Medical Mycology 2001, 39, Supplement 1, 111±121
The yeast cell-wall salvage pathway
L. POPOLO*, T. GUALTIERI & E. RAGNI
UniversitaÁ degli Studi di Milano, Dipartimento di Fisiologia e Biochimica Generali, Via Celoria 26, 20133 Milano, Italy
The integrity of the cell wall depends on the synthesis and correct assembly of its
individual components. Several environmental factors, such as temperature up-shift,
treatments with mating factors or with speciŽc cell wall-perturbing drugs, or genetic
factors, such as inactivation of cell wall-related genes (for example FKS1 or GAS1)
can impair construction of the cell wall. As the cell wall is essential for preserving the
osmotic integrity of the cell, several responses are triggered in response to cell-wall
damage. This review focuses on the activation of salvage pathways that guarantee
cell survival through remodeling of the extracellular matrix. These researches have
useful implication for the study of similar pathways in human fungal pathogens, and
for the evaluation of the efŽcacy of new antifungal drugs.
Keywords
Cell wall, signal transduction, stress response
Introduction
Yeast and fungal cells like bacteria and plant cells are
surrounded by an extracellular matrix that is essential for
their viability. In the budding yeast Saccharomyces
cerevisiae, the cell wall determines the typical ellipsoidal
shape, functions as a permeability barrier and preserves
the osmotic integrity of the cell by counteracting the high
turgor pressure that acts outwardly on the plasma
membrane. Thus, a weakening or a local loss of integrity
of the cell wall can severely threaten cell survival.
Because of its essential biological role and absence in
mammalian cells, the cell wall is regarded as an attractive
target for the development of new antifungal agents.
In recent years, several studies have focussed on the
biogenesis of the cell wall in its various aspects: (i) the
biosynthesis and assembly of the individual components
and (ii) the regulation of its construction. Cell wall
biogenesis in S. cerevisiae is better characterized than in
other Ascomycetes. For its versatility to genetic and
biochemical analysis, budding yeast has proved to be a
useful organism in the study of cell wall biogenesis, as it
has been used in the investigation of other biological
processes of eukaryotic cells such as the control of the
cell cycle.
ãã
` degli Studi di Milano,
Correspondence: L. Popolo, Universita
Dipartimento di Fisiologia e Biochimica Generali, Via Celoria 26,
20133 Milano, Italy. Tel.: ‡39-02-58354919; Fax: ‡39-02-58354912;
e-mail: [email protected].
2001
2001 ISHAM
ISHAM, Medical Mycology, 39, Supplement 1, 111±121
Several studies have shown that the yeast cell wall is
not a static shield, but a highly dynamic structure that
can change accordingly to the physiological needs of the
cell. For example, during the cell cycle, the cell wall has
to be remodelled to be more plastic in the point of bud
emergence where the growth takes place. Moreover, the
cell wall can change in composition and/or structure in
response to stimuli coming from the environment as in,
for example, mating or stress signals.
This review will focus on the current knowledge of the
cellular responses to conditions that damage the cell
wall. Several studies have provided evidence for the
existence of a salvage pathway that guarantees the
preservation of the cell integrity.
This knowledge is useful for exploring similar processes in human fungal pathogens and relevant for the
evaluation of the efŽcacy of new antifungal agents
directed against the cell wall.
Evidences for the existence of a salvage
pathway
The yeast cell wall is composed primarily of b (1,3)glucan, mannoproteins (cell wall proteins; CWPs),
b (1,6)-glucan and chitin, which have to be synthesized
and correctly assembled outside the cell. In particular,
b (1,6)-glucan plays an important role as a cross-linker
because it has been found to be covalently linked to
glycosylphosphatidylinositol (GPI)-CWPs through a
remnant of a GPI, to b (1,3)-glucan and also to chitin
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Popolo et al.
(for review see [1] and Klis et al. and Munrow & Gow,
this issue).
The existence of a compensatory response to cell wall
damage has been proposed on the basis of studies
performed on yeast cells harbouring null mutations in
genes encoding for cell wall-related enzymes, mainly
fks1 and gas1 mutations, or on wild-type cells subjected
to acute cell wall stresses.
gas D mutant
In yeast, GAS1 gene encodes a novel type of b (1,3)glucanosyltransferase that appears to play an important
cross-linking function. The Žrst biochemical proof of the
existence of such an activity has been carried out in
Aspergillus fumigatus and the protein endowed with this
activity has been designed Gel1p (glucan-elongating
protein) [2,3]. Subsequent studies have revealed the
presence of similar activities in other fungi and yeasts [3].
By exploiting the same biochemical assay used for
Gel1p, it has been shown that Gas1p catalyses in vitro
the hydrolysis of an internal b (1,3)-glycosidic linkage in
a b (1,3)-glucan and then the transfer of the new reducing
end to the non-reducing end of a b (1,3)-glucan with the
formation of a b (1,3)-glycosidic linkage [3]. It has been
proposed that in vivo these proteins could act as
assembly enzymes that transfer segments of b (1,3)glucan on branching points of other glucans, thus
creating multiple anchoring sites for mannoproteins,
chitin or for galactomannans of A. fumigatus [3].
Gas1p is a 125-kDa-extracellular protein that is
abundantly N- and O-mannosylated (for review, see
[4]). It belongs to a family of related yeast and fungal
proteins that share various degrees of amino acid
identities with Gas1p, and has a common means of
anchoring to the plasma membrane consisting of a GPI
[3,5]. This family includes four proteins of S. cerevisiae
encoded by homologs of the GAS1 gene detected in the
yeast genome (Gas2, Gas3, Gas4 and Gas5), proteins of
human fungal pathogens: A. fumigatus (Gel1, Gel2,
Gel3) [5], Candida albicans (Phr1, Phr2, Phr3) [6],
C. dubliniensis (Phr1, Phr2p) [7] and Pneumocystis
carinii (Phr1). These proteins, along with others of C.
maltosa (Epd1p and Epd2p) and three ORFs identiŽ ed
in the Schizosaccharomyces pombe genome sequencing
project, have been classiŽed as belonging to a new family
of glycoside hydrolases, Family 72. Interestingly, in C.
albicans the expression of the PHR1 and PHR2
(PHR ˆ pH-responsive gene) is regulated by the external pH being PHR1 expressed at pH values of 5¢5 or
higher and PHR2 at pH values below 5¢5 [8,9]. Despite
the fact that these proteins were found to be functionally
interchangeable, it has been proposed that their parti-
cular pattern of expression enhances adaptation of these
organisms to the different pH niches that this microorganism colonizes [10].
Out of the GAS subfamily of yeast proteins, only
Gas1p appears to be expressed during vegetative growth.
The lack of Gas1p cross-linking activity causes an
aberrant cell morphology that is also characteristic of
C. albicans mutants lacking Phr1 or Phr2 proteins
[8,9,11]. The gas1D cells become enlarged and rounded
and have defects in the maturation of the bud and in cell
separation [11]. Cell wall properties are also affected:
permeability to exogenous substances increases, the
resistance to hydrolytic enzymes (Zymolyase) in exponentially growing cells is abnormally high and similar to
that of stationary-phase cells and cells are highly
sensitive to Calcouor [12,13]. Moreover, cells lacking
Gas1p acquire an unusually high thermotolerance [14].
These defects reect gross changes in the cell wall and a
situation that demands a stress response. Indeed, gas1D
mutant shows a reduction of incorporation in the cell
wall of b (1,3)-glucan and b (1,6)-glucosylated GPImannoproteins that are detected in the culture medium
[15]. The cell wall content of glucan and in particular of
b (1,6)-glucan is reduced [12,16]. Other evidence suggests
that remarkable changes in the molecular architecture of
the cell wall occur in gas1 cells to counteract damage in
the construction of the cell wall. In wild-type cells, only
2% of all CWPs are linked directly to chitin. However,
this linkage is 20-fold more abundant in gas1 cells and
the level of bulk CWPs increases [17]. Moreover, while
chitin normally constitutes only 1–2% of the cell wall dry
weight, in gas1 cells a remarkable three- to Žvefold
increase in chitin is observed that is delocalized over the
lateral cell wall [12]. At a molecular level, the mutant
triggers the expression of Fks2p, the alternative subunit
of the b (1,3)-glucan synthase, which is usually expressed
during stationary phase, mating and sporulation. In
addition, the expression of the gene CWP1, encoding a
structural cell wall mannoprotein, is increased threefold
[15,17].
By taking advantage of the pH-conditional expression
of C. albicans PHR1 and PHR2 genes, a temporal
analysis of cell wall changes has been performed. This
study demonstrated that the phr1 null cells lost their
ability to incorporate b (1,6)-glucan into the glucan
matrix and that their chitin level increased [6]. This
remarkable increase in chitin and its cross-linking with
b (1,6)-glucosylated mannoproteins has been proposed to
be part of a secondary response activated in gas1 and
phr1 null mutants to prevent excessive release of
mannoproteins resulting from the inability of the cells
to establish cross-links between mannoproteins and
glucans [6,12,18].
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2001 ISHAM, Medical Mycology, 39, Supplement 1, 111±121
The yeast cell wall salvage pathway
Supporting the possibility that the synthesis of b (1,6)glucan and chitin hyperaccumulation could be part of a
complex compensatory response, a synthetic lethal
genetic interaction has been found between gas1 null
mutation and deletion of genes involved in chitin or
b (1,6)-glucan synthesis.
Three chitin synthases (CHSI, CHSII and CHSIII)
exist in S. cerevisiae (see article by Munro & Gow, this
issue). CHSII is required for the formation of the
primary septum, CHSIII is involved in the formation of
the chitin ring at the base of an emerging bud and
chitin interspersed in the cell wall whereas CHSI has a
repair function during cell separation [1]. CHSIII
activity is responsible for the hyperaccumulation of
chitin in gas1 mutants [19]. The deletion of CHS3,
encoding the catalytic component of CHSIII, severely
compromises viability of the gas1 cells since chs3 gas1
null mutants show a strong susceptibility to lyse and a
dramatic reduction of chitin content [19]. Moreover
gas1 cells are hypersensitive to nikkomycin Z, an in
vivo inhibitor of Chs3p [12]. On contrary, deletion of
CHS1 does not bring about any deleterious effect for
gas1 mutant and the chitin is only diminished slightly
[19]. Although the Chs1p content and the CHSI activity
increase in the gas1 null mutant, the in vivo role of this
repair enzyme seems to be irrelevant for the chitin
synthesis salvage pathway.
Sustained synthesis of the cell wall cross-linker
polymer b (1,6)-glucan is crucial for gas1 cells. A null
mutation in KRE6 gene in wild-type cells is known to
cause a 50% reduction of the b (1,6)-glucan level without
affecting growth. This is lethal in combination with the
gas1 mutation [12]. Thus, gas1D cells depend on chitin
and b (1,6)-glucan synthesis for their survival.
A general criterion to demonstrate that a phenotypic
trait is caused by a cell-wall defect is to test the
remediating effect of inclusion of 1 M sorbitol in the
growth medium. Sorbitol has a dual beneŽ cial effect: Žrst
it increases the external osmolarity reducing the outward
turgor pressure, and secondly it plays a beneŽ cial effect
by stabilizing the cell wall. Moreover, sorbitol is not
metabolized. The phenotypic defects of gas1 cells are
alleviated by the presence of 1 M sorbitol: cells are less
swollen, cell wall properties revert to normal and chitin
levels are partially reduced [20].
fks D mutant
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FKS1 gene encodes a 215-kDa protein corresponding to
the catalytic subunit of the plasma membrane biosynthetic enzyme b (1,3)-glucan synthase. In fks1D, the
b (1,3)-glucan level is reduced by 50% relative to the
control isogenic strain. Some aspects of the resulting
2001 ISHAM, Medical Mycology, 39, Supplement 1, 111±121
113
phenotype are similar to those of gas1 [16]. Cells lacking
Fks1p have a higher chitin and mannoprotein content,
and increased cross-linking between mannoproteins and
chitin [15,17]. The fks1 null mutant is also more
thermotolerant than wild-type but less so than in the
gas1D strain [14]. As observed in the gas1 null mutant,
fks1 strain triggers Fks2p expression and a threefold
increase in the level of CWP1 mRNA [15,17]. Despite
the induction of Fks2p, fks1 cells cannot produce as
much glucan as the wild type and their cell integrity is
compromised. Thus, the presence of certain common
responses in gas1 and fks1 provides support for the
existence of a compensatory mechanism responding to
defects in assembly or synthesis of cell wall.
Also in fks1 mutant, Chs3p is again responsible for
chitin hyperaccumulation [21]. The FKS1 gene was
identiŽed in a search for synthetic lethal mutations in a
chs3D strain. This Žnding strengthens the hypothesis that
stress-related chitin synthesis is essential in fks1 mutants
[22].
It is not yet known how chitin synthesis is upregulated
in fks1 and gas1 cells. Chs3p activity and intracellular
transport is controlled by several genes. The products of
CHS7 (required for the export of Chs3p from ER),
CHS5 (required for the vesicle transport from Golgi),
CHS4 (a putative activator of Chs3p) were found to be
required for stress-related chitin synthesis both in fks1
and gas1 cells, whereas the product of CHS6 (involved in
the anterograde transport of Chs3p) is required in gas1
but not in fks1 mutants [22 and unpublished data of the
authors]. CHSIII activity is higher in membrane preparation of fks1 and in permeabilized gas1 cells than in
the wild type [21]. Nevertheless the level of Chs3p is not
appreciably affected in both mutants whereas the
localization is abnormal. Also, the level of CHS4 mRNA
in gas1 (L. Popolo, unpublished data) and of Chs4
protein in fks1 cells do not change [21]. Chs7p increases
in fks1 and could promote an increased exit of Chs3p
from ER. The data suggests that activation of Chs3p
occurs via a post-translational regulation mechanism and
by control of enzyme localization but the mechanism of
upregulation of Chs3p activity remains obscure.
Other cell wall perturbing conditions
Some experimental conditions used to perturb the cell
wall are: temperature (thought to indirectly affect cell
wall by inuencing the activity of plasma membrane
enzymes through its primary effect on membrane
uidity), mild treatment with b (1,3)-glucanase (Zymolyase 100-T), addition of Calcouor (an agent that
interferes with chitin molecules crystallization and
induces an increase of chitin synthesis) and use of
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Popolo et al.
sodium dodecyl sulphate (SDS), an anionic detergent
that destabilize the cell wall at very low concentration
[14,23]. All these treatments have the advantage of
allowing the analysis of the time-course responses to
acute treatments and have been proved to be very useful
in discerning which are the early primary responses to
cell wall damage. With the use of these types of acute
perturbing conditions it has been shown that cell wall
stress depolarizes the actin cytoskeleton transiently and
also induces a transient depolarized distribution of Fks1p
and its regulatory subunit Rho1p, possibly as a general
mechanism of repair [23].
The MAP kinase pathways in yeast
Cells sense and respond to environmental stresses via
signalling pathways. Several signal transduction pathways in yeast are based on a conserved module of
protein kinases Žrst identiŽ ed in mammalian cells. An
input signal induces a kinase cascade, constituted by a
module of three protein kinases, that culminates in the
activation of a mitogen-activated protein kinase
(MAPK) or extracellular signal-regulated kinase
(ERK). The MAPK is activated by a so-called MAP/
ERK kinase (MEK or MAPKK) which in turn is
activated by a MEK kinase (MEKK or MAPKKK).
The MEK is a dual-speciŽ city protein kinase that
phosphorylates MAPK on two highly conserved threonine and tyrosine residues, which can be recognized by
Western analysis using speciŽc antibodies, and determines a strong increase of its kinase activity. This
cascade of protein kinases culminates in the phosphor-
ylation in serine or threonine of many different proteins
including nuclear transcriptional factors that mediate the
cellular response. The activation of the pathway is
usually transient and regulated by protein phosphatases
that recognize the dual phosphorylated form of the
MAPK and switch off the pathway when the cell has
responded to the extracellular stimulus or has adapted to
the onset of the new condition.
At least six MAPK (Fus3p, Kss1p, Hog1p, Slt2p-also
called Mpk1p-, Smk1p and Mlp1) are encoded by the
S. cerevisiae genome. As shown in Figure 1, four of them
mediate the response to speciŽc stimuli and are
implicated in generating specialized biological responses
[for reviews, see 24,25]. Fus3p is necessary for mating,
Kss1p regulates the invasive growth both in haploid or
diploid cells, Hog1p is necessary for the response to
hypertonic stress, Slt2 controls cell integrity in response
to hypotonic stress and heat shock, Kss1p also mediates
cell integrity in response to defects in protein glycosylation, and a Žfth one, Smk1p, is implicated in sporulation
(this last pathway is not shown in the Žgure). The role of
Mlp1p is still unclear. Yeast cells of S. cerevisiae carrying
deletion of all six genes encoding MAPKs are viable but
sterile and severally affected in the response to stress
conditions like sudden changes in external salinity or
decreased nutrient availability [26].
The fact that some pathways share the same elements
poses a problem on the speciŽcity. This theme is
intriguing and cannot be treated exhaustively in this
context (see also Navarro-Garciá et al., this issue). The
speciŽcity of the response appears to be regulated by
multiple mechanisms. One of these is the presence of
Fig. 1 Summary of Žve MAPK cascades that regulate growth and differentiation in haploid S. cerevisiae. Elements shared by different
pathways are shown in bold.
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2001 ISHAM, Medical Mycology, 39, Supplement 1, 111±121
The yeast cell wall salvage pathway
speciŽc upstream elements (receptors, osmosensors, etc.)
and downstream transcription factors that route the
input signal on speciŽc pathways. For example, mating
factor receptors, G protein and Ste5 scaffold protein are
speciŽc for the mating factor response and Tec1
transcription factor is speciŽc for invasive growth.
Moreover, the pathways cross-regulate one another to
ensure that they are not activated by the wrong signal.
Also, Fus3p inhibits misactivation of the invasive growth
pathway by mating pheromone, whereas Hog1p inhibits
misactivation of the mating pathway by hypertonic stress
[25]. However, it is also important to recall that certain
stimuli may require more than one pathway to yield the
full biological response. The fact that a signal is
transmitted by a cascades of phosphorylation events
and other reactions is already an indication of possibilities for cross-talk between the various pathways that
are necessary to produce a co-ordinated cellular response. For instance, a -factor activates the MAP kinase
pathway for mating in the short term but later also
activates the PKC1–MAP kinase pathway. At the
moment, the formation of shmoo projections requires
an extensive cell wall remodelling and the two pathways
must be co-ordinated [27].
As shown in Figure 1, two MAPK pathways, the
PKC1–MAPK and the Sho1–Kss1 pathways, have so far
shown to be involved in controlling cell integrity.
Whereas the former has been characterized extensively,
the later has been only recently identiŽ ed by genetic
screens originally designed to identify negative regulators of the pheromone response signalling or mutations
that determine synthetic lethality in combination with
the ste11D mutation [28,29]. The Ste20p, Ste11p and
Ste7p enzymes of this pathway are in common with the
mating pathway but Sho1p is associated with the HOG
pathway and Kss1p with the invasive growth pathway (in
bold in Fig. 1). The activation of this later pathway leads
to the Kss1p-mediated activation of FUS1 transcription,
a reporter characteristic of the mating pathway [29]. This
pathway functions during vegetative growth although its
role is more evident under stress conditions. FUS1
promoter activity is increased strongly by mutations in
genes affecting protein glycosylation (och1, pmi40,
dpm1), GPI anchor synthesis (spt14) or moderately
enhanced in cells lacking GAS1. Moreover all these
mutants require the functionality of this pathway as
shown by lethality or severe worsening of the phenotypes
if elements of the Sho1-pathway are inactivated [29].
The PKC1±MAP kinase pathway
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The activation of the MAP kinase module constituted by
Bck1p–Mkk1/Mkk2–Mpk1 (Slt2) is mediated by Pkc1p,
2001 ISHAM, Medical Mycology, 39, Supplement 1, 111±121
115
the yeast homologue of mammalian protein kinase C.
This pathway is activated by high temperature, hypoosmotic shock, a -factor at the onset of the formation of the
mating projections and during the cell cycle at the
initiation of budding [for review, see 25,30].
PKC1 is an essential gene and pkc1 null mutants are
strictly dependent on the presence of osmotic support for
growth. Also, the loss of the downstream elements
results in a lytic phenotype. This is attenuated as it is
manifest at 37 oC, suggesting that this pathway may
branch [30]. The characteristic osmotic-remedial phenotype of mutants of the PKC1–MAPK pathway suggested
that it is required for cell-wall construction, and for this
reason it has also been called cell integrity pathway.
Indeed, the basal level of expression of some cell wallrelated genes (FKS1, MNN1 and CHS3) depends on the
functionality of this pathway [31].
The PKC1–MAPK pathway becomes activated in
response to cell-wall damage. The Žrst clue for this was
provided by the Žnding that this pathway was activated
by high temperature and at the time of formation of
mating projections in a -factor treated cells that requires
extensive cell wall remodelling [25]. In addition, a
persistent activation of the cell integrity pathway was
found in gas1 and fks1 null mutants [14]. Two different
readouts were used to monitor the activation of the
pathway: (i) the detection of the phosphorylated form of
Slt2p by antibodies that recognize the two highly
conserved phosphorylated p44/p42 mammalian MAPK
residues (Thr202 and Tyr204 in the human form)
corresponding to Thr190 and Tyr192 in the yeast
Slt2p(Mpk1p), and (ii) the expression of a FKS2–LacZ
reporter gene [14,32]. Indeed, both fks1D and gas1D
mutants show a higher level of the phosphorylated form
of Slt2p than their wild-type counterparts. The presence
of 1 M sorbitol in the growth medium greatly reduces the
increase in Slt2p-phosphorylation in these cells [14].
Examples of Slt2p activation are shown in Figure 2.
Combined deletions of FKS1 or GAS1 genes with
inactivation of elements of the pathway leads to lethality
[12,33]. Therefore, it has been proposed that the PKC1–
MAP kinase pathway may be essential in gas1 and fks1
null mutants, because it is necessary to rescue the cells in
response to cell-wall stress.
In a wild-type cell the acquisition of thermal tolerance
was reduced greatly by the slt2 null mutation and slt2
cells were also highly sensitive to Zymolyase. These
results suggest that also the increased thermal resistance
and the resistance to Zymolyase of gas1 and fks1
mutants depend on Slt2p-activation [14].
The recent Žnding that the Sho1–Kss1 pathway is
involved in the response to cell-wall defects accounts for
the Žnding that another MAPK is activated in gas1D cells
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Popolo et al.
Fig. 2 High level of Slt2p-phosphorylation in heat-shocked cells
and in gas1D cells. Upper panels: immunoblot analysis with antiphospho-p42/p44 MAPK antibody of total yeast extracts prepared
as described in [19], in the presence of protease and phosphatase
inhibitors [32]. Lower panels: identical extracts were analysed with
anti-Slt2 antibodies. Equal amounts of total proteins were loaded
on each lane. a, W303-1A cells were cultured to mid-log phase in
YNB-glucose at 24 o C and then transferred at 37 o C for 1 h. b,
W303-1B strain and its gas1D derivative were cultured to mid-log
phase in YNB-glucose at 30 o C in the absence or presence of 1 M
sorbitol. Slt2p-phosphorylationwas almost undetectable in the wildtype strain and was high in gas1D cells with respect to the isogenic
strain. Slt2p level doubled in the gas1D mutant. The presence of 1 M
sorbitol reduced both the activation and the doubling of Slt2p.
(p48 in Fig. 2b). The identiŽcation of p48 is currently
underway in our laboratory.
The activation of PKC1–MAPK pathway was also
detected in wild-type cells treated with agents that
perturb the cell wall. Calcouer induces an increase in
the phosphorylation of Slt2p that remains high even
150 min after drug addition. Furthermore, mild treatment with Zymolyase or vanadate, an agent that
presumably affects cell-wall stability, generates a similar
response [14].
Among the ultimate targets of the cell integrity
pathway that have so far been identiŽ ed are the Rlm1p,
a member of the MADS family, SBF transcription factor,
a heterodimer of Swi4p and Swi6p, required for
expression of G1 cyclin genes, and proteins of the
HMG1-group of chromatin-associated proteins that
could affect gene transcription indirectly. The role of
these factors is reviewed in [25] and [30].
Perception and transmission of cell-wall
stress to the PKC1±MAP kinase pathway
The mechanism by which information regarding the state
of the cell wall is transmitted to the intracellular
signalling apparatus is still an open question. However,
two hypotheses have been formulated that converge on
the activation of the PKC1–MAP kinase pathway. The
Žrst proposes that cell wall damage mimics a condition of
hypo-osmotic shock. Stretching of the plasma membrane
could be induced by the inability of weakened cell wall
to effectively counteract turgor pressure. In this regard,
it has been demonstrated that hypotonic shock generates
a stretch-activated channel-dependent calcium pulse in
yeast [34]. The increase of the cytosolic calcium
concentration could then activate Pkc1p. It has been
found that the Pkc1ts phenotype is suppressed by growth
in high levels of calcium. Nevertheless, in vitro activity of
soluble Pkc1p in yeast is calcium-independent but the
interpretation of this data is hindered by the fact that the
fraction of soluble tested Pkc1p molecules is small [25].
The mating pheromone response provides an example
of ion ux activation by a mechanosensor. Pheromone
stimulates Ca2‡ inux through a calcium channel
complex whose components are Cch1p, the putative
true channel, and Mid1p that may confer the property of
stretch activation to Cch1p [35,36]. Mid1p is an integral
membrane protein with a single transmembrane domain
and associates with Cch1p that has 16 transmembrane
domains [36,37]. Mid1p expressed in Chinese hamster
ovary cells mediates Ca2‡ inux in response to mechanical stress. Moreover, the channel is inhibited by
gadolinium, a blocker of stretch-activated cation channels [38].
A second hypothesis proposes that a recently identiŽed redundant family of transmembrane proteins could
function as sensors of the state of the cell wall. Mid2p
and Slg1p, with their homologs Mtl1p (MID2-like 1) and
Wsc2-3-4 proteins, are putative cell wall sensors involved
in remodelling cell wall during vegetative growth or
pheromone-induced morphogenesis. SLG1 gene was
isolated several times and variously designated HCS77
[39], WSC1 [40] or SLG1 [41]. MID2 was isolated by
complementation of a mutation that causes defects in the
mating response [42]. Interestingly, MID2 (mating
induced death) was also isolated in several genetic
screens as a multicopy suppressor of defects in microtubule organization, in actin polymerization and as
activator of Skn7 transcription factor [reviewed in 43].
Since SLG1 is a multicopy suppressor of mid2D mutant
phenotype and MID2 of the slg1D phenotype, Slg1p and
Mid2p play at least partially redundant functions [43,44].
Mid2p and Slg1p has the major in vivo role, as the
different combinations of deletions of their homologues
have only a minor effect on the phenotype displayed by
the single slg1 or mid2 null mutants. The slg1 null
mutants show a sorbitol-remedial lysis phenotype at high
temperature (39 oC), similarly to PKC1–MAPK pathway
mutants, and a higher sensitivity to different drugs [39–
41]. The mid2D grows normally and shows lysis defects
only when exposed to mating factors. This phenotype
can be prevented by increase of external calcium
suggesting a possible role of this protein in the
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The yeast cell wall salvage pathway
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transmission of calcium signalling [37,42]. A peculiar
phenotype of mid2 null mutants is the resistance to
Calcouor [43]. The mid2D cells have the same chitin
level as the wild type but the extent of chitin synthesis
induced by Calcouor or a -factor treatments is reduced.
Thus it has been proposed that Mid2p is a regulator of
chitin synthesis under stress conditions [43].
Slg1 and Mid2 proteins have a common structure as
Type I transmembrane proteins consisting of an extracellular domain rich in serine and threonine residues, a
transmembrane domain and a relatively short cytoplasmic tail. The extracellular domain of Slg1p and Mid2p is
extensively modiŽed by O-mannosylation that causes an
abnormal migration of these proteins in SDS gels [43,45].
Despite the similarity shared in the overall structure,
Slg1p and Mid2p have dissimilar primary structures. A
potential Ca2‡ binding motif is present in the intracellular domain of Mid2p and a cysteine-rich region in the
extracellular domain of Slg1p [42].
By use of the gene fusion with the green uorescent
protein (GFP), it has been shown that both Mid2p and
Slg1p are distributed evenly at the cell periphery and
fractionation experiments, indicated that these proteins
behave as integral membrane proteins [42–44]. A HA
tagged-Slg1p expressed from a centromeric plasmid
appeared to localize in the growing bud [23]. Following
cell wall stress, Slg1p was Žrst depolarized and then
repolarized at the bud apex [23].
The structure of Mid2 and Slg1 suggests these proteins
are structurally analogous to the integrins of mammalian
cells, a class of receptors that are not endowed with any
intrinsic biochemical activity but bind ligands present in
the extracellular matrix and activate signalling pathways
responsible for changes in the actin cytoskeleton. This is
consistent with the observation that Slg1p, like mammalian integrins, both controls and responds to the actin
cytoskeleton [23].
It is not yet understood how cell-wall defects are
perceived by these sensor proteins. It seems to be
unlikely that Mid2p and Slg1p bind to speciŽc ligands of
the extracellular matrix as do integrins. However, their
extracellular domain is essential for their role as sensors.
In particular, the inhibition of O-mannosylation by a null
mutation in PMT2 gene, encoding an enzyme involved in
the Žrst step of O-mannosylation, impairs the sensor
function of Mid2p, suggesting a possible novel role of the
O-linked oligosaccharide chains in such surface proteins
[45]. It has been proposed that the high degree of Omannosylation of the serine and threonine residues
induces the polypeptide chain to assume an extended
and stiffened conformation that could span the cell wall
and enter the physico-chemical region of the extracellular matrix [43–45].
2001 ISHAM, Medical Mycology, 39, Supplement 1, 111±121
117
Several lines of evidences indicate that Slg1 and Mid2p
may act as upstream activators of the PKC1–MAPK
pathway [14,43,44]. The absence of Mid2p reduces the
level of Slt2-phosphorylation in response to a shift to
high temperature (39 oC), a -factor, Calcouor and
vanadate treatment, whereas the absence of Slg1p results
only in a weak reduction of heat-induced phosphorylation [14,43]. In conclusion, Mid2p appears to be involved
more in signalling of stress conditions, and Slg1p in
condition of normal vegetative growth.
How these proteins activate the cell integrity pathway
has been only recently elucidated. It has been shown by
two-hybrid system experiments that the C-terminal
domains of Slg1p and Mid2p interact with Rom2p, one
of the two guanine nucleotide exchange factors (GEFs)
for Rho1p, the main regulator of the PKC1–MAP kinase
pathway in yeast [45] (Fig. 3). Rho1p is a member of a
GTPase family and a key protein for yeast morphogenesis. Rho1p can cycle between an inactive (bound to
GDP) and an active state (bound to GTP). Transition is
regulated by Rom1 and Rom2p and the hydrolysis of
GTP is stimulated by two potential GAP proteins,
Bem2p and Sac7p [32]. Slg1p and Mid2p regulate GEF
activity for Rho1p. Extracts from a slg1D mutant display
a 50% reduction in the exchange activity towards Rho1p,
whereas those from a mid2D mutant only result in a 20%
reduction consistent with the relatively minor role that
Mid2p has in vegetative growth [45]. The activation of
Rho1p via GEF Rom2p occurs in response to certain cell
wall stress conditions such as treatment with SDS and in
the cell wall defective mutants gas1, fks1 and cwh41 [46].
Mid2p and Slg1p could mediate these reactions. Indeed,
an increase in the exchange activity for Rho1p was found
following Calcouor treatment [45].
Fig. 3 Model of Rho1-activation by Mid2p and Wsc1p (Slg1p) via
Rom2p and of the various cellular activities co-ordinated by Rho1p.
118
Popolo et al.
Rho1p has several effectors: Pkc1p, Fks1-Fks2p, the
catalytic subunits of b (1,3)-glucan synthase, the nonessential formin-like protein Bni1p, the two-component
signalling protein Skn7p and the protein Sec3 involved in
the polarized transport of the exocytotic vesicles [47; see
also citations in 23]. An important role of Rho1p resides
in the control of actin cytoskeleton organization. Rho1p
can affect the organization of the cytoskeleton both by
interacting with Bni1p or indirectly through the activation of Slt2p as in Tor2p signalling [48]. Rho1p mediates
the actin cytoskeleton, repolarization through PKC1–
MAP kinase pathway after a cell-wall stress [23]. Figure
3 illustrates the cellular activities controlled by Rho1p.
However, the mechanism by which Mid2p and Slg1p
transmits information concerning cell-wall perturbations
to Rom proteins remains obscure.
The calcineurin pathway
In S. cerevisiae, calcineurin (Ca2‡/calmodulin dependent
phosphatase: PP2B) plays a key role in mediating several
important physiological processes. These include NaCl
tolerance, mating intracellular calcium buffering and
construction of the cell wall [reviewed in 25]. Calcineurin
is not essential for normal growth. It is a heterodimer of
CNA1 and CNA2-encoded redundant catalytic subunits
and a regulatory subunit encoded by the CNB1 gene
[49].
The b (1,3)-glucan synthase complex is responsible for
the synthesis of the most abundant glucan of yeast cell
wall and Fks1/2p are its catalytic components. Deletion
of single FKS1 or FKS2 does not affect viability but the
removal of both is lethal. Therefore, in the absence of
FKS1, FKS2 becomes essential. It has been found that
cells with compromised cell integrity due to the lack of
FKS1 gene or mutations in the PKC1–MAPK pathway,
depend on calcineurin for growth [33,49]. Moreover,
fks1D and gas1D mutants display an increased sensitivity
to the immunosuppressants FK506 and cyclosporin A
that bind to their receptors, FKBP12 or cyclophilin,
thereby inhibiting calcineurin [33; J. Arroyo and
L. Popolo, unpublished data].
The Žnding that FKS2 expression is under the control
of calcineurin and that its lack or inhibition causes a
deŽciency of Fks2p suggests that calcineurin is a
component of the compensatory response [50].
Large-scale approaches to the study of the
cell wall stress response
Powerful technologies such as DNA microarrays and
proteomics have made possible the simultaneous analysis
of the expression levels of many genes of an organism. In
an organism whose genome is completely sequenced
such as S. cerevisiae, it is possible to examine which
genes are up- or downregulated under given experimental conditions.
To create a condition of strong activation of the
PKC1–MAPK pathway, a gain-of-function allele of
MKK1 (MKKS368P) was placed under the control of
the galactose-inducible promoter. Following a shift from
glucose to galactose, the transcript proŽles of cells
expressing the wild-type or the hyperactive allele were
compared using a Žlter hybridization technique [51]. The
artiŽcial activation of the pathway led to an increase in
the transcript level of 20 genes and a decrease of 5 genes
[51] (Table 1). Most of the genes identiŽed are known or
suspected to encode CWPs or proteins involved in cellwall biogenesis. The transcript levels of several genes
were further analysed separately by northern blot
analysis, and for each transcript the behaviour in a wild
type and a rlm1D strain was compared. With the
exception of FKS2, all the regulated genes were
apparently dependent on Rlm1p [52] and had Rlm1p
putative binding elements in their promoter regions. The
expression of CHS3, FKS1 and MNN1 previously
reported to be regulated by the PKC1–MAP kinase
pathway was not identiŽ ed in this genome wide screen.
However, when tested separately these transcripts
appeared to be weakly regulated and MNN1 not to be
regulated at all under these conditions.
In another recent study, the expression proŽle of a
fks1D mutant is described [53]. The transcriptional
regulation was investigated using microarrays and tested
subsequently by fusing the 50 non-coding region from
each gene to the Escherichia coli LacZ reporter gene:
22 genes were upregulated (Table 1). Among these,
three genes encoding GPI-proteins were in common
with those reported in the previous microarray survey
(PST1, CRH1, CWP1). These genes may be direct
targets of the constitutive activation of the PKC1–
MAPK pathways that occurs in fks1 mutant. Other
genes as FKS2 and CHS1 were also in common in both
array experiments. Interestingly the transcript of SLT2
was itself upregulated, in agreement with other experimental data (Fig. 2b). These results further support the
existence of a feedback positive loop of phosphorylated
Slt2p on its own expression [51]. The persistent activation of the pathway by cell wall disturbance could lead
not only to the activation of Slt2p-phosphorylation but
also to an increase of the level of this protein that could
be required for a more efŽcient response to the stress.
Conclusions
The cell wall is essential for the survival of yeast and
fungal cells. These organisms have developed several
ã
2001 ISHAM, Medical Mycology, 39, Supplement 1, 111±121
The yeast cell wall salvage pathway
Table 1
119
Comparison between genes up-regulated in response to activation of Slt2p and in fks1D mutant*
Category
Gene name
ORF
Cell
integrity
signalling
GPI proteins
PST1
SED1
CRH1
CWP1
PRY2
YPS3
SSR1
YDR055W
YDR077W
YGR189C
YKL096W
YKR013W
YLR121C
YLR194C
YLR390W-a
>
>
>
>
—
—
>
>
>
—
>
>
>
>
—
—
Unknown
Structural cell-wall protein of stationary phase
Putative b (1,3)-b (1,4)-transglucosidase
Structural cell-wall protein
Unknown
Plasma membrane aspartyl protease
Unknown function
Structural cell-wall protein
PIR family
PIR1 (CCW6)
PIR2 (HSP150)
PIR3
CIS3 (CCW5)
YKL164C
YJL159W
YKL163W
YKL158C
>
>
>
>
—
—
—
—
Structural
Structural
Structural
Structural
Other cell wall proteins
BGL2
YGR282C
>
—
b (1,3)-endoglucanase/glucanosyltransferase
FKS1
FKS2
CHS3
CHS1
CHS7
GFA1
KTR2
YJL342W
YGR032W
YBR023C
YNL192W
YHR142W
YKL104C
YKR061W
>
>
>
>
—
—
—
N.A.
>
>
>
>
>
>
Catalytic component b (1,3)-glucan synthase
Alternative component b (1,3)-glucan synthase
Chitin synthase III
Chitin synthase I
Export Chs3p from ER
Glucosamin-fructose-6-phosphate aminotransferase
Mannosyltransferase
SLT2 (MPK1)
MLP1
PTP2
YHR030C
YKL161C
YOR208W
>
>
—
>
—
>
S/T protein kinase MAP kinase
S/T protein kinase MAP kinase
protein tyrosine phosphatase
SEC28
DFG5
YIL076W
YMR238W
YIL117C
YNL058C
YER133C
YNL285W
YPL163C
YPL221W
YPR057W
YAL053W
YBR071W
YPL067C
>
>
>
>
—
—
—
—
—
—
—
—
—
—
—
—
>
>
>
>
>
>
>
>
Vesicle coat protein
Unknown
Unknown
Unknown
Unknown regulator of early meiotic gene expression
G1/S-phase cyclin
S-T rich protein
Unknown
RNA-binding protein involved in snRNP biogenesis
Unknown
Unknown
Unknown
Cell wall biogenesis
proteins
Regulatory proteins
Other not related
to cell wall
PMD1
PCL1
SVS1
BOP1
BRR1
fks1D
mutant
Biochemical function/cellular role
cell-wall
cell-wall
cell-wall
cell-wall
protein
protein
protein
protein
* adapted from [51] and [53]; N.A., not applicable.
ã
pathways to respond to imposed environmental stress
and damage, such as changes in external osmolarity, the
action of glucanases or chitinases secreted by competing
organisms or by the defence reaction of plants, or
changes in temperature or nutrient availability. The
salvage pathway relies on elements that are conserved in
all the eukaryotes, and yeast is a valuable organism for
the molecular investigation in this Želd.
The future progress on the molecular characterization
of the salvage pathway in yeast will provide useful
2001 ISHAM, Medical Mycology, 39, Supplement 1, 111±121
information for the evaluation of the degree of damage
caused by new antifungal drugs directed against the cell
wall, and will help the developments of tools for
monitoring the cellular responses. Furthermore, the
salvage pathway may have critical roles in the responses
and adaptation of fungal pathogens during infection, for
example on the surface mucosae or in the phagolysosome of macrophages. The results generated to date also
underline the conclusion that the cell wall of fungi is a
highly dynamic structure whose composition and struc-
120
Popolo et al.
ture is responsive to both endogenous constraints (cellcycle regulation, changes in turgour) and exogenous
challenges.
13
Acknowledgements
We wish to thank R. Zippel and M. Vai for critical
reading of the manuscript, M. Molina for Slt2 antibodies
and helpful discussions and J. Arroyo for having
communicated unpublished data on gas1 sensitivity to
FK506. We apologize to the yeast people for possible
omissions in pertinent literature.
The research from our laboratory reported in this
review was supported by MURST 1999 to L.P., EC
contract QLK3-CT-2000-01537 to L.P. and AzioniIntegrate Italia-Spagna 1999–2000.
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