The yeast cell-wall salvage pathway

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 itsindividual components. Several environmental factors, such as temperature up-shift,treatments with mating factors or with speci�c cell wall-perturbing drugs, or geneticfactors, 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 theosmotic integrity of the cell, several responses are triggered in response to cell-walldamage. This review focuses on the activation of salvage pathways that guaranteecell survival through remodeling of the extracellular matrix. These researches haveuseful implication for the study of similar pathways in human fungal pathogens, andfor the evaluation of the ef�cacy of new antifungal drugs.

Keywords Cell wall, signal transduction, stress response

Introduction

Yeast and fungal cells like bacteria and plant cells aresurrounded by an extracellular matrix that is essential fortheir viability. In the budding yeast Saccharomycescerevisiae, the cell wall determines the typical ellipsoidalshape, functions as a permeability barrier and preservesthe osmotic integrity of the cell by counteracting the highturgor pressure that acts outwardly on the plasmamembrane. Thus, a weakening or a local loss of integrityof the cell wall can severely threaten cell survival.Because of its essential biological role and absence inmammalian cells, the cell wall is regarded as an attractivetarget for the development of new antifungal agents.

In recent years, several studies have focussed on thebiogenesis of the cell wall in its various aspects: (i) thebiosynthesis and assembly of the individual componentsand (ii) the regulation of its construction. Cell wallbiogenesis in S. cerevisiae is better characterized than inother Ascomycetes. For its versatility to genetic andbiochemical analysis, budding yeast has proved to be auseful organism in the study of cell wall biogenesis, as ithas been used in the investigation of other biologicalprocesses of eukaryotic cells such as the control of thecell cycle.

Several studies have shown that the yeast cell wall isnot a static shield, but a highly dynamic structure thatcan change accordingly to the physiological needs of thecell. For example, during the cell cycle, the cell wall hasto be remodelled to be more plastic in the point of budemergence where the growth takes place. Moreover, thecell wall can change in composition and/or structure inresponse to stimuli coming from the environment as in,for example, mating or stress signals.

This review will focus on the current knowledge of thecellular responses to conditions that damage the cellwall. Several studies have provided evidence for theexistence of a salvage pathway that guarantees thepreservation of the cell integrity.

This knowledge is useful for exploring similar pro-cesses in human fungal pathogens and relevant for theevaluation of the ef�cacy of new antifungal agentsdirected against the cell wall.

Evidences for the existence of a salvagepathway

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 synthesizedand correctly assembled outside the cell. In particular,b (1,6)-glucan plays an important role as a cross-linkerbecause it has been found to be covalently linked toglycosylphosphatidylinositol (GPI)-CWPs through aremnant of a GPI, to b (1,3)-glucan and also to chitin

ã 2001 ISHAM, Medical Mycology, 39, Supplement 1, 111±121

Medical Mycology 2001, 39, Supplement 1, 111±121

Correspondence: L. Popolo, Universita degli Studi di Milano,Dipartimento di Fisiologia e Biochimica Generali, Via Celoria 26,20133 Milano, Italy. Tel.: ‡39-02-58354919; Fax: ‡39-02-58354912;e-mail: [email protected].

ã 2001 ISHAM

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(for review see [1] and Klis et al. and Munrow & Gow,this issue).

The existence of a compensatory response to cell walldamage has been proposed on the basis of studiesperformed on yeast cells harbouring null mutations ingenes encoding for cell wall-related enzymes, mainlyfks1 and gas1 mutations, or on wild-type cells subjectedto 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 importantcross-linking function. The �rst biochemical proof of theexistence of such an activity has been carried out inAspergillus fumigatus and the protein endowed with thisactivity has been designed Gel1p (glucan-elongatingprotein) [2,3]. Subsequent studies have revealed thepresence of similar activities in other fungi and yeasts [3].By exploiting the same biochemical assay used forGel1p, it has been shown that Gas1p catalyses in vitrothe hydrolysis of an internal b (1,3)-glycosidic linkage ina b (1,3)-glucan and then the transfer of the new reducingend to the non-reducing end of a b (1,3)-glucan with theformation of a b (1,3)-glycosidic linkage [3]. It has beenproposed that in vivo these proteins could act asassembly enzymes that transfer segments of b (1,3)-glucan on branching points of other glucans, thuscreating multiple anchoring sites for mannoproteins,chitin or for galactomannans of A. fumigatus [3].

Gas1p is a 125-kDa-extracellular protein that isabundantly N- and O-mannosylated (for review, see[4]). It belongs to a family of related yeast and fungalproteins that share various degrees of amino acididentities with Gas1p, and has a common means ofanchoring to the plasma membrane consisting of a GPI[3,5]. This family includes four proteins of S. cerevisiaeencoded by homologs of the GAS1 gene detected in theyeast genome (Gas2, Gas3, Gas4 and Gas5), proteins ofhuman fungal pathogens: A. fumigatus (Gel1, Gel2,Gel3) [5], Candida albicans (Phr1, Phr2, Phr3) [6],C. dubliniensis (Phr1, Phr2p) [7] and Pneumocystiscarinii (Phr1). These proteins, along with others of C.maltosa (Epd1p and Epd2p) and three ORFs identi�edin the Schizosaccharomyces pombe genome sequencingproject, have been classi�ed as belonging to a new familyof glycoside hydrolases, Family 72. Interestingly, in C.albicans the expression of the PHR1 and PHR2(PHR ˆ pH-responsive gene) is regulated by the ex-ternal pH being PHR1 expressed at pH values of 5¢5 orhigher and PHR2 at pH values below 5¢5 [8,9]. Despitethe fact that these proteins were found to be functionallyinterchangeable, it has been proposed that their parti-

cular pattern of expression enhances adaptation of theseorganisms to the different pH niches that this micro-organism colonizes [10].

Out of the GAS subfamily of yeast proteins, onlyGas1p appears to be expressed during vegetative growth.The lack of Gas1p cross-linking activity causes anaberrant cell morphology that is also characteristic ofC. albicans mutants lacking Phr1 or Phr2 proteins[8,9,11]. The gas1D cells become enlarged and roundedand have defects in the maturation of the bud and in cellseparation [11]. Cell wall properties are also affected:permeability to exogenous substances increases, theresistance to hydrolytic enzymes (Zymolyase) in expo-nentially growing cells is abnormally high and similar tothat of stationary-phase cells and cells are highlysensitive to Calco�uor [12,13]. Moreover, cells lackingGas1p acquire an unusually high thermotolerance [14].These defects re�ect gross changes in the cell wall and asituation that demands a stress response. Indeed, gas1D

mutant shows a reduction of incorporation in the cellwall of b (1,3)-glucan and b (1,6)-glucosylated GPI-mannoproteins that are detected in the culture medium[15]. The cell wall content of glucan and in particular ofb (1,6)-glucan is reduced [12,16]. Other evidence suggeststhat remarkable changes in the molecular architecture ofthe cell wall occur in gas1 cells to counteract damage inthe construction of the cell wall. In wild-type cells, only2% of all CWPs are linked directly to chitin. However,this linkage is 20-fold more abundant in gas1 cells andthe level of bulk CWPs increases [17]. Moreover, whilechitin normally constitutes only 1–2% of the cell wall dryweight, in gas1 cells a remarkable three- to �vefoldincrease in chitin is observed that is delocalized over thelateral cell wall [12]. At a molecular level, the mutanttriggers the expression of Fks2p, the alternative subunitof the b (1,3)-glucan synthase, which is usually expressedduring stationary phase, mating and sporulation. Inaddition, the expression of the gene CWP1, encoding astructural cell wall mannoprotein, is increased threefold[15,17].

By taking advantage of the pH-conditional expressionof C. albicans PHR1 and PHR2 genes, a temporalanalysis of cell wall changes has been performed. Thisstudy demonstrated that the phr1 null cells lost theirability to incorporate b (1,6)-glucan into the glucanmatrix and that their chitin level increased [6]. Thisremarkable increase in chitin and its cross-linking withb (1,6)-glucosylated mannoproteins has been proposed tobe part of a secondary response activated in gas1 andphr1 null mutants to prevent excessive release ofmannoproteins resulting from the inability of the cellsto establish cross-links between mannoproteins andglucans [6,12,18].

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Supporting the possibility that the synthesis of b (1,6)-glucan and chitin hyperaccumulation could be part of acomplex compensatory response, a synthetic lethalgenetic interaction has been found between gas1 nullmutation and deletion of genes involved in chitin orb (1,6)-glucan synthesis.

Three chitin synthases (CHSI, CHSII and CHSIII)exist in S. cerevisiae (see article by Munro & Gow, thisissue). CHSII is required for the formation of theprimary septum, CHSIII is involved in the formation ofthe chitin ring at the base of an emerging bud andchitin interspersed in the cell wall whereas CHSI has arepair function during cell separation [1]. CHSIIIactivity is responsible for the hyperaccumulation ofchitin in gas1 mutants [19]. The deletion of CHS3,encoding the catalytic component of CHSIII, severelycompromises viability of the gas1 cells since chs3 gas1null mutants show a strong susceptibility to lyse and adramatic reduction of chitin content [19]. Moreovergas1 cells are hypersensitive to nikkomycin Z, an invivo inhibitor of Chs3p [12]. On contrary, deletion ofCHS1 does not bring about any deleterious effect forgas1 mutant and the chitin is only diminished slightly[19]. Although the Chs1p content and the CHSI activityincrease in the gas1 null mutant, the in vivo role of thisrepair enzyme seems to be irrelevant for the chitinsynthesis salvage pathway.

Sustained synthesis of the cell wall cross-linkerpolymer b (1,6)-glucan is crucial for gas1 cells. A nullmutation in KRE6 gene in wild-type cells is known tocause a 50% reduction of the b (1,6)-glucan level withoutaffecting growth. This is lethal in combination with thegas1 mutation [12]. Thus, gas1D cells depend on chitinand b (1,6)-glucan synthesis for their survival.

A general criterion to demonstrate that a phenotypictrait is caused by a cell-wall defect is to test theremediating effect of inclusion of 1 M sorbitol in thegrowth medium. Sorbitol has a dual bene�cial effect: �rstit increases the external osmolarity reducing the outwardturgor pressure, and secondly it plays a bene�cial effectby stabilizing the cell wall. Moreover, sorbitol is notmetabolized. The phenotypic defects of gas1 cells arealleviated by the presence of 1 M sorbitol: cells are lessswollen, cell wall properties revert to normal and chitinlevels are partially reduced [20].

fks D mutant

FKS1 gene encodes a 215-kDa protein corresponding tothe catalytic subunit of the plasma membrane biosyn-thetic enzyme b (1,3)-glucan synthase. In fks1D, theb (1,3)-glucan level is reduced by 50% relative to thecontrol isogenic strain. Some aspects of the resulting

phenotype are similar to those of gas1 [16]. Cells lackingFks1p have a higher chitin and mannoprotein content,and increased cross-linking between mannoproteins andchitin [15,17]. The fks1 null mutant is also morethermotolerant than wild-type but less so than in thegas1D strain [14]. As observed in the gas1 null mutant,fks1 strain triggers Fks2p expression and a threefoldincrease in the level of CWP1 mRNA [15,17]. Despitethe induction of Fks2p, fks1 cells cannot produce asmuch glucan as the wild type and their cell integrity iscompromised. Thus, the presence of certain commonresponses in gas1 and fks1 provides support for theexistence of a compensatory mechanism responding todefects in assembly or synthesis of cell wall.

Also in fks1 mutant, Chs3p is again responsible forchitin hyperaccumulation [21]. The FKS1 gene wasidenti�ed in a search for synthetic lethal mutations in achs3D strain. This �nding strengthens the hypothesis thatstress-related chitin synthesis is essential in fks1 mutants[22].

It is not yet known how chitin synthesis is upregulatedin fks1 and gas1 cells. Chs3p activity and intracellulartransport is controlled by several genes. The products ofCHS7 (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 berequired for stress-related chitin synthesis both in fks1and gas1 cells, whereas the product of CHS6 (involved inthe anterograde transport of Chs3p) is required in gas1but not in fks1 mutants [22 and unpublished data of theauthors]. CHSIII activity is higher in membrane pre-paration of fks1 and in permeabilized gas1 cells than inthe wild type [21]. Nevertheless the level of Chs3p is notappreciably affected in both mutants whereas thelocalization is abnormal. Also, the level of CHS4 mRNAin gas1 (L. Popolo, unpublished data) and of Chs4protein in fks1 cells do not change [21]. Chs7p increasesin fks1 and could promote an increased exit of Chs3pfrom ER. The data suggests that activation of Chs3poccurs via a post-translational regulation mechanism andby control of enzyme localization but the mechanism ofupregulation of Chs3p activity remains obscure.

Other cell wall perturbing conditions

Some experimental conditions used to perturb the cellwall are: temperature (thought to indirectly affect cellwall by in�uencing the activity of plasma membraneenzymes through its primary effect on membrane�uidity), mild treatment with b (1,3)-glucanase (Zymo-lyase 100-T), addition of Calco�uor (an agent thatinterferes with chitin molecules crystallization andinduces an increase of chitin synthesis) and use of

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sodium dodecyl sulphate (SDS), an anionic detergentthat destabilize the cell wall at very low concentration[14,23]. All these treatments have the advantage ofallowing the analysis of the time-course responses toacute treatments and have been proved to be very usefulin discerning which are the early primary responses tocell wall damage. With the use of these types of acuteperturbing conditions it has been shown that cell wallstress depolarizes the actin cytoskeleton transiently andalso induces a transient depolarized distribution of Fks1pand its regulatory subunit Rho1p, possibly as a generalmechanism of repair [23].

The MAP kinase pathways in yeast

Cells sense and respond to environmental stresses viasignalling pathways. Several signal transduction path-ways in yeast are based on a conserved module ofprotein kinases �rst identi�ed in mammalian cells. Aninput signal induces a kinase cascade, constituted by amodule of three protein kinases, that culminates in theactivation 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 isactivated by a MEK kinase (MEKK or MAPKKK).The MEK is a dual-speci�city protein kinase thatphosphorylates MAPK on two highly conserved threo-nine and tyrosine residues, which can be recognized byWestern analysis using speci�c antibodies, and deter-mines a strong increase of its kinase activity. Thiscascade of protein kinases culminates in the phosphor-

ylation in serine or threonine of many different proteinsincluding nuclear transcriptional factors that mediate thecellular response. The activation of the pathway isusually transient and regulated by protein phosphatasesthat recognize the dual phosphorylated form of theMAPK and switch off the pathway when the cell hasresponded to the extracellular stimulus or has adapted tothe onset of the new condition.

At least six MAPK (Fus3p, Kss1p, Hog1p, Slt2p-alsocalled Mpk1p-, Smk1p and Mlp1) are encoded by theS. cerevisiae genome. As shown in Figure 1, four of themmediate the response to speci�c stimuli and areimplicated in generating specialized biological responses[for reviews, see 24,25]. Fus3p is necessary for mating,Kss1p regulates the invasive growth both in haploid ordiploid cells, Hog1p is necessary for the response tohypertonic stress, Slt2 controls cell integrity in responseto hypotonic stress and heat shock, Kss1p also mediatescell integrity in response to defects in protein glycosyla-tion, and a �fth one, Smk1p, is implicated in sporulation(this last pathway is not shown in the �gure). The role ofMlp1p is still unclear. Yeast cells of S. cerevisiae carryingdeletion of all six genes encoding MAPKs are viable butsterile and severally affected in the response to stressconditions like sudden changes in external salinity ordecreased nutrient availability [26].

The fact that some pathways share the same elementsposes a problem on the speci�city. This theme isintriguing and cannot be treated exhaustively in thiscontext (see also Navarro-Garcia et al., this issue). Thespeci�city of the response appears to be regulated bymultiple mechanisms. One of these is the presence of

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Fig. 1 Summary of �ve MAPK cascades that regulate growth and differentiation in haploid S. cerevisiae. Elements shared by differentpathways are shown in bold.

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speci�c upstream elements (receptors, osmosensors, etc.)and downstream transcription factors that route theinput signal on speci�c pathways. For example, matingfactor receptors, G protein and Ste5 scaffold protein arespeci�c for the mating factor response and Tec1transcription factor is speci�c for invasive growth.Moreover, the pathways cross-regulate one another toensure that they are not activated by the wrong signal.Also, Fus3p inhibits misactivation of the invasive growthpathway by mating pheromone, whereas Hog1p inhibitsmisactivation of the mating pathway by hypertonic stress[25]. However, it is also important to recall that certainstimuli may require more than one pathway to yield thefull biological response. The fact that a signal istransmitted by a cascades of phosphorylation eventsand other reactions is already an indication of pos-sibilities for cross-talk between the various pathways thatare necessary to produce a co-ordinated cellular re-sponse. For instance, a -factor activates the MAP kinasepathway for mating in the short term but later alsoactivates the PKC1–MAP kinase pathway. At themoment, the formation of shmoo projections requiresan extensive cell wall remodelling and the two pathwaysmust be co-ordinated [27].

As shown in Figure 1, two MAPK pathways, thePKC1–MAPK and the Sho1–Kss1 pathways, have so farshown to be involved in controlling cell integrity.Whereas the former has been characterized extensively,the later has been only recently identi�ed by geneticscreens originally designed to identify negative regula-tors of the pheromone response signalling or mutationsthat determine synthetic lethality in combination withthe ste11D mutation [28,29]. The Ste20p, Ste11p andSte7p enzymes of this pathway are in common with themating pathway but Sho1p is associated with the HOGpathway and Kss1p with the invasive growth pathway (inbold in Fig. 1). The activation of this later pathway leadsto the Kss1p-mediated activation of FUS1 transcription,a reporter characteristic of the mating pathway [29]. Thispathway functions during vegetative growth although itsrole is more evident under stress conditions. FUS1promoter activity is increased strongly by mutations ingenes affecting protein glycosylation (och1, pmi40,dpm1), GPI anchor synthesis (spt14) or moderatelyenhanced in cells lacking GAS1. Moreover all thesemutants require the functionality of this pathway asshown by lethality or severe worsening of the phenotypesif elements of the Sho1-pathway are inactivated [29].

The PKC1±MAP kinase pathway

The activation of the MAP kinase module constituted byBck1p–Mkk1/Mkk2–Mpk1 (Slt2) is mediated by Pkc1p,

the yeast homologue of mammalian protein kinase C.This pathway is activated by high temperature, hypoos-motic shock, a -factor at the onset of the formation of themating projections and during the cell cycle at theinitiation of budding [for review, see 25,30].

PKC1 is an essential gene and pkc1 null mutants arestrictly dependent on the presence of osmotic support forgrowth. Also, the loss of the downstream elementsresults in a lytic phenotype. This is attenuated as it ismanifest at 37 oC, suggesting that this pathway maybranch [30]. The characteristic osmotic-remedial pheno-type of mutants of the PKC1–MAPK pathway suggestedthat it is required for cell-wall construction, and for thisreason it has also been called cell integrity pathway.Indeed, the basal level of expression of some cell wall-related genes (FKS1, MNN1 and CHS3) depends on thefunctionality of this pathway [31].

The PKC1–MAPK pathway becomes activated inresponse to cell-wall damage. The �rst clue for this wasprovided by the �nding that this pathway was activatedby high temperature and at the time of formation ofmating projections in a -factor treated cells that requiresextensive cell wall remodelling [25]. In addition, apersistent activation of the cell integrity pathway wasfound in gas1 and fks1 null mutants [14]. Two differentreadouts were used to monitor the activation of thepathway: (i) the detection of the phosphorylated form ofSlt2p by antibodies that recognize the two highlyconserved phosphorylated p44/p42 mammalian MAPKresidues (Thr202 and Tyr204 in the human form)corresponding to Thr190 and Tyr192 in the yeastSlt2p(Mpk1p), and (ii) the expression of a FKS2–LacZreporter gene [14,32]. Indeed, both fks1D and gas1D

mutants show a higher level of the phosphorylated formof Slt2p than their wild-type counterparts. The presenceof 1 M sorbitol in the growth medium greatly reduces theincrease in Slt2p-phosphorylation in these cells [14].Examples of Slt2p activation are shown in Figure 2.Combined deletions of FKS1 or GAS1 genes withinactivation 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 fks1null mutants, because it is necessary to rescue the cells inresponse to cell-wall stress.

In a wild-type cell the acquisition of thermal tolerancewas reduced greatly by the slt2 null mutation and slt2cells were also highly sensitive to Zymolyase. Theseresults suggest that also the increased thermal resistanceand the resistance to Zymolyase of gas1 and fks1mutants depend on Slt2p-activation [14].

The recent �nding that the Sho1–Kss1 pathway isinvolved in the response to cell-wall defects accounts forthe �nding that another MAPK is activated in gas1D cells



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(p48 in Fig. 2b). The identi�cation of p48 is currentlyunderway in our laboratory.

The activation of PKC1–MAPK pathway was alsodetected in wild-type cells treated with agents thatperturb the cell wall. Calco�uer induces an increase inthe phosphorylation of Slt2p that remains high even150 min after drug addition. Furthermore, mild treat-ment with Zymolyase or vanadate, an agent thatpresumably affects cell-wall stability, generates a similarresponse [14].

Among the ultimate targets of the cell integritypathway that have so far been identi�ed are the Rlm1p,a member of the MADS family, SBF transcription factor,a heterodimer of Swi4p and Swi6p, required forexpression of G1 cyclin genes, and proteins of theHMG1-group of chromatin-associated proteins thatcould affect gene transcription indirectly. The role ofthese factors is reviewed in [25] and [30].

Perception and transmission of cell-wallstress to the PKC1±MAP kinase pathway

The mechanism by which information regarding the stateof the cell wall is transmitted to the intracellularsignalling apparatus is still an open question. However,two hypotheses have been formulated that converge onthe activation of the PKC1–MAP kinase pathway. The�rst proposes that cell wall damage mimics a condition ofhypo-osmotic shock. Stretching of the plasma membrane

could be induced by the inability of weakened cell wallto effectively counteract turgor pressure. In this regard,it has been demonstrated that hypotonic shock generatesa stretch-activated channel-dependent calcium pulse inyeast [34]. The increase of the cytosolic calciumconcentration could then activate Pkc1p. It has beenfound that the Pkc1ts phenotype is suppressed by growthin high levels of calcium. Nevertheless, in vitro activity ofsoluble Pkc1p in yeast is calcium-independent but theinterpretation of this data is hindered by the fact that thefraction of soluble tested Pkc1p molecules is small [25].

The mating pheromone response provides an exampleof ion �ux activation by a mechanosensor. Pheromonestimulates Ca2‡ in�ux through a calcium channelcomplex whose components are Cch1p, the putativetrue channel, and Mid1p that may confer the property ofstretch activation to Cch1p [35,36]. Mid1p is an integralmembrane protein with a single transmembrane domainand associates with Cch1p that has 16 transmembranedomains [36,37]. Mid1p expressed in Chinese hamsterovary cells mediates Ca2‡ in�ux in response to mechan-ical stress. Moreover, the channel is inhibited bygadolinium, a blocker of stretch-activated cation chan-nels [38].

A second hypothesis proposes that a recently identi-�ed redundant family of transmembrane proteins couldfunction as sensors of the state of the cell wall. Mid2pand Slg1p, with their homologs Mtl1p (MID2-like 1) andWsc2-3-4 proteins, are putative cell wall sensors involvedin remodelling cell wall during vegetative growth orpheromone-induced morphogenesis. SLG1 gene wasisolated several times and variously designated HCS77[39], WSC1 [40] or SLG1 [41]. MID2 was isolated bycomplementation of a mutation that causes defects in themating response [42]. Interestingly, MID2 (matinginduced death) was also isolated in several geneticscreens as a multicopy suppressor of defects in micro-tubule organization, in actin polymerization and asactivator of Skn7 transcription factor [reviewed in 43].Since SLG1 is a multicopy suppressor of mid2D mutantphenotype and MID2 of the slg1D phenotype, Slg1p andMid2p play at least partially redundant functions [43,44].

Mid2p and Slg1p has the major in vivo role, as thedifferent combinations of deletions of their homologueshave only a minor effect on the phenotype displayed bythe single slg1 or mid2 null mutants. The slg1 nullmutants show a sorbitol-remedial lysis phenotype at hightemperature (39 oC), similarly to PKC1–MAPK pathwaymutants, and a higher sensitivity to different drugs [39–41]. The mid2D grows normally and shows lysis defectsonly when exposed to mating factors. This phenotypecan be prevented by increase of external calciumsuggesting a possible role of this protein in the

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Fig. 2 High level of Slt2p-phosphorylation in heat-shocked cellsand in gas1D cells. Upper panels: immunoblot analysis with anti-phospho-p42/p44 MAPK antibody of total yeast extracts preparedas described in [19], in the presence of protease and phosphataseinhibitors [32]. Lower panels: identical extracts were analysed withanti-Slt2 antibodies. Equal amounts of total proteins were loadedon each lane. a, W303-1A cells were cultured to mid-log phase inYNB-glucose at 24 oC and then transferred at 37 oC for 1 h. b,W303-1B strain and its gas1D derivative were cultured to mid-logphase in YNB-glucose at 30 oC in the absence or presence of 1 M

sorbitol. Slt2p-phosphorylationwas almost undetectable in the wild-type strain and was high in gas1D cells with respect to the isogenicstrain. Slt2p level doubled in the gas1D mutant. The presence of 1 M

sorbitol reduced both the activation and the doubling of Slt2p.

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transmission of calcium signalling [37,42]. A peculiarphenotype of mid2 null mutants is the resistance toCalco�uor [43]. The mid2D cells have the same chitinlevel as the wild type but the extent of chitin synthesisinduced by Calco�uor or a -factor treatments is reduced.Thus it has been proposed that Mid2p is a regulator ofchitin synthesis under stress conditions [43].

Slg1 and Mid2 proteins have a common structure asType I transmembrane proteins consisting of an extra-cellular domain rich in serine and threonine residues, atransmembrane domain and a relatively short cytoplas-mic tail. The extracellular domain of Slg1p and Mid2p isextensively modi�ed by O-mannosylation that causes anabnormal migration of these proteins in SDS gels [43,45].Despite the similarity shared in the overall structure,Slg1p and Mid2p have dissimilar primary structures. Apotential Ca2‡ binding motif is present in the intracel-lular domain of Mid2p and a cysteine-rich region in theextracellular domain of Slg1p [42].

By use of the gene fusion with the green �uorescentprotein (GFP), it has been shown that both Mid2p andSlg1p are distributed evenly at the cell periphery andfractionation experiments, indicated that these proteinsbehave as integral membrane proteins [42–44]. A HAtagged-Slg1p expressed from a centromeric plasmidappeared to localize in the growing bud [23]. Followingcell wall stress, Slg1p was �rst depolarized and thenrepolarized at the bud apex [23].

The structure of Mid2 and Slg1 suggests these proteinsare structurally analogous to the integrins of mammaliancells, a class of receptors that are not endowed with anyintrinsic biochemical activity but bind ligands present inthe extracellular matrix and activate signalling pathwaysresponsible for changes in the actin cytoskeleton. This isconsistent with the observation that Slg1p, like mamma-lian integrins, both controls and responds to the actincytoskeleton [23].

It is not yet understood how cell-wall defects areperceived by these sensor proteins. It seems to beunlikely that Mid2p and Slg1p bind to speci�c ligands ofthe extracellular matrix as do integrins. However, theirextracellular domain is essential for their role as sensors.In particular, the inhibition of O-mannosylation by a nullmutation in PMT2 gene, encoding an enzyme involved inthe �rst step of O-mannosylation, impairs the sensorfunction of Mid2p, suggesting a possible novel role of theO-linked oligosaccharide chains in such surface proteins[45]. It has been proposed that the high degree of O-mannosylation of the serine and threonine residuesinduces the polypeptide chain to assume an extendedand stiffened conformation that could span the cell walland enter the physico-chemical region of the extracel-lular matrix [43–45].

Several lines of evidences indicate that Slg1 and Mid2pmay act as upstream activators of the PKC1–MAPKpathway [14,43,44]. The absence of Mid2p reduces thelevel of Slt2-phosphorylation in response to a shift tohigh temperature (39 oC), a -factor, Calco�uor andvanadate treatment, whereas the absence of Slg1p resultsonly in a weak reduction of heat-induced phosphoryla-tion [14,43]. In conclusion, Mid2p appears to be involvedmore in signalling of stress conditions, and Slg1p incondition of normal vegetative growth.

How these proteins activate the cell integrity pathwayhas been only recently elucidated. It has been shown bytwo-hybrid system experiments that the C-terminaldomains of Slg1p and Mid2p interact with Rom2p, oneof the two guanine nucleotide exchange factors (GEFs)for Rho1p, the main regulator of the PKC1–MAP kinasepathway in yeast [45] (Fig. 3). Rho1p is a member of aGTPase family and a key protein for yeast morphogen-esis. Rho1p can cycle between an inactive (bound toGDP) and an active state (bound to GTP). Transition isregulated by Rom1 and Rom2p and the hydrolysis ofGTP is stimulated by two potential GAP proteins,Bem2p and Sac7p [32]. Slg1p and Mid2p regulate GEFactivity for Rho1p. Extracts from a slg1D mutant displaya 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 thatMid2p has in vegetative growth [45]. The activation ofRho1p via GEF Rom2p occurs in response to certain cellwall stress conditions such as treatment with SDS and inthe 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 foundfollowing Calco�uor treatment [45].



Fig. 3 Model of Rho1-activation by Mid2p and Wsc1p (Slg1p) viaRom2p and of the various cellular activities co-ordinated by Rho1p.

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Rho1p has several effectors: Pkc1p, Fks1-Fks2p, thecatalytic subunits of b (1,3)-glucan synthase, the non-essential formin-like protein Bni1p, the two-componentsignalling protein Skn7p and the protein Sec3 involved inthe polarized transport of the exocytotic vesicles [47; seealso citations in 23]. An important role of Rho1p residesin the control of actin cytoskeleton organization. Rho1pcan affect the organization of the cytoskeleton both byinteracting with Bni1p or indirectly through the activa-tion of Slt2p as in Tor2p signalling [48]. Rho1p mediatesthe actin cytoskeleton, repolarization through PKC1–MAP kinase pathway after a cell-wall stress [23]. Figure3 illustrates the cellular activities controlled by Rho1p.

However, the mechanism by which Mid2p and Slg1ptransmits information concerning cell-wall perturbationsto Rom proteins remains obscure.

The calcineurin pathway

In S. cerevisiae, calcineurin (Ca2‡/calmodulin dependentphosphatase: PP2B) plays a key role in mediating severalimportant physiological processes. These include NaCltolerance, mating intracellular calcium buffering andconstruction of the cell wall [reviewed in 25]. Calcineurinis not essential for normal growth. It is a heterodimer ofCNA1 and CNA2-encoded redundant catalytic subunitsand a regulatory subunit encoded by the CNB1 gene[49].

The b (1,3)-glucan synthase complex is responsible forthe synthesis of the most abundant glucan of yeast cellwall and Fks1/2p are its catalytic components. Deletionof single FKS1 or FKS2 does not affect viability but theremoval of both is lethal. Therefore, in the absence ofFKS1, FKS2 becomes essential. It has been found thatcells with compromised cell integrity due to the lack ofFKS1 gene or mutations in the PKC1–MAPK pathway,depend on calcineurin for growth [33,49]. Moreover,fks1D and gas1D mutants display an increased sensitivityto the immunosuppressants FK506 and cyclosporin Athat bind to their receptors, FKBP12 or cyclophilin,thereby inhibiting calcineurin [33; J. Arroyo andL. Popolo, unpublished data].

The �nding that FKS2 expression is under the controlof calcineurin and that its lack or inhibition causes ade�ciency of Fks2p suggests that calcineurin is acomponent of the compensatory response [50].

Large-scale approaches to the study of thecell wall stress response

Powerful technologies such as DNA microarrays andproteomics have made possible the simultaneous analysisof the expression levels of many genes of an organism. Inan organism whose genome is completely sequenced

such as S. cerevisiae, it is possible to examine whichgenes are up- or downregulated under given experi-mental conditions.

To create a condition of strong activation of thePKC1–MAPK pathway, a gain-of-function allele ofMKK1 (MKKS368P) was placed under the control ofthe galactose-inducible promoter. Following a shift fromglucose to galactose, the transcript pro�les of cellsexpressing the wild-type or the hyperactive allele werecompared using a �lter hybridization technique [51]. Thearti�cial activation of the pathway led to an increase inthe transcript level of 20 genes and a decrease of 5 genes[51] (Table 1). Most of the genes identi�ed are known orsuspected to encode CWPs or proteins involved in cell-wall biogenesis. The transcript levels of several geneswere further analysed separately by northern blotanalysis, and for each transcript the behaviour in a wildtype and a rlm1D strain was compared. With theexception of FKS2, all the regulated genes wereapparently dependent on Rlm1p [52] and had Rlm1pputative binding elements in their promoter regions. Theexpression of CHS3, FKS1 and MNN1 previouslyreported to be regulated by the PKC1–MAP kinasepathway was not identi�ed in this genome wide screen.However, when tested separately these transcriptsappeared to be weakly regulated and MNN1 not to beregulated at all under these conditions.

In another recent study, the expression pro�le of afks1D mutant is described [53]. The transcriptionalregulation was investigated using microarrays and testedsubsequently by fusing the 50 non-coding region fromeach gene to the Escherichia coli LacZ reporter gene:22 genes were upregulated (Table 1). Among these,three genes encoding GPI-proteins were in commonwith those reported in the previous microarray survey(PST1, CRH1, CWP1). These genes may be directtargets of the constitutive activation of the PKC1–MAPK pathways that occurs in fks1 mutant. Othergenes as FKS2 and CHS1 were also in common in botharray experiments. Interestingly the transcript of SLT2was itself upregulated, in agreement with other experi-mental data (Fig. 2b). These results further support theexistence of a feedback positive loop of phosphorylatedSlt2p on its own expression [51]. The persistent activa-tion of the pathway by cell wall disturbance could leadnot only to the activation of Slt2p-phosphorylation butalso to an increase of the level of this protein that couldbe required for a more ef�cient response to the stress.

Conclusions

The cell wall is essential for the survival of yeast andfungal cells. These organisms have developed several

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pathways to respond to imposed environmental stressand damage, such as changes in external osmolarity, theaction of glucanases or chitinases secreted by competingorganisms or by the defence reaction of plants, orchanges in temperature or nutrient availability. Thesalvage pathway relies on elements that are conserved inall the eukaryotes, and yeast is a valuable organism forthe molecular investigation in this �eld.

The future progress on the molecular characterizationof the salvage pathway in yeast will provide useful

information for the evaluation of the degree of damagecaused by new antifungal drugs directed against the cellwall, and will help the developments of tools formonitoring the cellular responses. Furthermore, thesalvage pathway may have critical roles in the responsesand adaptation of fungal pathogens during infection, forexample on the surface mucosae or in the phagolyso-some of macrophages. The results generated to date alsounderline the conclusion that the cell wall of fungi is ahighly dynamic structure whose composition and struc-



Table 1 Comparison between genes up-regulated in response to activation of Slt2p and in fks1D mutant*

Category Gene name ORF

Cellintegritysignalling

fks1D

mutant Biochemical function/cellular role

GPI proteins PST1 YDR055W > > UnknownSED1 YDR077W > — Structural cell-wall protein of stationary phaseCRH1 YGR189C > > Putative b (1,3)-b (1,4)-transglucosidaseCWP1 YKL096W > > Structural cell-wall proteinPRY2 YKR013W — > UnknownYPS3 YLR121C — > Plasma membrane aspartyl protease

YLR194C > — Unknown functionSSR1 YLR390W-a > — Structural cell-wall protein

PIR family PIR1 (CCW6) YKL164C > — Structural cell-wall proteinPIR2 (HSP150) YJL159W > — Structural cell-wall proteinPIR3 YKL163W > — Structural cell-wall proteinCIS3 (CCW5) YKL158C > — Structural cell-wall protein

Other cell wall proteins BGL2 YGR282C > — b (1,3)-endoglucanase/glucanosyltransferase

Cell wall biogenesisproteins FKS1 YJL342W > N.A. Catalytic component b (1,3)-glucan synthase

FKS2 YGR032W > > Alternative component b (1,3)-glucan synthaseCHS3 YBR023C > > Chitin synthase IIICHS1 YNL192W > > Chitin synthase ICHS7 YHR142W — > Export Chs3p from ERGFA1 YKL104C — > Glucosamin-fructose-6-phosphate aminotransferaseKTR2 YKR061W — > Mannosyltransferase

Regulatory proteins SLT2 (MPK1) YHR030C > > S/T protein kinase MAP kinaseMLP1 YKL161C > — S/T protein kinase MAP kinasePTP2 YOR208W — > protein tyrosine phosphatase

Other not relatedto cell wall SEC28 YIL076W > — Vesicle coat protein

DFG5 YMR238W > — UnknownYIL117C > — UnknownYNL058C > — Unknown

PMD1 YER133C — > Unknown regulator of early meiotic gene expressionPCL1 YNL285W — > G1/S-phase cyclinSVS1 YPL163C — > S-T rich proteinBOP1 YPL221W — > UnknownBRR1 YPR057W — > RNA-binding protein involved in snRNP biogenesis

YAL053W — > UnknownYBR071W — > UnknownYPL067C — > Unknown

* adapted from [51] and [53]; N.A., not applicable.

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ture is responsive to both endogenous constraints (cell-cycle regulation, changes in turgour) and exogenouschallenges.

Acknowledgements

We wish to thank R. Zippel and M. Vai for criticalreading of the manuscript, M. Molina for Slt2 antibodiesand helpful discussions and J. Arroyo for havingcommunicated unpublished data on gas1 sensitivity toFK506. We apologize to the yeast people for possibleomissions in pertinent literature.

The research from our laboratory reported in thisreview was supported by MURST 1999 to L.P., ECcontract QLK3-CT-2000-01537 to L.P. and Azioni-Integrate Italia-Spagna 1999–2000.

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The yeast cell-wall salvage pathway