Genome-wide survey of yeast mutations leading to activation of the yeast cell integrity MAPK pathway: novel insights into diverse MAPK outcomes.

Abstract

The yeast cell wall integrity mitogen-activated protein kinase (CWI-MAPK) pathway is the main regulator of adaptation responses to cell wall stress in yeast. Here, we adopt a genomic approach to shed light on two aspects that are only partially understood, namely, the characterization of the gene functional catalog associated with CWI pathway activation and the extent to which MAPK activation correlates with transcriptional outcomes.
A systematic yeast mutant deletion library was screened for constitutive transcriptional activation of the CWI-related reporter gene MLP1. Monitoring phospho-Slt2/Mpk1 levels in the identified mutants revealed sixty-four deletants with high levels of phosphorylation of this MAPK, including mainly genes related to cell wall construction and morphogenesis, signaling, and those with unknown function. Phenotypic analysis of the last group of mutants suggests their involvement in cell wall homeostasis. A good correlation between levels of Slt2 phosphorylation and the magnitude of the transcriptional response was found in most cases. However, the expression of CWI pathway-related genes was enhanced in some mutants in the absence of significant Slt2 phosphorylation, despite the fact that functional MAPK signaling through the pathway was required. CWI pathway activation was associated to increased deposition of chitin in the cell wall - a known survival compensatory mechanism - in about 30% of the mutants identified.
We provide new insights into yeast genes related to the CWI pathway and into how the state of activation of the Slt2 MAPK leads to different outcomes, discovering the versatility of this kind of signaling pathways. These findings potentially have broad implications for understanding the functioning of other eukaryotic MAPKs.

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RESEARCH ARTICLEOpen Access
Genome-wide survey of yeast mutations leading to
activation of the yeast cell integrity MAPK pathway:
Novel insights into diverse MAPK outcomes
Patricia Arias, Sonia Díez-Muñiz, Raúl García, César Nombela, José M Rodríguez-Peña*and Javier Arroyo
Abstract
Background: The yeast cell wall integrity mitogen-activated protein kinase (CWI-MAPK) pathway is the main
regulator of adaptation responses to cell wall stress in yeast. Here, we adopt a genomic approach to shed light on
two aspects that are only partially understood, namely, the characterization of the gene functional catalog
associated with CWI pathway activation and the extent to which MAPK activation correlates with transcriptional
outcomes.
Results: A systematic yeast mutant deletion library was screened for constitutive transcriptional activation of the CWI-
related reporter gene MLP1. Monitoring phospho-Slt2/Mpk1 levels in the identified mutants revealed sixty-four deletants
with high levels of phosphorylation of this MAPK, including mainly genes related to cell wall construction and
morphogenesis, signaling, and those with unknown function. Phenotypic analysis of the last group of mutants suggests
their involvement in cell wall homeostasis. A good correlation between levels of Slt2 phosphorylation and the
magnitude of the transcriptional response was found in most cases. However, the expression of CWI pathway-related
genes was enhanced in some mutants in the absence of significant Slt2 phosphorylation, despite the fact that functional
MAPK signaling through the pathway was required. CWI pathway activation was associated to increased deposition of
chitin in the cell wall - a known survival compensatory mechanism - in about 30% of the mutants identified.
Conclusion: We provide new insights into yeast genes related to the CWI pathway and into how the state of
activation of the Slt2 MAPK leads to different outcomes, discovering the versatility of this kind of signaling
pathways. These findings potentially have broad implications for understanding the functioning of other eukaryotic
MAPKs.
Background
The capacity to respond properly to external stimuli or
environmental conditions is essential for homeostasis in
eukaryotes. Signaling pathways, in particular those
mediated by mitogen-activated protein kinases (MAPKs),
play a key role in these processes. A significant amount
of research has been conducted in recent years to charac-
terize these signal transduction pathways, based largely
on the model yeast Saccharomyces cerevisiae (also known
as baker’s or budding yeast). Since MAPK pathways are
evolutionarily conserved, insights gained from yeast con-
tribute to a better understanding of orthologous path-
ways in higher organisms. The basic assembly of MAPK
pathways is a three-component module conserved from
yeast to humans, consisting of three kinases that establish
a sequential activation pathway by means of phosphoryla-
tion events [1]. The regulation of MAPK signal transduc-
tion depends on a variety of mechanisms including
scaffold proteins, subcellular localization of different ele-
ments and the action of protein phosphatases [2].
In yeast, six MAPK cascades have been identified that
mediate the response to different stimuli: (i) pheromones
(pheromone response pathway); (ii) nitrogen starvation
(filamentous growth pathway); (iii) hyperosmolarity (high
osmolarity/glycerol pathway); (iv) STE vegetative growth
pathway (SVG) [3]; (v) nutrient starvation (spore wall
assembly pathway); and (vi) cell wall stress (CWI: cell wall
integrity pathway) (see an extensive review of MAPK path-
ways in [2]). The CWI pathway is essential for maintaining
* Correspondence: josemanu@farm.ucm.es
Departamento de Microbiología II, Facultad de Farmacia, Universidad
Complutense de Madrid, IRYCIS, 28040 Madrid, Spain
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© 2011 Arias et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
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cellular integrity. Therefore, mutations affecting different
elements of the pathway lead to cell lysis [4,5]. The main-
tenance of cellular integrity and morphology, as well as
the protection of the cell from adverse environmental con-
ditions, depends on the cell wall, an essential structure
that has been characterized extensively in Saccharomyces
cerevisiae (reviewed in [6,7]). It has three major compo-
nents: an inner layer of glucans (b-1,3 and b-1,6-glucan),
chitin and an outer layer of mannoproteins. These compo-
nents must be correctly assembled in order to build a fully
functional structure [6,8,9].
The essentiality of the cell wall for fungal viability makes
it one of the most attractive targets for therapeutic inter-
vention against fungal pathogens [10]. Treatment with cell
wall-perturbing agents such as the chitin-binding dyes
Congo red and Calcofluor white or zymolyase, which
degrades the b-1,3-glucan network, elicits a cellular survi-
val response known as “compensatory mechanism” [11].
This adaptive response includes changes in the yeast tran-
scriptional program that we and other groups have charac-
terized, not only in yeast mutants affected at different
stages of cell wall biosynthesis [12], but also in wild-type
yeast cells growing under different conditions causing
transient cell wall damage [13-18]. The compensatory
response leads to, among other effects, an increase in the
amount of b-glucan and chitin, the production of several
cell wall proteins and changes in the cross-linking between
cell wall polymers [19]. Although several signaling path-
ways contribute to the regulation of cell wall remodeling
in order to ensure cell integrity, the regulation of this com-
pensatory response is controlled mainly by the MAPK
Slt2p/Mpk1p (hereafter noted as Slt2p) through the cell
wall integrity pathway (for a review, see [20,21]). The CWI
pathway is regulated through the cell cycle, being also acti-
vated in response to a variety of external stimuli and mor-
phological events that cause cell wall stress, such as heat
stress, hypo-osmotic shock, mating pheromones, oxidative
stress, actin depolymerization, cell wall-related mutations,
cell wall-stressing agents, alkaline stress and endoplasmic
reticulum (ER) stress [20,22-25].
Several cell membrane proteins (Mid2, Wsc1-4 and
Mtl1) [26-28] act as sensors of the CWI pathway. For
further intracellular transduction of the activation signal,
these sensors interact with the guanine nucleotide
exchange factor (GEF) Rom2, activating the small GTPase
Rho1, which then activates the yeast protein kinase C
(Pkc1). The main role of activated Pkc1 is to trigger a
MAPK module. Upon phosphorylation, the MAPKKK
Bck1 activates a pair of redundant MAPKKs (Mkk1 and
Mkk2), which phosphorylate the MAPK Slt2. Slt2 is a
functional homolog of human ERK5 [29], a MAPK that is
activated in response to both growth factors and physical
and chemical stresses [30,31]. The dually phosphorylated
(Thr190/Tyr192) form of Slt2 activates two transcription
factors: the MADS-box transcription factor Rlm1 [32] and
SBF, a heterodimeric complex of two proteins, Swi4 and
Swi6, which are mainly involved in the regulation of gene
expression in G1/S transition [33]. Although Rlm1, acti-
vated through phosphorylation by Slt2 [34], is responsible
for the transcriptional activation of the majority of the
genes induced in CWI adaptation responses, a non-cataly-
tic mechanism of transcriptional activation mediated by
SBF through Slt2 has recently been described [35]. This
alternative mechanism extends the regulatory roles of the
MAPK cascade.
In accordance with the complexity of the cellular pro-
cesses related to cell wall homeostasis in yeast, cross-
talk between distinct MAPK pathways has recently been
described. This complicates the simple linear “top-
down” concept of signaling pathways. For example, Slt2
is activated in response to hyperosmotic shock through
the HOG1 MAPK pathway [36]. Similarly, our group
has shown that treating yeast cells with zymolyase also
activates Slt2 in a Hog1 pathway-dependent manner
[17,37].
Considering that the CWI pathway is activated under
cell wall stress, our working hypothesis is that yeast
strains lacking genes functionally relevant for cell wall
biogenesis or pathway regulation should present a con-
stitutive activation of this route. In this paper, we
describe the development of a genomic-wide screening
in order to identify genes whose absence produces a
functional constitutive activation of the CWI pathway of
Saccharomyces cerevisiae under vegetative (non-stressed)
growth conditions. As a result, we have identified, for
the first time at genomic scale, a map of genes that
could be functionally related to the CWI pathway.
Furthermore, this report gives new insights into the link
between the magnitude of MAPK activation and tran-
scriptional induction or cell wall remodeling events.
This information can be extended to pathogenic fungi,
being useful for future therapeutic purposes.
Results and discussion
Large-scale identification of gene deletions that activate
the cell wall integrity pathway in yeast
The CWI pathway of Saccharomyces cerevisiae, governed
by the MAPK Slt2, is triggered under conditions that com-
promise the integrity of the cell wall. Signaling through
this pathway can be monitored by taking advantage of
reporters driven by Rlm1-responsive promoters. We have
recently developed a transcriptional reporter system, espe-
cially suitable for large-scale studies, that potentially allows
detecting the functional activation of the CWI pathway
[38]. Essentially, this system is based on a plasmid con-
struction (pJS05) that includes a transcriptional fusion of
the promoter region of the MLP1 gene, one of the main
effectors of the transcriptional up-regulation response
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under cell wall stress, and the coding sequence of the
NAT1 gene, which encodes resistance to the antibiotic
nourseothricin. In other words, the expression of NAT1 is
controlled by the MLP1 promoter; therefore, yeast
mutants with a constitutive activation of the CWI pathway
transformed with this construction should be able to grow
in the presence of higher concentrations of the antibiotic
than a wild-type strain.
Following this approach, we have designed a large-
scale screening to identify yeast deletions associated
with Slt2-driven transcriptional activation. To achieve
this goal, we used the collection of haploid mutant
strains in all non-essential genes of Saccharomyces
cerevisiae (~4800 strains). As represented in Figure 1,
we proceeded to transform this collection into a 96-
well microplate format with the plasmid containing
MLP1P-NAT1, and the transformed strains were
screened for their ability to grow in the presence of
300 μg/ml nourseothricin. This inhibitory concentra-
tion of antibiotic was previously determined using
selected mutants in which basal levels of Slt2 activa-
tion were similar to those found in a wild-type strain
(data not shown). After 48-72 hours of growth in a
selective medium, 174 mutant strains with hyper-resis-
tance to nourseothricin were identified and selected
for further characterization.
Slt2 phosphorylation levels in nourseothricin-resistant
mutants identified in the screening
A reliable method for monitoring signaling through the
CWI pathway is to follow the activation state of the
MAPK Slt2 by using commercially available antibodies
that recognize the dual phosphorylation of conserved
threonine and tyrosine residues within the activation loop
of Slt2, analogous to Thr202/Tyr204of mammalian p44/p42
MAP Kinase (ERK) [39]. In order to associate the phos-
phorylation status of Slt2 to MLP1 up-regulation, the 174
deletant strains formerly selected were examined following
this approach. After densitometric quantification of the
phospho-Slt2 bands obtained by Western blotting analysis
of the total protein extracts from each mutant strain,
64 mutants recorded higher Phospho-Slt2 levels (at least
twofold) than the wild-type strain. The relative amount of
phospho-Slt2 in these mutants was distributed over a wide
range of values (from twice to thirtyfold) (Table 1). Repre-
sentative examples of Slt2 activation in different mutants
are shown in Figure 2. The complete data set (Western
blots) for all the selected mutants is presented in Addi-
tional file 1. These data indicate different levels of CWI
pathway activation for each mutant, suggesting that yeast
cells modulate pathway activation as required by specific
stimuli. Remarkably, more than 20% of the mutations
identified in a large-scale analysis revealing synthetic lethal
interaction with slt2 Δ [40] have also been isolated in our
screening (Table 1).
As shown in Table 1, the functional categorization of
mutants identified for constitutive Slt2 activation
revealed the most representative functional groups
according to the BIOBASE Knowledge Library Proteome
and Saccharomyces Genome Database classifications,
being those involved in cell wall organization and mor-
phogenesis (28%), genes of unknown function (17%),
signal transduction (17%), transport (11%) and transcrip-
tion (9%). The remaining functional categories comprise
mutants linked to metabolism (principally of RNA and
proteins) and other cellular functions with lower repre-
sentation in the screening (see Table 1). This distribu-
tion is consistent with putative inputs of the pathway,
namely, cell wall alterations or regulatory proteins.
An analysis was conducted of predicted and known
interactions between the whole set of genes identified in
the screening using the STRING web resource (http://
string-db.org) [41]. This tool is very useful for the retrie-
val of an overall perspective of interacting genes/proteins.
As shown in Figure 3, a large number of genes (38 out of
64) showed functional interactions between them. Inter-
estingly, the interaction network was clustered again in
the three main functional nodes (cell wall and morpho-
genesis, signal transduction and transcription) described
above. From the large group of 18 mutants related to cell
wall and morphogenesis, different subgroups can be
Figure 1 Overview of the screening strategy designed to
identify yeast mutants with constitutive CWI pathway
activation. Yeast mutant strains, transformed with the plasmid
pJS05 (MLP1P-NAT1), in which the expression of MLP1 is higher
compared to the wild-type strain, were identified as resistant to
nourseothricin.
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Table 1 S. cerevisiae mutants with increased levels of dually phosphorylated MAP Kinase Slt2
ORFGeneP-Slt2Functional GroupDescription
YAL058W
YBL007C
YBR078W
YCR088W
YDL095W
YDR349C
YJL062W
YJL099W
YJR075W
YLR319C
YLR337C
YLR350W
YLR370C
YMR307W
YNL079C
YNL116W
YNL192W
YOR002W
YER037W
YJL137C
YHL023C
YPR031W
YBR101C
YDR069C
YFL007W
YBL024W
YPL029W
YPL213W
YDL047W
YDR162C
YDR389W
YER155C
YHR082C
YHR206W
YJR074W
YKL126W
YLR371W
YNL053W
YNR047W
YLR242C
YER139C
YMR136W
YNL025C
YPL042C
YPR065W
YPR115W
YDR200C
YFL048C
YGR028W
YHR032W
YJL133W
CNE1
SLA1
ECM33
ABP1
PMT1
YPS7
LAS21
CHS6
HOC1
BUD6
VRP1
ORM2
ARC18
GAS1
TPM1
DMA2
CHS1
ALG6
PHM8
GLG2
RMD11
NTO1
FES1
DOA4
BLM10
NCL1
SUV3
LEA1
SIT4
NBP2
SAC7
BEM2
KSP1
SKN7
MOG1
YPK1
ROM2
MSG5
FPK1
ARV1
RTR1
GAT2
SSN8
SSN3
ROX1
16.4
8.1
14.4
2.3
10.8
16.6
3.6
2.9
2.1
2.4
8.6
2.6
5.5
27.4
7.2
3.4
2.0
6.3
4.0
2.3
2.9
2.0
2.8
2.6
4.0
5.8
2.0
9.0
4.3
16.9
7.5
2.4
2.9
2.5
5.4
2.3
2.5
5.4
3.9
8.6
5.2
10.7
2.2
3.9
4.7
3.6
3.9
4.1
2.1
3.5
5.2
Cell wall and morphogenesis
Cell wall and morphogenesis
• Cell wall and morphogenesis
Cell wall and morphogenesis
Cell wall and morphogenesis
• Cell wall and morphogenesis
Cell wall and morphogenesis
Cell wall and morphogenesis
• Cell wall and morphogenesis
Cell wall and morphogenesis
Cell wall and morphogenesis
Cell wall and morphogenesis
Cell wall and morphogenesis
• Cell wall and morphogenesis
• Cell wall and morphogenesis
Cell wall and morphogenesis
Cell wall and morphogenesis
Cell wall and morphogenesis
Metabolism
Metabolism
Nuclear
Nuclear
Protein metabolism
Protein metabolism
Protein metabolism
RNA metabolism
RNA metabolism
RNA metabolism
Signal transduction
Signal transduction
Signal transduction
• Signal transduction
Signal transduction
Signal transduction
Signal transduction
Signal transduction
Signal transduction
Signal transduction
Signal transduction
Sphingolipid metabolism
Transcription
Transcription
Transcription
Transcription
Transcription
Transcription
Transport
Transport
Transport
Transport
Transport
Calnexin 1. may be involved in a QC process for secretory pathway proteins
Functions in the assembly of cortical actin cytoskeleton
GPI-anchored protein required for CW integrity and mannoprotein biosynthesis
Actin binding protein that functions in clathrin- and actin-mediated endocytosis
Mannosyltransferase, first step in O-glycosylation
GPI-anchored aspartyl protease involved in maintaining cell wall integrity
Protein required for addition of a side chain to the GPI core structure
Protein involved in chitin synthase Chs3p activity
Alpha-1,6-mannosyltransferase activity
Budding 6, required for bipolar budding and involved in bud site selection
Proline-rich protein verprolin, involved in cytoskeletal organization
Putative transmembrane protein that may function in the CWI pathway
Component of the ARP2/3 actin-organizing complex, involved in actin assembly
GPI-anchored protein with 1,3-beta-glucanosyltransferase activity
Tropomyosin, functions in a variety of processes involving the actin cytoskeleton
Functions in spindle positioning and septin ring assembly
Chitin synthase I, has a repair function during cell separation
Dolichyl-phosphate-mannose-protein mannosyltransferase activity
Lysophosphatidic acid phosphatase
Transferase activity, transferring hexosyl groups
Protein possibly involved in meiotic nuclear division
May be involved in chromatin-mediated transcription regulation
NEF for the cytosolic chaperone Ssa1p. Response to oxidative stress
Ubiquitin-specific protease that acts in recycling ubiquitin
Proteasome assembly
tRNA (cytosine-5-)-methyltransferase activity
Mitochondrial RNA helicase of the DEAD box family
RNA splicing factor activity
Serine/threonine phosphatase involved in cell cycle regulation/ion homeostasis
Negative regulator of the HOG pathway
GTPase-activating protein for Rho1p
GTPase-activating (GAP) protein that regulates Rho1p
Serine/threonine kinase involved in filamentous growth
Transcription factor involved in the oxidative and osmotic stress responses
Involved in nuclear protein import, plays a role in osmoregulation via SLN1-SKN7
Putative S/T protein kinase possibly related with the CWI and sphingolipids
GDP-GTP exchange factor for Rho1p
Dual-specificity protein tyrosine phosphatase involved in response to pheromone
Serine/threonine protein kinase that regulates phospholipid asymmetry
Protein involved in sterol uptake required for normal sphingolipid metabolism
Protein required for growth at high temperature, RNA polymerase II factor
GATA zinc finger transcription factor
RNA polymerase II transcription mediator
Cyclin-dependent serine/threonine protein kinase of the RNA polymerase II
Transcriptional repressor of hypoxic genes
Phosphoinositide binding. Response to oxidative stress
Class B vacuolar sorting protein involved in Prc1p trafficking/a-factor secretion
Golgi and ER membrane protein involved in glycoprotein secretion
Intra-mitochondrial sorting protein, member of the AAA family of ATPases
Member of the MatE family. Transporter activity
Mitochondrial carrier (MCF, family of membrane transporters). Iron transport
VPS64
EMP47
MSP1
ERC1
MRS3
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highlighted. The first subgroup comprises seven genes
(SLA1, ABP1, BUD6, VRP1, ARC18, TPM1, DMA2)
related to actin cytoskeleton organization. Actin cytoske-
leton disruption has been shown to activate the CWI
pathway, probably due to the induction of cell wall stress,
but the precise molecular mechanism by which Slt2 is sti-
mulated has not been fully established [42]. Moreover,
related to this group we found the Doa4 deubiquitinating
enzyme (Figure 3), which has been associated with cell
morphology and actin cytoskeleton defects. In fact,
DOA4 had a synthetic genetic interaction with SLA1 [43].
A second subgroup encompasses six mutants, including
structural cell wall-related proteins. Three of them are
glycosylphosphatidylinositol-anchored proteins (GPI-
APs) on the cell surface: Gas1, which is a b-1,3-glucano-
syltransferase; Ecm33, which is linked to cell wall mainte-
nance; and Yps7, an aspartyl protease. The corresponding
deletant strains have severe cell wall defects that may
explain their high basal Slt2 activation. In fact, the com-
pensatory response elicited in a gas1Δ strain has pre-
viously been characterized, and it involves a significant
induction in MLP1 expression [12]. In agreement with
this, gas1Δ and ecm33Δ mutants have previously been
shown to have a constitutively high level of Slt2
phosphorylation [44,45]. Moreover, within this second
subgroup we found Las21, an ER membrane protein
involved in the synthesis of the GPI core structure. The
absence of this protein leads to global cell wall defects.
Functionally linked to Las21, the protein Arv1 was identi-
fied (Figure 3). This protein is involved in sphingolipid
metabolism and has recently been related to the process
of GPI synthesis and anchoring [46]. Finally, two proteins
related to chitin metabolism, Chs6, which is involved in
chitin synthase Chs3 activity, and Chs1 (chitin synthase
I), which is required for repairing the chitin septum dur-
ing cytokinesis [7] were uncovered in our screening. The
identification of these mutants is significant since, due to
functional redundancy and the existence of gene families,
the deletion of certain individual genes encoding cell
wall-related proteins does not usually lead to observable
phenotypes. A third subset includes pmt1Δ, hoc1Δ and
alg6Δ strains. All of them encode mannosyltransferase
activities, and their selection in the screening is consis-
tent with the finding that protein glycosylation of cell
surface proteins is important for cell wall assembly
[7,47]. Interestingly, transcriptional responses to O- and
N-glycosylation defects in yeast include the fingerprint
of the cell wall damage transcriptional profile [48,49].
Table 1 S. cerevisiae mutants with increased levels of dually phosphorylated MAP Kinase Slt2 (Continued)
YJR152W
YLR292C
YCR090C
YDL173W
YDR290W
YGR022C
YLR250W
YLR338W
YMR119W-A
YNL058C
YNL105W
YNR014W
YPL158C
DAL5
SEC72
5.7
2.9
2.0
4.1
4.2
3.9
5.2
7.2
3.2
3.0
5.5
2.5
3.0
Transport
Transport
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Member of the allantoate family of the major facilitator superfamily (MFS)
Component of ER protein-translocation subcomplex.
Unknown function
Unknown function
Unknown function
Unknown function
Unknown function
Unknown function
Unknown function
Unknown function
Unknown function
Unknown function
Unknown function
SSP120
Levels of Slt2 phosphorylation (P-Slt2) correspond to the fold-change in each mutant strain relative to the wild-type calculated as described in Methods.
Representative data of at least replicated experiments are shown. Functional categories and descriptions were assigned based on the information provided by
the BIOBASE Knowledge Library Proteome. Genes were grouped together on the basis of their functional category. Those gene deletions that have been
reported to be synthetic lethal with slt2Δ are labeled with a black dot.
Figure 2 Representative examples of yeast mutants with increased levels of Slt2 phosphorylation. Exponential phase cultures of the
indicated yeast strains were taken and processed for immunoblotting as described under “Methods”. Western blots detecting the
phosphorylated form of Slt2 (P-Slt2) and actin as loading control are shown. Numbers correspond to the P-Slt2 fold-change obtained from
densitometric quantification of the P-Slt2 bands from Western blots normalized with respect to the actin bands, using the values of the wild-
type strain as reference (fold-change set to 1.0).
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