p kinase activity [2]. Also arrest at the G2/M
transition has been demonstrated via inhibition of Clb2pCdc28p kinase [3]. It is unknown why yeast cells arrest
progression through the cell cycle. Most likely this is
part of a strategic re-setting mechanism required for proper adjustment to the new growth conditions.
Secondly, the glycerol channel Fps1p closes [1]. Glycerol
is the major compatible solute of Saccharomyces. By accumulating internal glycerol cells accomplish regain of turgor.
Thirdly, the high-osmolarity glycerol (HOG) mitogenactivated protein (MAP) kinase pathway is triggered [4].
This results in the rapid phosphorylation and nuclear
translocation of MAP kinase Hog1p. Nuclear Hog1p mediates regulation, both repression and activation, of gene
expression. For instance, Hog1p phosphorylates and hence
inactivates the transcriptional repressor Sko1p, thereby
1567-1356 / 02 / $22.00 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 1 6 - 2
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inducing transcription of, among others, the sodium
pump-encoding ENA1 gene [5]. In addition, the transcriptional activator Hot1p is a substrate for Hog1p. Hot1p
regulates transcription of the GPD1 gene encoding glycerol 3-phosphate dehydrogenase [6]. So, apart from preventing glycerol e¥ux as mentioned above, also increasing its
synthesis contributes to the intracellular glycerol accumulation. Understanding the ins and outs of glycerol biosynthesis and accumulation is of major commercial relevance
for controlling the performance of yeast in dough preparation or in wine and beer fermentation (e.g. [7]).
Recently, Hog1p has been reported to associate with the
transcription machinery via stress-mediating transcriptional activator proteins, and may modulate promoter binding
of (some of) these factors [8]. Hog1p likely plays a role in
the cytoplasm as well. For instance it has been demonstrated to activate Rck2p, a putative calmodulin protein
kinase that supposedly inhibits protein biosynthesis [9,10].
This makes sense : upon stress exposure protein synthesis
is transiently repressed.
Part of the primary response to hyperosmotic stress
overlaps with the response to other types of stress : the
so-called general stress response. Transcription factors
Msn2/Msn4p are the mediators of the general stress response [11]. Under non-stress conditions these factors
are localized in the cytoplasm but upon stress exposure
they rapidly translocate to the nucleus where they bind
to the STRE-control elements in the promoters of a large
number of stress-responsive genes. This results in a drastic,
temporary change in the transcript pro¢le of yeast cells
[12]. Cellular localization of Msn2/4p is regulated by the
activity of cAMP-dependent protein kinase A (PKA):
under conditions of high PKA the factors remain in the
cytoplasm whereas under low PKA conditions they are
mainly nuclear [11]. How general stress conditions promote the PKA-dependent nuclear translocation of Msn2/
4p is as yet unknown.
(iii) The sustained phase of the response, generally spoken, is the sum of the (metabolic) processes evoked in the
primary phase of the response. Indeed, genomic transcript
pro¢ling has revealed that gene expression re-sets to a new
steady state [12]. The same most likely holds for cellular
levels of metabolic and regulatory activities. Intracellular
glycerol accumulates by the combined e¡ects of diminished e¥ux and enhanced synthesis. This will ultimately
lead to intake of water and swelling of the cell to a size
which remains smaller than before the osmotic challenge
[1]. When the critical cell size is attained, growth resumes
and cells continue to divide. Cytoskeleton is repaired and
displays actin repolarization. Interestingly, in addition
cell-wall organization is adjusted to the new environmental
circumstances (see below).
Here we focus on the role of the HOG pathway in the
hyperosmotic stress response, on the implications of the
osmo-stress response for the maintenance of cellular integrity and on the intriguing role of glycerol.
2. Activation of the HOG pathway
In Saccharomyces so far two proteins that can serve as
upstream receptors of the HOG MAP kinase pathway
have been postulated [4] (see Fig. 1). Sln1p is part of a
two-component class of signalling systems, in this case de
facto acting as a three-component system. Sln1p, the signal receiver, is autophosphorylated at an internal histidine.
This phosphate is intramolecularly transferred to an aspartic acid residue, and then to a histidine in Ypd1p. Subsequently phosphotransfer occurs to an aspartic residue in
the ‘response regulator’ protein Ssk1p. Ssk1p in its nonphosphorylated form activates the functionally redundant
MAP kinase kinase kinases (MAPKKKs) Ssk2/Ssk22p.
These MAPKKKs activate MAP kinase kinase Pbs2p,
which in turn dually phosphorylates and activates MAP
kinase Hog1p. The other osmo-signalling branch of the
HOG pathway was until recently thought to be triggered
by the putative osmo-sensor Sho1p, which physically interacts with Pbs2p via its SH3 domain. Recent elegant
experiments using chimeric genes and truncated alleles of
Sho1p however have revealed that its transmembrane part
is not speci¢cally required for stimulating HOG signalling
[13]. In addition, a membrane-targeted version of Pbs2p
has been found to be su⁄cient to do the job [13]. Therefore, Sho1p likely has a membrane-docking rather than a
sensing function. Present insight suggests that an unknown
sensor protein transmits its signal via the adaptor protein
Sho1p. In signal transduction via this route a series of
upstream components are involved which re£ect the need
to recruit the osmo-signalling complex to the plasma membrane: Cdc42p, Ste20p, Ste50p and Ste11p [14]. It is likely
that the small GTPase Cdc42p plays a role in recruiting
the Pbs2p complex to the site where the cell wall/membrane structure is supposed to be most susceptible to osmotic stress, for instance at the site of bud emergence
(where polarized growth occurs) [14]. The p21-activatedkinase Ste20p is activated by Ste50p kinase and in its turn
phosphorylates Ste11p, also known as the MAPKKK of
the mating pheromone pathway [4]. Thus, Ste20p can be
considered the MAPKKKK of the HOG pathway. Phosphorylation of Ste11p by Ste20p leads to release of the
inhibitory N-terminal domain of Ste11p from its catalytic
C-terminal domain [15].
We have recently demonstrated that yeast mutants defective in both signalling branches of the HOG pathway,
due to the deletion of Sho1p, Ssk2p and Ssk22p or Ste11p,
Ssk2p and Ssk22p, still show Hog1p activation under severe osmotic stress conditions ( s 1.2 M sorbitol) [16]. This
activation is Pbs2p-dependent. The components involved
in this alternative signal transduction have not yet been
identi¢ed. The alternative activation of Hog1p in the sho1
ssk2 ssk22 background under severe osmotic stress turned
out to be abolished after additional disruption of BCK1,
encoding the MAPKKK of the PKC pathway (our own
unpublished results). This may re£ect activation of Pbs2p
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253
Fig. 1. Hyperosmotic stress induces signalling through the HOG MAP kinase pathway. Two signal input branches, via Sho1p and Sln1p^Ypd1p^Ssk1p,
respectively, can be distinguished. Details are described in the text. Exposure of sho1 ssk2 ssk22 or ste11 ssk2 ssk22 mutant strains to severe osmotic
stress revealed occurrence of yet another, up to now unknown, manner of Hog1p activation. Both cytoplasmic and nuclear targets of Hog1p have been
identi¢ed. Among others, HOG pathway activation contributes to enhancement of GPD1 expression and, hence, to increase in glycerol biosynthesis. In
addition, under hyperosmotic stress conditions, the glycerol transport facilitator Fps1p closes, via an as yet unknown mechanism.
by Bck1p directly or it reveals the need for PKC pathway
signalling and, thus, is an indirect e¡ect.
The HOG pathway is deactivated by dephosphorylation
of Hog1p by the Ser/Thr phosphatases Ptc1, 2 and 3p [18]
and the Tyr phosphatases Ptp2p and Ptp3p [19]. Under
physiological conditions this deactivation is essential : constitutive Hog1p activation is lethal. The phosphatases involved play a rather pleiotropic role in regulation of MAP
kinase signalling in yeast.
3. Osmo-stress and cell wall integrity
Thus far surprisingly little attention has been paid to the
possible involvement of the yeast cell wall in sensing and
signalling of osmotic stress. The yeast cell wall is a dynamic structure [20]. It is organized in two layers, built-up of
four classes of covalently bound macromolecules: mannoproteins (or cell-wall proteins, some of which contain a
glycosyl-phosphatidylinositol anchor), L-1,3 and L-1,6 glucan and chitin (L-1,4-N-acetyl-glucosamine polymer). The
actual composition of the cell wall may di¡er signi¢cantly
according to the cellular demands. S. cerevisiae requires
cell-wall reorganization at di¡erent stages of its biological
cycle, for instance for bud formation during vegetative
growth, and for developmental processes like mating or
sporulation.
The PKC MAP kinase pathway has been described as
the most important signal transduction route involved in
maintenance of cell-wall integrity ([4] ; see Fig. 2). It is
activated under conditions of hypo-osmotic stress and at
elevated temperature. In addition, cell-wall damage evokes
activation of the pathway leading to compensatory alterations in the cell wall [21]. Upstream components of the
PKC MAP kinase cascade are the plasma membrane proteins Wsc1,2,3,4p and Mid2p [4]. These putative signal
receptors elicit signal transduction via a GTPase-switch
mechanism operated by Rom2p (a GDP^GTP exchange
protein) and Rho1p (a small GTPase), Tor2p (a phosphatidylinositol kinase homolog) being a regulator [4]. Sac7p
and Bem2p are activators (GAPs) of Rho1p-GTPase. Subsequently Pkc1p is activated, which in its turn serves as an
upstream kinase of the cascade Bck1p^Mkk1/2p^Mpk1p
[4]. Activated Mpk1p triggers activity of several transcription factors, amongst other, SBF (consisting of Swi14p
and Swi6p), involved in transcription of cell cycle genes,
and Rlm1p, implicated in cell-wall gene transcription [4].
In addition, Rho1p serves as a regulatory subunit of the
glucan synthase complex [20].
Recently, a novel signal transduction route implicated in
the maintenance of cellular integrity has been postulated:
the SVG (‘sterile vegetative growth’) pathway [22,23]. This
SVG pathway makes use of the major components of the
¢lamentous growth MAP kinase module, viz. Ste11p,
Ste7p and Fus3p (see Fig. 2). Upstream components
have not yet been identi¢ed. The SVG pathway is sup-
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Fig. 2. Three MAP kinase pathways, viz. PKC, SVG and HOG, mediate signalling ensuring maintenance of cell-wall integrity (see text). Under hyperosmotic stress conditions, temperature-induced PKC signalling is inhibited. A mutant strain lacking STE11, SSK2 and SSK22 is not able to perform SVG
and HOG signalling as well, leading to growth impairment under these conditions (37‡C, 0.6 M sorbitol).
posed to be active at normal vegetative growth, under
conditions where cell-wall defects are present (e.g. at the
level of glycosylated components). Recently we have obtained evidence that the SVG pathway plays a part under
certain osmo-stress conditions as well [24]. Evidence came
from analysis of HOG pathway mutants challenged with
hyperosmolarity at elevated growth temperature.
In general, HOG pathway mutants are osmo-sensitive,
but strikingly enough osmo-sensitivity is largely alleviated
by growth at 37‡C ([25]; see Fig. 3A). The ste11 ssk2 ssk22
strain however shows an exception to this rule: this triple
mutant is even more osmo-sensitive at 37‡C than at 24‡C.
This growth defect appears to be suppressed by overexpression of, amongst others, LRE1, HLR1 and WSC3
genes putatively involved in cell-wall biosynthesis ([24];
see Fig. 3B). LRE1 has been identi¢ed as a gene that
upon overexpression confers laminarinase (L-glucanase)
resistance. HLR1 is its homolog on the yeast genome.
Lre1p has been demonstrated to interact with Cbk1p, a
conserved PAK-kinase involved in control of cellular morphogenesis, and to be linked with the chitinase CTS1 gene
expression via transcription factor Ace2p [26,27,28]. LRE1
and HLR1, but not WSC3 overexpression also suppresses
osmo-sensitivity of pbs2 and hog1 at 24‡C [24].
Our work model to explain the phenotype is illustrated
in Fig. 2. We propose that all three parallel MAP kinase
pathways (PKC, SVG and HOG) contribute to ensure cell
integrity. Under hyperosmotic conditions activation of the
PKC pathway, which normally occurs at elevated temperature, is repressed. In wild-type cells the SVG pathway
may take over in triggering the necessary modi¢cations.
However, in the ste11 ssk2 ssk22 mutant strain due to the
disruption of STE11 also the SVG pathway is not functional, whereas in addition HOG signalling is absent. This
may lead to the observed growth defect. Blocking the SVG
and HOG pathways in a di¡erent way, e.g. by disruption
of STE7 and PBS2, gives rise to the same phenotype [24].
The phenotype of the ste11 ssk2 ssk22 strain has disclosed
the intricate interplay of the respective MAP kinase pathways in control of cellular integrity. It is likely that also
under iso-osmotic conditions Hog1p plays a part in regulation of cell-wall structure. Indeed, hog1 and pbs2 strains
are sensitive to zymolyase (a glucan-degrading enzyme
preparation), whereas suppression occurs by overexpression of LRE1 and HLR1. In addition, hog1 and pbs2
strains are resistant to the chitin-binding antifungal drug
calco£uorwhite [29]. The data discussed above make clear
that it is very worthwhile to continue and extend studies of
cell-wall structure and formation under hyperosmotic
stress conditions, in order to elucidate the role of the
cell wall under these conditions. Meanwhile it has become
very likely that at the level of cell-wall architecture major
adjustments to the new physiological demands occur. In
addition, it is tempting to speculate that the cell wall is not
only a target of the osmo-stress response but that also part
of the osmo-stress-induced signalling events is evoked at
the level of the cell wall. This would be consistent with
predicted protein structure elements of some upstream
components of signalling pathways. Evidence for the latter
presumption, however, is still lacking and probably has to
await insight into the actual mechanism by which yeast
senses osmotic stress.
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Fig. 3. HOG pathway mutants display decreased sensitivity to high osmolarity at 37‡C (A). Ste11 ssk2 ssk22 cells, however, are hypersensitive (A) as
explained by the model depicted in Fig. 2. Among the multicopy suppressors of this phenotype are LRE1, HLR1 and WSC3, involved in cell-wall formation (B) and GPD1 and FPS1, involved in glycerol accumulation (C).
4. The intriguing role of glycerol
In Fig. 4 the major mechanisms involved in control of
the intracellular glycerol level are depicted. As discussed
above, glycerol is the major compatible solute of yeast
[30]. In addition, glycerol metabolism plays an important
part in redox-balancing [31] and of course in phospholipid
biosynthesis. In agreement with the role of glycerol as
osmolyte, osmo-sensitivity of the hog1 strain has been explained by the reduced intracellular glycerol concentration. However, it is not easy to understand how the
HOG pathway which is transiently activated under hyperosmotic stress conditions is able to regulate intracellular
glycerol under steady-state (adapted) growth conditions.
We recently have found evidence that glycerol may play
a more general role in cellular functioning [25]. The reduced osmo-sensitivity of HOG pathway mutants at elevated growth temperature appears to be parallelled by
(two-fold) enhanced intracellular glycerol. The importance
of glycerol for growth at increased temperature is also
manifest from the temperature-sensitive phenotype of the
gpd1 gpd2 and gpp1 gpp2 double disruption strains [25].
The molecular mechanism underlying the increase in internal glycerol at 37‡C in the absence of Hog1p remains to
be elucidated; GPD1 transcription is only transiently elevated after a shift to higher temperature. Consistent with
the ¢nding of the higher basal level of glycerol in the
‘temperature-remedial osmo-resistant’ HOG pathway mu-
tants, the hypersensitive ste11 ssk2 ssk22 strain contains a
low level of glycerol. In agreement with this, in our screening FPS1 was found as a suppressor of the hypersensitive
phenotype of ste11 ssk2 ssk22, and subsequently also
GPD1 overexpression turned out to mediate suppression
(see Fig. 3C). In conclusion, the suppressors found in the
screen for osmo-resistance of ste11 ssk2 ssk22 at elevated
temperature fall into di¡erent categories. Therefore, an
appealing hypothesis is that suppression of the ste11
ssk2 ssk22 growth de¢ciency can be accomplished in several ways, which have in common that they lead to restoration of signalling competence of the cell. Obviously,
growth under hyperosmotic conditions, as well as growth
at elevated temperature, requires cell-wall modi¢cations. A
critical threshold in turgor pressure may be necessary to
evoke the proper signalling processes. Turgor, hence,
might play a part not only in HOG signalling but in
PKC and SVG signalling as well.
Acknowledgements
Parts of the reported studies have been ¢nancially supported by the EU TMR Grant ERB-FMRX-CT96-0007
to W.H.M. We gratefully acknowledge the contributions
to the work performed in our group by our coworkers
Iwona Wojda, Rebeca Alonso Monge, Eliana Real and
JanPaul Bebelman.
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Fig. 4. Glycerol synthesis in Saccharomyces occurs by a two-step conversion from dihydroxyacetonephosphate (DHAP ; from glycolysis), which is catalyzed by Gpd1, Gpd2p and Gpp1, Gpp2p consecutively. In particular GPD1 and GPP2 gene expression is induced upon a hyperosmotic stress challenge. Under hyperosmotic conditions glycerol accumulation is accomplished by the decrease of e¥ux via the glycerol transport facilitator Fps1p and increase in synthesis. Gut1p and Gut2p are glycerol kinase and mitochondrial glycerol 3-phosphate dehydrogenase, respectively. Glycerol uptake is
depicted as a mere di¡usion process. Genes encoding putative glycerol permeases have been identi¢ed, but their physiological relevance is as yet obscure.
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