Molecular Microbiology (2016) 00(00), 00–00 䊏
13416
The Saccharomyces cerevisiae Ptc1 protein phosphatase
attenuates G2-M cell cycle blockage caused by activation
of the cell wall integrity pathway
Laura Tatjer, Asier Gonza lez,†
Albert Serra-Cardona, Anna Barcelo  ,‡
Antonio Casamayor and Joaquın Arin  ~ o*
Institut de Biotecnologia i Biomedicina and
Departament de Bioquımica i Biologia Molecular,
Universitat Auto noma de Barcelona, Cerdanyola del
Vallès 08193, Barcelona, Spain.
Summary
Lack of the yeast Ptc1 Ser/Thr protein phosphatase
results in numerous phenotypic defects. A parallel
search for high-copy number suppressors of three of
these phenotypes (sensitivity to Calcofluor White,
rapamycin and alkaline pH), allowed the isolation of
25 suppressor genes, which could be assigned to
three main functional categories: maintenance of cell
wall integrity (CWI), vacuolar function and protein
sorting, and cell cycle regulation. The characteriza-
tion of these genetic interactions strengthens the rel-
evant role of Ptc1 in downregulating the Slt2-
mediated CWI pathway. We show that under stress
conditions activating the CWI pathway the ptc1
mutant displays hyperphosphorylated Cdc28 kinase
and that these cells accumulate with duplicated DNA
content, indicative of a G2-M arrest. Clb2-associated
Cdc28 activity was also reduced in ptc1 cells. These
alterations are attenuated by mutation of the MKK1
gene, encoding a MAP kinase kinase upstream Slt2.
Therefore, our data show that Ptc1 is required for
proper G2-M cell cycle transition after activation of
the CWI pathway.
Accepted 5 May, 2016. *For correspondence. E-mail Joaquin.
Arino@uab.es; Tel. 34-93-5811315; Fax 34-93-5812011. Present
addresses: †Biozentrum, University of Basel, Switzerland ‡Servei
de Genomica i Bioinforma
tica, Universitat Auto
noma de Barcelona,
Spain.
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doi:10.1111/mmi.
First published online 2016
Introduction
Protein phosphatases (PPases) are key regulators of
cellular homeostasis. Three major classes of so-called
classical Ser/Thr protein phosphatases exist, differing in
their primary structures: phosphoprotein phosphatases
(PPP), metal-dependent protein phosphatases (PPM)
and RNA polymerase II carboxy-terminal domain phos-
phatase (Cohen, 2004). PPMs, also known as type 2C
Ser/Thr protein phosphatases, are evolutionarily con-
served and, in contrast with other PPase families, are
usually monomeric enzymes, lacking regulatory or tar-
geting subunits. Therefore, to achieve functional special-
ization most organisms express a relatively large
number of diverse isoforms. In the yeast Saccharomy-
ces cerevisiae, type 2C Ser/Thr protein phosphatases
are encoded by seven genes (PTC1-7), which have
been characterized in more or less detail [see (Arino
et al., 2011) for a review].
Ptc1 is the closest yeast homolog of human Wip1, a
phosphatase that may have oncogenic properties and is
implicated in diverse physiological and pathological con-
ditions (Lammers and Lavi, 2007; Zhu and Bulavin,
2012). Ptc1 is involved in a large diversity of cellular
processes that are not shared by other Ptc family mem-
bers, as it is deduced from the large number of specifc
phenotypes and the characteristic changes in the tran-
scriptomic profile derived from deletion of the gene
(Gonzalez et al., 2006). A role for Ptc1 in the negative
regulation of the HOG pathway by dephosphorylating
Hog1 was early identified (Maeda et al., 1993; Maeda
et al., 1994; Warmka et al., 2001). However, many other
defects caused by the absence of Ptc1 activity cannot
be attributed to deregulation of the HOG pathway. For
instance, ptc1 mutants are sensitive to diverse cations,
including calcium, which causes hyperactivation of the
calcineurin phosphatase (Gonzalez et al., 2006), and
lithium, likely due to a Hog1-independent decrease in
the expression of the Na1-ATPase ENA1 gene (Ruiz
et al., 2006). Ptc1-deficient cells also display fragmented
vacuoles, mimicking class B vps (vacuolar protein-sort-
ing) mutants (Sambade et al., 2005; Bonangelino et al.,
2 L. Tatjer et al. 䊏
2002; Seeley et al., 2002; Gonzalez et al., 2006), and
these strains manifest Hog1-independent defects in cor-
rect inheritance of diverse organelles (Roeder et al.,
1998; Du et al., 2006; Jin et al., 2009). A role for Ptc1 in
the dephosphorylation of the Snf1 kinase has been
recently documented (Ruiz et al., 2013). Deletion of
PTC1 results in intense sensitivity to rapamycin, an
inhibitor of the TORC1 pathway, (Parsons et al., 2004;
Xie et al., 2005), and it has been shown that Ptc1 is
required for normal TORC1 signaling by regulating, in a
HOG-independent manner, a step upstream the Sit4
phosphatase (Gonzalez et al., 2009).
Early evidence (Huang and Symington, 1995) sug-
gested a link between Ptc1 and the cell wall integrity
(CWI) pathway, which includes the Pkc1 kinase as the
upstream element of a MAPK cascade composed by
the MAPKKK Bck1, two similar MAPKK (Mkk1 and
Mkk2), and the MAPK Slt2 [see (Levin, 2011) for a
review]. In agreement with the involvement of Ptc1 in
the CWI pathway, the ptc1 mutation is synthetically
lethal with mutations in genes such as FKS1, GAS1, or
SMI1, which are crucial for cell wall construction (Les-
age et al., 2004). Cells lacking Ptc1 are also sensitive to
diverse cell wall antagonists, such as Calcofluor White
(CFW) or caspofungin (Ram et al., 1994; Markovich
et al., 2004), or to conditions that activate the CWI path-
way, such as alkaline pH (Serrano et al., 2004; Serrano
et al., 2006). These cells display higher-than-normal
amounts of active Slt2 (Du et al., 2006; Gonzalez et al.,
2006) and show increased expression of diverse genes
(MLP1, CRH1, SED1) positively regulated by the Slt2
MAPK module (Gonzalez et al., 2006). Although it is
known that a cross-talk between the Slt2 and HOG
pathways exists (Hahn and Thiele, 2002; Bermejo et al.,
2008), the hypersensitivity of the ptc1 strain to cell wall-
damaging agents is not caused by hyperactivation of
Hog1 (Gonzalez et al., 2006). Importantly, whereas Ptc2
and Ptc3 are capable of dephosphorylating the cyclin-
dependent kinase (CDK) Cdc28 at Thr169, a residue
essential for its activity as CDK, Ptc1 was found to have
little or none effect in dephosphorylating Cdc28 in vitro
and in vivo (Cheng et al., 1999), and hence its role in
cell cycle regulation has been questioned.
In spite of the considerable effort dedicated to the
study of Ptc1 functions, the question concerning
whether the large variety of phenotypes derived from
lack of Ptc1 function are caused by deregulation of only
a few key proteins or derive from a direct effect on multi-
ple cellular targets, remained unsolved for many years.
In a very recent work, we demonstrated that many ptc1
defects can be attributed to the hyperactivation of the
CWI pathway and, specifically, to the inability to properly
dephosphorylate and inactivate the MAPKK Mkk1 (Tatjer
et al., 2016). Here we present a parallel search for high-
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copy suppressors of three different phenotypic defects
of the ptc1 mutant: sensitivity to CFW, rapamycin, and
alkaline pH, resulting in the isolation of 25 suppressor
genes. The characterization of these genetic interactions
allows pointing to a role of Ptc1 on the G2-M transition
of the cell cycle, likely by contributing to the necessary
downregulation of the CWI pathway upon stress.
Results and discussion
Identification of high-copy suppressors of three ptc1
phenotypes
Upon transformation of the ptc1 strain with the genomic
libraries, clones containing plasmids were recovered in
liquid media and plated on plates containing CFW (5 or
10 mg ml21, 60,000 clones), rapamycin (5 or 10 ng
ml21, 50,000 clones), or adjusted to alkaline pH (8.0 or
8.2, 60,000 clones). These numbers of clones represent
a genomic coverage > 99.999% for each screen. After
2–3 days of incubation, clones able to generate macro-
scopic colonies were selected and tested for the pres-
ence of plasmid-borne PTC1 gene (which was positive
in around 50% of these clones), Restriction mapping of
these plasmids revealed up to six different restriction
patterns, indicating that different PTC1-containing clones
were retrieved. Plasmids negative for the presence of
PTC1 were reintroduced into the ptc1 strain and tested
again for increased tolerance to the specific condition.
Inserts from representative plasmids were subjected to
end-sequencing and positioned in the corresponding
chromosomal loci. The information gathered is pre-
sented in Supporting Information Table S1.
The specific gene responsible for the gain in tolerance
was isolated from each genomic insert by digestion with
the appropriate restriction enzymes (Supporting Infor-
mation Table S2). The screen for CFW sensitivity sup-
pression yielded 13 genes, whereas the screen for high
pH and rapamycin sensitivity produced 11 and 5 genes
respectively. Three genes (VPS73, MIH1 and CLN1)
appeared from both CFW and high pH screens,
whereas one gene (VPS70) was found both in the high
pH and rapamycin screens, thus producing a total of 25
suppressor genes (Table 1 and Fig. 1A). Gene Ontology
analysis of the suppressor genes indicated that the
majority of them can be placed in two categories: cell
cycle (CLN1, CLB1, CLB6, MIH1, TAF10, CDC37,
ZDS1, PPH22 and PIN4), and vacuolar function and
protein sorting (VRG4, OST5, VPS70, VPS73, RCR2,
SSH4, VOA1 and SMY2). Three genes (GAS1, KRE6
and BCK1) can be clearly assigned to cell wall mainte-
nance, although three of the genes included in the
vacuolar function and protein sorting category (VRG4,
OST5 and RCR2) are also related to cell wall
 Ptc1 relieves cell cycle blockage upon cell wall stress 3

Table 1. Suppressor genes isolated in the triple screen.

Systematic name Gene name Screen Description

YBL051C PIN4 CFW Involved in G2/M transition and DNA damage response
YBR172C SMY2 Rap GYF domain protein, involved in COPII vesicle formation
YDL188C PPH22 Rap Catalytic subunit of protein phosphatase 2A (PP2A)
YDR003W RCR2 pH Vacuolar protein
YDR167W TAF10 CFW Subunit (145 kDa) of TFIID and SAGA complexes
YDR168W CDC37 CFW Essential Hsp90p co-chaperone
YGL104C VPS73 CFW/pH Mitochondrial protein, mutation affects vacuolar protein sorting
YGL225W VRG4 pH Golgi GDP-mannose transporter
YGL226C-A OST5 pH Zeta subunit of the oligosaccharyltransferase complex of the ER lumen
YGR106C VOA1 CFW ER protein that functions in assembly of the V0 sector of V-ATPase
YGR108W CLB1 CFW B-type cyclin involved in cell cycle progression
YGR109C CLB6 CFW B-type cyclin involved in DNA replication during S phase
YIR018W YAP5 CFW Basic leucine zipper (bZIP) iron-sensing transcription factor
YJL095W BCK1a Rap MAPKKK acting in the protein kinase C signaling pathway
YJL110C GZF3 pH GATA zinc finger protein
YJR126C VPS70 pH/Rap Protein of unknown function involved in vacuolar protein sorting
YKL124W SSH4 pH Specificity factor required for Rsp5p-dependent ubiquitination
YMR036C MIH1 CFW/pH Protein tyrosine phosphatase involved in cell cycle control
YMR199W CLN1 CFW/pH G1 cyclin involved in regulation of the cell cycle
YMR273C ZDS1 Rap Protein with a role in regulating Swe1p-dependent polarized growth
YMR307W GAS1 CFW b-1,3-glucanosyltransferase, required for cell wall assembly
YOR028C CIN5 CFW Basic leucine zipper (bZIP) transcription factor of the yAP-1 family
YPL104W MSD1 pH Mitochondrial aspartyl-tRNA synthetase
YPR124W CTR1 pH High-affinity copper transporter of the plasma membrane
YPR159W KRE6 CFW Type II integral membrane protein, putative b-glucan synthase

Rap, Rapamycin; pH, alkaline pH

a. As three different C-terminally truncated versions.

biogenesis (see below). It must be noted that our screen
yielded three different C-terminally truncated forms of
the BCK1 gene, encoding the MAPKKK upstream the
Slt2 MAP kinase (Supporting Information Table S1). In
all cases, the truncation affects the catalytic domain and
thus the protein should lack protein kinase activity. Two
suppressor genes draw immediately our attention: CTR1
and PPH22. CTR1 was isolated in the alkaline pH
screen. It encodes a high affinity copper transporter
that, together with FET4, was identified by our group as
one of the only two genes able to increase high pH tol-
erance in a wild type strain (Serrano et al., 2004). FET4
codes for a low affinity iron permease, also able to
transport other transition metal ions such as copper and
zinc. Therefore, we decided to incorporate FET4 into
subsequent analyses. PPH22 encodes a catalytic subu-
nit of the type 2A protein phosphatase. S. cerevisiae
contains a highly similar gene (PPH21), and Pph21 and
Pph22 are thought to have redundant functions (Zab-
rocki et al., 2002). For this reason, we also incorporated
PPH21 in our work.
The set of 25 genes, plus FET4 and PPH21, was sub-
jected to detailed characterization with regard their
potency in suppressing characteristic ptc1 phenotypes.
Conditions tested were: growth on non-fermentable car-
bon source (ethanol), diverse alkaline pHs (from 7.8 to
8.2), and an array of concentrations of CFW, caffeine,
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rapamycin, CaCl2, LiCl, ZnCl2 and hydrogen peroxide.
In addition, the ability of each gene in normalizing
vacuolar morphology was also tested. The integration of
the data allowed assigning a suppression score for each
gene and each condition, ranging from 1 (behavior
equivalent to the ptc1 strain carrying an empty plasmid,
that is, no suppressor effect) to 7, where score 6 was
attributed to suppressors able to restore the wild type
phenotype and score 7 (only very few cases) to those
able to increase tolerance to a given condition above
wild type level. The score matrix was then subjected to
clustering analysis for both genes and conditions tested.
As it can be observed (Fig. 1B), clustering analysis
reveals a number of common features among the sup-
pressor genes. For instance, cluster 1 contains only two
genes, VPS70 and VPS73, both required for normal
protein sorting. Four out of the five members of cluster
2 are directly involved or required for normal cell cycle
progression (CLB1, CLB6, MIH1 and TAF10). Cluster 3
contains genes GAS1 and KRE6, which code for
enzymes that synthesize components of the yeast cell
wall. It is worth noting that FET4, although not found in
the screen, appears linked to CTR1 in cluster 4. Simi-
larly, the PPH22 and PPH21 pair is found in cluster 5.
Interestingly, this cluster also includes ZDS1, encoding
a protein that interacts with and regulates specific PP2A
functions (Wicky et al., 2011; Queralt and Uhlmann,
4 L. Tatjer et al. 䊏

Fig. 1. Characterization of high-copy suppressors of ptc1 phenotypes.

A. Venn diagram showing the suppressors genes identified in the screens for CFW, alkaline pH and rapamycin tolerance. Note that three
common suppressors were isolated in the CFW and high pH screens and one in the high pH and rapamycin screens.
B. The 25 suppressors (plus the FET4 and PPH22 genes) were transformed into ptc1 cells and their ability to rescue 10 different ptc1
phenotypes was scored (1 indicates no suppressor effect and seven maximum effect, see text for details). The asterisk in BCK1 denotes that
the suppressor clone is a truncation of the BCK1 gene. The heat-map depicts the clustering analysis of the data matrix generated (similarity
metrics, correlation centered; clustering method, average linkage). Numbers identify clusters discussed in the text. Abbreviations for gene
functions are: CC, cell cycle; CWI, cell wall integrity; VPS, vacuolar protein sorting.

2008). Indeed, all three genes have in common a very
strong suppressor capacity when ptc1 cells are chal-
lenged with rapamycin. Finally, cluster 6 includes two
related transcription factors, CIN5/YAP4 and YAP5,
belonging to the yAP-1 family. These two members are
induced by many different environmental stresses and,
among the eight components of this family in yeast
(YAP1-YAP8), they seem to constitute a specific func-
tional subgroup that share many common targets
(Rodrigues-Pousada et al., 2010).
Suppression by CTR1 or CIN5 is likely not related to
Ptc1-specific functions
To explore the possibility that a given suppressor effect
could be not specifically related to the ptc1 defect, we
transformed the wild type BY4741 strain with each of
the 27 suppressor genes and transformants were sub-
jected to phenotypic analysis. In this case, phenotypes
were scored from 0 to 5, being score 0 attributed to
strains in which overexpression of the gene worsened
growth, score 1 to those in which growth was not
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altered, and scores 2 to 5 according to the improvement
observed. As shown in Supporting Information Fig. S1,
in most cases overexpression of the suppressors in the
wild type background was without effect. However,
expression of CTR1 and FET4 greatly improved growth
under high pH stress and when ethanol was the only
carbon source. This was not unexpected, since both
genes were described by our laboratory as able to con-
fer higher tolerance to alkaline pH to wild type cells due
to their role in the uptake of iron, which becomes limiting
under alkaline stress (Serrano et al., 2004). In fact, the
beneficial effect of overexpression of CTR1 in wild type
cells is abolished by deletion of FET3, a copper-
containing protein that is a component of the high affin-
ity iron transport system (Serrano et al., 2004). As
shown in Fig. 2A, the effect of overexpressing CTR1 in
the ptc1 mutant is also lost when FET3 is absent. Simi-
larly, addition of micromolar amounts of copper or iron
ions improves growth of the ptc1 strain at alkaline pH,
being copper more effective than iron, whereas a combi-
nation of both metals further improved growth (Fig. 2B).
Again, this reproduces the behavior previously reported
 Ptc1 relieves cell cycle blockage upon cell wall stress 5

Fig. 2. Suppression of high pH sensitivity in the ptc1 mutant by CTR1 and FET4. The indicated strains were transformed with the high-copy
empty vector (YEp195), or the same vector carrying the CTR1 (pCTR1) or FET4 (pFET4) genes and spotted (3 ml, OD 5 0.05) on YPD plates
adjusted at the indicated pHs (OD 5 0.5 for plates at pH 8.1). Plates were incubated for 48 h prior documentation. B) Cultures of strain
MAR143 (ptc1) were inoculated in YPD-50 mM TAPS adjusted to pH 7.6 in the absence or the presence of the indicated concentrations of
metal cations. Growth was monitored by determining the A660 of the cultures after 20 h at 288C. Data are mean 6 SEM from 5 to 10
determinations.

for the wild type strain (Serrano et al., 2004). These
results, and the fact that the relatively moderate sup-
pressor potencies of CTR1 and FET4 were mainly
restricted to the high pH-related phenotype (Fig. 1B),
suggest that the observed amelioration is not specific
for the effect(s) of the ptc1 mutation.
Similarly, overexpression of CIN5 in wild type cells
caused increased tolerance to LiCl, CFW, rapamycin
and, to some extent, to hydrogen peroxide. The effect of
CIN5 in LiCl is not unexpected, since its alias (HAL6) is
reminiscent of its discovery as able, when in high-copy
number, to increase tolerance to LiCl and NaCl (Mendi-
zabal et al., 1998). It has been reported that CIN5 is
involved in the transcriptional response of yeast cells
exposed to the glucosamine polymer chitosan and that
cin5 mutants are sensitive to this compound (Zakrzew-
ska et al., 2005). Since chitosan seems to elicit the acti-
vation of the CWI pathway (Zakrzewska et al., 2005) as
CFW does, overexpression of CIN5 might confer advant-
age when cells are confronted with cell wall damaging
agents (Supp. Fig. 1). Overexpression of CIN5 also
increases tolerance to rapamycin in the wild type strain.
Although the reason for this is unclear, it might be
related to the observation that exposure to rapamycin
affects the localization of this transcription factor, which
shifts from the nucleus to the cytoplasm (Shin et al.,
2009). In any case, all these results suggest that most
of the observed effects for CIN5 are not Ptc1-specific.
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Suppression by VPS73 may be related to its putative
transport activity
Vps73 is a 486-residues protein with 10–12 predicted
transmembrane domains and likely mitochondrial sub-
cellular localization. Very little is known about its bio-
chemical activity. Similarity analysis reveals that Vps73
is reminiscent to known sugar transporters from very
diverse organisms from bacteria to human (Fig. 3A and
B). In S. cerevisiae, Vps73 and its paralog Ybr241c are
highly similar to several hexose transporters, such as
Hxt4, Hxt2, Hxt10 or Gal2, and show a number of highly
conserved residues. In this regard, Glu at position 169
in Vps73, located in the short intracellular loop between
TM4 and TM5, drew our attention because mutation of
the equivalent residue (E153) to Ala completely abol-
ished the activity of the bacterial transporter (Sun et al.,
2012). This is relevant because mutations in the corre-
sponding Glu in human GLUT1 (Glu146) have been
found in patients with the glucose transporter-1 defi-
ciency syndrome (Wang et al., 2000). Therefore, we
mutated Glu169 to Ala and tested the ability of the
mutated Vps73 version in alleviating the ptc1 defect at
high pH or elevated calcium concentrations (which
VPS73 does with rather high efficiency). As shown in
Fig. 3C, mutation of Glu169 completely abolishes the
effect of VPS73 overexpression. This suggests that
VPS73 might encode a functional transporter and that
its transport ability would be beneficial in the absence of
6 L. Tatjer et al. 䊏

Fig. 3. Effect of VPS73 as suppressor of ptc1 phenotypes.

A. Multiple protein sequences alignment of Vps73-related proteins. The analysis tool ClustalW2 (Larkin et al., 2007) was used to align the full
protein sequences of several transporters of the facilitator superfamily using the default settings. Residues conserved in all the analyzed
sequences are specified below the alignment. The figure shows the sequence surrounding the conserved Glu169 of Vps73 (denoted with an
asterisk), corresponding to Glu146 in GLUT1, around the transmembrane 4 and 5 regions (TM4 and TM5 in the cartoon). The sequences
analyzed were: Vps73 (gb EWH18 624.1), Ybr241c (NP 009800.1), Hxt4 (NP 011960.2), Gal2 (NP 013182.1) and Rtg2 (NP 010143.1) from
S. cerevisiae; GLUT1 (NP 006507.2), GLUT3 (NP 008862.1), GLUT 4 (NP 001033.1) and Fruct (gb AAB60641.1) from H. sapiens and xylE
(gb EFF03252.1) from E. coli.
B. Phylogenetic tree of Vps73-related proteins. The tree was based on the multiple protein sequences alignment described in 3A provided by
ClustalW2 (default settings) and was drawn using the TreeView software 1.6.6 (Page, 2002).
C. The wild-type BY4741 strain and its ptc1 derivative were transformed with the empty plasmid (YEp195) or the same plasmid carrying the
native version of VPS73, the VPS73 version carrying the Glu179!Ala mutation (YEp-VPS73E169A) or the paralog gene YBR241c (YEp-
YBR241c). Two dilutions of the cultures were spotted on the indicated YPD plates and incubated for 2 (CaCl2) or 4 (pH) days prior
documentation.

Ptc1 under certain stress situations such as high pH.
This possibility is suggestive, since it is known that
exposure to high pH triggers a transcriptional response
that includes induction of diverse glucose transporters
and that, in some way, mimics that of glucose starvation
(Viladevall et al., 2004; Ruiz et al., 2008).
YBR241c encodes a protein slightly larger than
Vps73 (488 residues), for which 10–11 transmembrane
domains are also predicted. However, in spite of the
high resemblance between the two proteins (46% iden-
tity, 69% similarity), YBR241c was unable to improve
growth of the ptc1 strain at high pH or when the medium
was supplemented with calcium chloride. This was
somewhat surprising, since key residues appear con-
served in both proteins. However, analysis of their co-
expression pattern using the SPELL search engine
(Hibbs et al., 2007) showed very different results. Fur-
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thermore, we found no correlation between the genetic
interactions of YBR241c and VPS73 with other genes,
according to the DRYGIN database (Koh et al., 2010).
Therefore, even if VPS73 and YBR241c share certain
structural determinants, they likely function under differ-
ent cellular circumstances, thus explaining the lack of
ptc1 suppressor function for YBR241c.
It is clear from Fig. 1B that VPS70 not only clusters
with VPS73, but also that both genes display virtually
identical phenotypic suppressor potency for all condi-
tions examined. Vps70 contains a transmembrane
domain and shares with two other yeast proteins, Tre1
and Tre2, a transferrin receptor-like dimerization and an
N-acetylated–alpha-linked acidic dipeptidase domain.
Our results suggest that Vps70 and Vps73 could per-
form similar or identical functions, which would be inter-
esting because, besides its requirement for normal

vacuolar protein sorting, the specific cellular role of
Vps70 remains unknown. VPS70 is one of the genes
with higher correlation with VPS73 in the DRYGIN data-
base. In addition, Vps70 is one of the four VPS genes,
together with VPS73, VPS62 and VPS74, whose
absence produces increased resistance to lactic acid
(Kawahata et al., 2006; Suzuki et al., 2013;), and cells
lacking VPS70 and VPS73 share several phenotypes,
such similar levels of secretion of CPY (Bonangelino
et al., 2002). All these cumulative evidences point to a
common function for Vps70 and Vps73.
The suppressor screens support a functional link
between Ptc1, the CWI pathway and cell cycle
regulation
Our screen identified several suppressor genes involved
in CWI, such as GAS1, encoding a cell wall-bound 1,3-
b-glucanosyltransferase required for the formation and
maintenance of the major polysaccharide of the cell
wall, 1,3-b-glucan, and KRE6, coding for a putative b-
glucan synthase required for 1,6-b-glucan biosynthesis.
It is worth noting that a ptc1 mutant shows decreased
levels of 1,6-b-glucan and, as a consequence is hyper-
tolerant to the killer toxin (Jiang et al., 1995). The finding
of GAS1 as suppressor (mainly under CFW stress) fits
with the negative genetic interactions found between the
gas1 and ptc1 mutations (Lesage et al., 2004; Tong
et al., 2004). In contrast, as far as we know, no genetic
interactions between KRE6 and PTC1 have been
described. In addition, recent evidence indicates that
other ptc1 suppressors could also be linked to the CWI
pathway (Fig. 1B). Thus, lack of PIN4 (also known as
MDT1), which was initially related to DNA damage
responses, causes hypersensitivity to CFW and caffeine,
and this mutation presents severe synthetic sickness
with mutations in the BCK1 and SLT2 genes (Traven
et al., 2010). The product of the essential VRG4 gene, a
GDP-mannose transporter into the lumen of Golgi appa-
ratus, is also required for maintenance of CWI (Ellerton
et al., 2008). Cells carrying the vrg4-2 allele have
defects in cell wall structure, being hypersensitive to
Congo red. Finally, OST5 encodes a subunit of the OST
(oligosaccharyltransferase) complex of the ER lumen
that catalyzes N-linked glycosylation of proteins, which
could be functionally related to the integrity of the cell
wall (Janik et al., 2012).
Interestingly, our screen recovered three fragments
containing different versions of the BCK1 gene, encod-
ing the MAPKKK acting in the CWI pathway, with sub-
stantial truncations at the C-terminal end of the protein.
These truncations affect exclusively the catalytic domain
of the kinase, which lies between residues 1175 and
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Ptc1 relieves cell cycle blockage upon cell wall stress 7
1439 (Fig. 4A), while the regulatory moiety would be
unaffected. Cells lacking Ptc1 display larger amounts of
active Slt2 (Gonzalez et al., 2006; Du et al., 2006) and
there are diverse evidences supporting that ptc1 strains
have a hyperactive CWI pathway [see (Arino et al.,
2011) for a review]. Thus, it was conceivable that the
truncated, catalytically inactive versions of Bck1 might
be acting in a dominant negative fashion, thus down-
regulating the Pkc1-Slt2 module (expression of the
hyperactive BCK1-20 allele did not result in any sup-
pressor effect, not shown). As observed in Fig. 4B, the
increased phosphorylation of Slt2 caused by deletion of
PTC1 was substantially decreased by expression of all
three truncated versions of Bck1. This would fit with the
relatively broad spectra of phenotypes alleviated by
these suppressors (Figs. 1B and 4C), compared with
that of GAS1 or KRE6, which likely counteract specifi-
cally the cell wall damage caused by exposure to CFW
(Fig. 1B), and reinforces the notion that hyperactivation
of the Pkc1-Slt2 module would be at the basis of a sig-
nificant number of phenotypic defects derived from the
absence of Ptc1. This view is supported by our observa-
tions that deletion of SLT2 or BCK1 in the ptc1 back-
ground aggravates the growth defects of the ptc1 strain
under cell wall stress (Gonzalez et al., 2006), but
improves growth of the ptc1 mutant under other stress
conditions, such as the presence of LiCl, CaCl2 or rapa-
mycin (Fig. 4D), and perfectly fits with our recent pro-
posal that the Mkk1 MAPKK is a major target for Ptc1
(Tatjer et al., 2016).
In contrast with Ptc2 and Ptc3, which were defined as
the major CDK phosphatase activities in yeast by
dephosphorylating Cdc28 at Thr169 (Cheng et al.,
1999), a direct effect of Ptc1 in the dephosphorylation of
immediate components of the cell cycle machinery has
not been reported. However, our screen recovered sev-
eral genes known to directly control cell cycle progres-
sion, such as CLN1, CLB1, CLB6, MIH1, CDC37, ZDS1
and PPH22. CLN1 encodes a G1 cyclin, whereas CLB6
encodes a B-type cyclin important at the end of G1 to
promote initiation of DNA synthesis. On the contrary,
CLB1 (and CLB2) associate to the Cdc28 kinase to pro-
mote the transition from G2 to M phase of the cell cycle.
Progress in mitosis is controlled by the phosphorylation
state of the Cdc28-cyclin complex. Whereas phosphoryl-
ation by the tyrosine kinase Swe1 inhibits the Cdc28-
cyclin complex, the action of Mih1 (the budding yeast
Cdc25 Tyr phosphatase) reverses such phosphorylation
and promotes progress through the cell cycle (see Fig.
5A). Interestingly, activation of the Slt2 pathway under
certain forms of stress has been reported to promote
cell cycle blockage at the G2-M transition by activating
Swe1 (Mizunuma et al., 1998; Zhang and Rao, 2008) or
inhibiting Mih1 (Harrison et al., 2001; Martinez-Anaya
8 L. Tatjer et al. 䊏

Fig. 4. Attenuation of the Slt2 MAP kinase CWI pathway rescues ptc1 phenotypic defects.
A. Schematic depiction of the three suppressors containing a C-terminally truncated version of Bck1. The black segment denotes the catalytic
domain and its boundaries. Numbers on the right indicate the expected length of the expressed protein.
B. Attenuation of Slt2 hyperphosphorylation in the ptc1 mutant by expression of C-terminally truncated forms of Bck1. Wild type strain
BY4741(1) or MAR143 (ptc1, -) were transformed with an empty episomal plasmid (YEp-Ø) or plasmids carrying the indicated versions of
Bck1 (see panel A), and protein extracts prepared. The presence of active phosphorylated (P-Slt2) and total Slt2 protein was determined by
Western blot analysis. The histogram at the bottom shows the P-Slt2/Slt2 ratio relative to the wild type strain carrying the empty plasmid
obtained by integration of the signals from two independent experiments, and is presented as the mean 6 SD.
C. Wild-type BY4741 cells and its ptc1 derivative were transformed with the vector (YEp24) or construct carrying the different suppressor
alleles of the BCK1 genes. Two dilutions of the cultures were spotted on YPD plates containing LiCl (100 mM), rapamycin (2.5 ng ml21),
CaCl2 (150 mM), ZnCl2 (4 mM) or caffeine (5 mM). Plates were documented after 2 days of incubation. D) The indicated strains were spotted
on YPD plates containing LiCl (100 mM), rapamycin (5 ng ml21), CaCl2 (200 mM), ZnCl2 (2 mM) or caffeine (5 mM). Photographs were taken
after 2 days of incubation.

et al., 2003). In this regard, the isolation of MIH1 as a
high-copy suppressor in two of our screens (CFW and
high pH) suggests that the overexpression of the phos-
phatase might, up to some extent, override the possible
inhibition caused by Slt2 hyperactivation. The functional
interaction between Mih1 and Ptc1 was reinforced by
our observation that deletion of MIH1 aggravates the
sensitivity of the ptc1 mutant to CFW and calcium ions
(data not shown). Indeed, we observed that deletion of
PTC1 results in strong hyperphosphorylation of Cdc28
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in response to moderate amounts of CFW (Fig. 5B). In
addition, deletion of the negative regulator SWE1 in the
ptc1 background allowed growth in ethanol as only car-
bon source and clearly improved tolerance to lithium
and calcium cations, as well as to CFW (Fig. 5C), with a
less pronounced gain in tolerance to high pH or under
caffeine or ZnCl2 stress (not shown).
CDC37 is another gene required for normal cell cycle
progression. Cdc37 encodes an essential co-chaperone
of Hsp90, although it also has Hsp90-independent
 Ptc1 relieves cell cycle blockage upon cell wall stress 9

Fig. 5. Lack of Ptc1 impacts on G2-M cell cycle transition.

A. The cartoon highlights the role of Cdc28 phosphorylation in G2 to M transition, including the MIH1 suppressor found in this work.
B. Effect of the ptc1 mutation on the phosphorylation state of Cdc28. Wild type BY4741 and MAR143 (ptc1) strains were grown up to
OD600 5 0.6 and exposed to CFW (5 mg ml21) for the indicated times. Samples were processed for immunoblot using anti-phospho-Cdc28
antibodies as indicated in Materials & Methods and in parallel for actin detection.
C. Dilutions of the indicated strains were spotted on YPD plates containing LiCl (75 mM), CaCl2 (150 mM), CFW (2.5 lg ml21) or grown on
ethanol as only C source (YPEt). Pictures were taken after 2 days of incubation.
D. The ptc1 derivative was transformed with the empty plasmid YEp195 or the same plasmid carrying the wild type version of CDC37 (YEp-
CDC37) or a version carrying the carrying the Ser14!Ala mutation (YEp-CDC37S14A). Dilutions of the cultures were spotted on YPD plates
containing LiCl (125 mM), CaCl2 (100 mM), caffeine (5 mM), ZnCl2 (2 mM), CFW (2.5 mg ml21), adjusted to pH 8.0 or on plates containing
ethanol as only carbon source (YPEt).

functions (MacLean and Picard, 2003). Many protein
kinases, including Cdc28, are clients of Cdc37 (Mandal
et al., 2007). Cdc37 can be phosphorylated at Ser14 by
casein kinase II (Bandhakavi et al., 2003; Miyata and
Nishida, 2004), and such phosphorylation is required for
Cdc37 function on multiple kinases, although some
exception has been reported very recently (Millson
et al., 2014). Interestingly, a cdc37S14A mutant shows a
very severe phenotype, likely as result of limiting Cdc28
activity since the cdc37S14A phenotype could be rescued
not only by Cdc28 overexpression but also by loss of
Swe1 (Bandhakavi et al., 2003). We tested if the muta-
tion of Ser14 would abolish the suppressor effect of
overexpression of CDC37 in a Ptc1-deficient strain. As
shown in Fig. 5D, expression of CDC37 counteracts the
sensitivity of the ptc1 mutant to diverse stress condi-
tions, but this beneficial effect is fully lost when Ser14 is
mutated to Ala. Taken together, these results suggest
that Ptc1 could play a positive role in promoting G2-M
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transition and that Cdc28 activity could be limiting in
Ptc1-deficient cells when subjected to certain kind of
stress.
It is known that Mih1 is hyperphosphorylated during
interphase and that this phosphatase undergoes
dephosphorylation during entry into mitosis, which is
dependent on the PP2A-Cdc55 complex (Pal et al.,
2008). It has been also reported that the PP2A-Cdc55
complex is regulated by a pair of redundant proteins,
Zds1 and Zds2, that associate with PP2A-Cdc55 and
target the complex to Mih1 (Queralt and Uhlmann,
2008; Wicky et al., 2011; Yasutis et al., 2010). Remark-
ably, our screen identified not only a PP2A catalytic sub-
unit isoform (PPH22), but also ZDS1 as suppressors of
certain ptc1 defects. While PP2A activity has been
related also to regulation of other phases of the cell
cycle, as far as we know ZDS1 plays a role only in mito-
sis (Queralt and Uhlmann, 2008; Rossio and Yoshida,
2011; Baro et al., 2013). In our recent work we
10 L. Tatjer et al. 䊏

Fig. 6. Rescue of the G2-M cell cycle defect by ZDS1.

A. Exponential cultures of strains BY4741 (WT) or MAR143 (ptc1) transformed with plasmid YEplac195 (YEp-Ø) or the same plasmid bearing
the ZDS1 gene were treated with 5 lg ml21 CFW (or vehicle) for 120 min, or subjected ot pH 8.2 for 150 min, stained with propidium iodide
and processed for flow cytometry to monitor DNA content. Numbers indicate the percentage of cells at G2 and represent the mean 6 SEM
from 4 to 6 samples obtained in three independent experiments. Asterisks symbolize that the difference between the indicated means is
statistically significant (P < 0.005).
B. Supression of Cdc28 hyperphosphorylation by overexpression of ZDS1. The above mentioned strains were treated with 5 lg ml21 CFW for
the indicated times and samples taken to monitor Cdc28 phosphorylation state (Cdc28-P) as in Fig. 5B.

observed the accumulation of cells with duplicated DNA
when a ptc1 strain is subjected to a moderate dose of
CFW (Tatjer et al., 2016). We show here that alkaline
pH treatment also results in accumulation of ptc1 cells
with duplicated DNA content (Fig. 6A). Interestingly, in
both cases overexpression of ZDS1 normalizes consid-
erably the altered pattern of the ptc1 strain (Fig. 6A).
Overexpression of ZDS1 also decreased nearly to wild-
type levels the phosphorylation state of Cdc28 in the
ptc1 strain exposed to a sub-lethal dose of CFW (Fig.
6B). In contrast, overexpression of PPH21 or CDC37
had very little effect on the DNA content profile and the
phosphorylation state of Cdc28 (not shown), suggesting
that the beneficial effects of the expression of these pro-
teins must include additional targets (see below). This
differential effect could be explained considering that
Zds1 has been reported to act also as a negative regu-
lator of the CWI pathway signaling (Griffioen et al.,
2003). Therefore, overexpression of ZDS1 might differ
from the other suppressors in that it would have a dual
effect, both at the level of the Slt2 MAP kinase module
and downstream of it, regulating Cdc28 activity.
As shown in Fig. 7A, deletion of MKK1 in the ptc1
background abolished hyperphosphorylation of Cdc28
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upon CFW treatment, indicating that deregulation of
Cdc28 phosphorylation in the ptc1 mutant is a conse-
quence of the impact of the ptc1 mutation in the activity
of the Slt2 MAPK module at the level of the Mkk1
MAPKK. The involvement of Slt2 in mediating Cdc28
phosphorylation was evidenced expressing the hyperac-
tive BCK1-20 allele, which results in constitutive activa-
tion of the pathway. As shown in Supporting Information
Fig. S2, expression of such allele resulted in marked
Cdc28 phosphorylation in the ptc1 background even in
the absence of additional cell-wall stress (5 lg ml21
CFW) and this phosphorylation was substantially
reduced when MKK1 or SLT2 are mutated.
Phosphorylation of Cdc28 should lead to inhibition of
its kinase activity. We confirmed this point by immuno-
precipitation of the G2/M phase cyclin Clb2 from cells
before and after treatment with CFW (100 min) and
monitoring Clb2-bound Cdc28 activity using Histone H1
as substrate. Because the cell cycle arrest caused by
CFW treatment in the ptc1 mutant results in different
amounts of immunoprecipitated Clb2, we normalized the
data by calculating the ratio of phosphorylated H1 and
immunoprecipitated Clb2 signals for each strain and
condition, and represented the change caused by CFW
 Ptc1 relieves cell cycle blockage upon cell wall stress 11

Fig. 7. Deletion of MKK1 eliminates hyperphosphorylation and inactivation of Cdc28 produced by lack of Ptc1 function.
A. The indicated strains were exposed to 5 lg ml21 CFW for the specified times, extracts prepared and processed for immunoblot as in Fig. 5B.
B. Clb2-associated Cdc28 activity in ptc1 and ptc1 mkk1 strains. V5 epitope-tagged Clb2 was immunoprecipitated from the indicated strains
grown in the presence of 5 lg ml21 CFW for 100 min and the immunoprecipitated kinase activity monitored using histone H1 as substrate. The
mixture was resolved by SDS-PAGE and phospho-histone H1 revealed by ProQ Diamond staining. Aliquots were processed for immunoblot for
Clb2 quantification. H1 specific kinase activity was calculated as the ratio between the histone H1 phosphorylation signal and the amount of Clb2
for CFW-treated and untreated cells. The histogram represents H1 specific kinase activity as the ratio between treated and untreated cells. Data
are mean 6 SEM from three independent experiments. The asterisk denotes statistical significance of the difference between the means
(P < 0.001)

treatment. As shown in Fig. 7B, exposure to CFW
resulted in a drastic reduction of Clb2-associated Cdc28
activity in ptc1 cells when compared with the wild type
(about fivefold), and the additional mutation of MKK1
restored Cdc28 activity to values similar to those
observed in the wild type strain.
Conclusions
Our screen for high-copy suppressors of the growth
defect under three different stress conditions of yeast
cells deficient in Ptc1 activity yielded 25 suppressor
genes that showed different suppressor spectra and
potency when subjected to a comprehensive phenotypic
analysis. It is remarkable that, though the type 2C phos-
phatase family is composed of six additional members
(Ptc2 to Ptc7) we never recovered a PTC gene as ptc1
suppressor. This reinforces the notion that most biologi-
cal properties of Ptc1 are not shared by other members
of the gene family (Gonzalez et al., 2006; Gonzalez
et al., 2009; Sharmin et al., 2014). Although several of
the suppressor genes were able to alleviate many ptc1
related phenotypes (i.e. PPH22, VPS73, VPS70,
CDC37 or the truncated versions of BCK1), none of
them fully reverted the mutant to the wild type pheno-
type, indicating that a single gene function cannot fully
counteract the absence of Ptc1. A possible explanation
would be that Ptc1 has many different direct cellular tar-
gets whose hyperphosphorylation would collectively
result in the myriad of cellular defects caused by the
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mutation of PTC1. However, our finding that diverse
genes functionally related with the CWI pathway (KRE6,
GAS1, PIN4 and catalytically-impaired versions of Bck1,
acting as dominant negative), behave as high-copy sup-
pressors of ptc1 phenotypic defects provides further
support to the notion that the hyperactivation of the Slt2
MAP kinase module could be at the basis of many of
the phenotypes caused by the lack of Ptc1. The link
between Ptc1 and the CWI pathway might even explain
the recovery in the screens of apparently unrelated
genes, such as VPS73, since Vps73 function has been
recently related to Slt2 within the context of vacuolar
function and actin remodeling in S. cerevisiae mutants
affected by endogenous oxidative stress (Pujol-Carrion
et al., 2013). Given the extreme similarity of their pheno-
typic profiles, this link might be extended to VPS70.
Physical interaction between Ptc1 and Bck1, mediated
by the Nbp2 adaptor, was reported some time ago
(Stanger et al., 2012), but evidence for Bck1 being a
substrate for Ptc1 was lacking. Only very recently, our
laboratory has gathered genetic and biochemical evi-
dence specifically pointing to the Mkk1 kinase of the
Slt2 MAPK module as a major Ptc1 target (Tatjer et al.,
2016). Our observation that ptc1 cells accumulate at the
G2-M transition under certain stresses, coincident with
hyperphosphorylated Cdc28, and that mutation of MKK1
normalizes both the phosphorylation state of Cdc28
(Fig. 7A) and the Clb2-associated Cdc28 activity (Fig.
7B) strongly supports the notion that the delayed G2-M
transition observed in ptc1 cells is due to the hyperacti-
vation of the Slt2 pathway as a consequence of the
12 L. Tatjer et al. 䊏

Ptc1 function, would result in delayed G2 to M transition

and contribute to the characteristic growth defect of the
ptc1 strain under many diverse stress conditions.

Experimental procedures
Yeast strains and growth conditions
Yeast cells were grown at 288C in YPD medium (10 g l21 yeast
extract, 20 g l21 peptone, and 20 g l21 dextrose) or, when car-
rying plasmids, in synthetic complete (SC) medium (Adams
et al., 1997) containing 2% glucose and lacking the appropriate
selection requirements. E. coli DH5a cells were used as the
plasmid DNA host and were grown at 378C in LB broth supple-
mented with 50 mg ml21 ampicillin, when required. Bacterial
and yeast cells were transformed using standard methods.
Fig. 8. A model for the involvement of Ptc1 in CWI-mediated cell Standard recombinant DNA techniques were performed as
cycle regulation.
A. The cartoon highlights the proposed link between
described elsewhere (Sambrook and Russell, 2001). Yeast
hyperactivation of the Slt2 MAPK module of the CWI pathway strains used in this work derived from strain BY4741 (Mat a
caused by lack of Ptc1-mediated downregulation and the role of his3D1 leu2D0 met15D0 ura3D0) and are listed in Table 2.
Cdc28 in G2 to M phase transition. Several suppressors reported
in this work, such as PPH22, ZDS1 or MIH1 are indicated. The
reported role of Slt2 in post-translational regulation of Swe1
Gene disruptions and plasmid constructions
(Mizunuma et al., 1998) is omitted for clarity. See main text for
details. Gene disruptions were performed by transforming the 2.0
kb ptc1::nat1 disruption cassette described in (Ruiz et al.,
inability of this strain to properly downregulate the Mkk1 2006) into the following mutants in the BY4741 background:
MAPK kinase. Our results support a model (Fig. 8) in fet3::KanMX4 (to generate AGS62), swe1::KanMX4, to yield
which hyperactivation of the Slt2 module would have strain CCV122, rim15::KanMX4, for RP003, and mih1::-
KanMX4, to produce strain AGS48. Positive clones were
two major consequences. From one side, hyperactivated
selected in the presence of 100 mg ml21 nourseothricin
Slt2 would inhibit Mih1, as proposed in the past (Harri- (Werner BioAgents) and the correct insertion of the cas-
son et al., 2001; Martinez-Anaya et al., 2003), and/or sette was verified by PCR. The YBR241c gene was ampli-
activate Swe1 (Mizunuma et al., 1998). On the other fied by PCR from genomic DNA with oligos YBR241C_UP
side, it would increase intercellular calcium through reg- and YBR241C_DO. The amplification fragment was
ulation of the Mid1-Cch1 plasma-membrane calcium digested with HindIII and XbaI and the 2.0 kbp restriction
fragment cloned into these same sites of the multicopy
transporters, as previously suggested (Bonilla and
plasmid YEplac195. Specific primers used in this work are
Cunningham, 2003), which in turn would activate calci- listed in Supporting Information Table S3.
neurin. This link is supported by our early observation
that deletion of CNB1 fully blocks the increase in
expression of a calcineurin-responsive reporter, Table 2. Yeast strains employed in this work.
observed in ptc1 cells (Gonzalez et al., 2006), and by
Strain Genotype Source
the recent evidence that the same effect is observed by
deletion of CCH1, MID1 or SLT2 (Tatjer et al., 2016). BY4741 MATa his3D1 leu2D0 met15D0 Winzeler et al.
ura3D0 (1999)
Activation of calcineurin would thus lead, through MAR143 BY4741 ptc1::nat1 Ruiz et al. (2006)
diverse mechanisms, to increased Swe1 kinase activity AGS62 BY4741 fet3::KanMX4 ptc1::nat1 This work
(see (Miyakawa and Mizunuma, 2007) and references CCV122 BY4741 swe1::KanMX4 ptc1::nat1 Tatjer et al. (2016)
MAR154 BY4741 slt2::KanMX4 ptc1::nat1 Gonzalez et al.
therein). In this regard, a recent report (Juanes et al., (2006)
2013) supports a model in which the Rim15 protein MAR213 BY4741 bck1::KanMX4 ptc1::nat1 Gonzalez et al.
(2006)
kinase would contribute to activation of PP2A-Cdc55
RP003 BY4741 rim15::KanMX4 ptc1::nat1 Tatjer et al. (2016)
through phosphorylation of Igo1 and Igo2 (Fig. 8). In AGS48 BY4741 mih1::KanMX4 ptc1::nat1 This work
agreement with such model, we have observed that LTR028 BY4741 mkk1::kanMX4 ptc1::nat1 Tatjer et al. (2016)
ASC79 BY4741 CLB2:3xV5 This work
deletion of RIM15 aggravates most ptc1 phenotypes ASC80 MAR143 CLB2:3xV5 This work
(Supporting Information Fig. S3), suggesting that proper ASC82 LTR028 CLB2:3xV5 This work
activation of PP2A is important in cells lacking Ptc1.
All single kanMX4 deletions, not listed here, were from the EURO-
These combined effects, derived from hyperactivation of SCARF collection (Winzeler et al., 1999)
the Slt2 MAPK module as a consequence of lack of
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The Glu179!Ala mutation in Vps73 was carried out as
follows. The VPS73 gene, cloned into YEplac195 (see
Supporting Information Table S2), was used as template
in two parallel reactions using the pairs of oligos
VPS73_E169A_up/M13fw-110, and NEW_VPS73E169A_-
down/M13rev. A mixture of both amplification fragments
plus the YEplac195 vector (digested with SalI and BamHI)
was used to transform yeast cells to allow in vivo recombi-
nation. Similar strategy was used to mutate the Ser14 to
Ala in Cdc37. Yeast cells were transformed with two over-
lapping PCR products obtained with the CDC37_S14A_dup/
M13fw and CDC37_S14A_down/M13rev (using YEplac195-
CDC37 as a template; see Supporting Information Table
S2) plus the PstI/XbaI digested YEplac195 vector. Plasmid
pBCK1-20 is a pRS316-based plasmid carrying the consti-
tutive BCK1-20 allele (Martin et al., 2000).
C-terminal tagging of Clb2 with the V5 epitope was car-
ried out by transforming cells with a 1.4 kbp
CLB223xV5:HIS3MX6 cassette, amplified with oligonucleo-
tides ET164 and ET165 using plasmid E133 as template (a
generous gift from Dr. E. Queralt, IDIBELL, Spain). Putative
positives clones were confirmed by genomic PCR and
immunoblot using anti-V5 antibodies.
Screening for suppressors of ptc1 phenotypes
Strain MAR143 (ptc1) was transformed with a high-copy
plasmid yeast genomic DNA library based on plasmid
YEp24 (a gift from Ramo n Serrano, U. Polite
cnica Valencia,
Spain) or a genomic DNA library based on the plasmid
YEp13 (ATCC, number 37415). Transformants (around
60,000 for CFW and alkaline pH screens, and 30,000
(YEp24) or 20,000 (YEp13) for the rapamycin screen, were
selected for the presence of plasmid in synthetic media
plates lacking uracil or leucine as required. In all cases, the
colonies that appeared after 3 days of incubation were
resuspended in 1 ml of selection medium. The appropriate
dilutions were then dispersed on agar plates (around 1500
colonies/plate) containing 5 or 10 mg ml21 of CFW, adjusted
at alkaline pH with TAPS (8.0 or 8.2) or containing 5 or 10
ng ml21 of rapamycin. Macroscopic colonies observed after
2-3 days of incubation were considered as positive, recov-
ered, and checked for the presence of PTC1-bearing plas-
mids by colony-PCR. Clones that appeared negative for
PTC1-containing insert were given a code number and sub-
jected to further characterization. Plasmid DNA from these
cells was isolated, amplified in bacteria, and subjected to
restriction mapping with EcoRI to identify identical inserts.
Representative clones were introduced again into strain
MAR143 to confirm suppressor capacity. The genomic frag-
ment contained in each clone was identified by DNA
sequencing using specific primers priming to regions
nearby the cloning site of the vector. Identification of the
specific ORF responsible for suppression of the ptc1 phe-
notype was carried out by subcloning each gene into
YEplac195 (Gietz and Sugino, 1988) and testing its ability
to act as a suppressor in comparison with the parental
clone. The subcloning strategies, size and genomic boun-
daries of each gene are listed in Supporting Information
Table S1.
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Ptc1 relieves cell cycle blockage upon cell wall stress 13
Determination of suppressor potencies
The relative potency of the suppressors in relieving different
ptc1 phenotypes was quantified in YPD plates containing a
wide range of distinct compounds or agents: CFW (2.5, 5,
7.5, 10 and 15 mg ml21), caffeine (1, 2.5, 5, 7.5 and
10 mM), alkaline pH (containing 50 mM TAPS adjusted to
7.8, 7.9, 8.0, 8.1 and 8.2), rapamycin (2.5, 5, 7.5, 10 and
15 ng ml21), CaCl2 (50, 100, 150, 200 and 400 mM), LiCl
(50, 75, 100, 150 and 200 mM), ZnCl2 (1, 2, 4 and 6 mM)
and growth on non-fermentable carbon source (YP plates
containing 2% of ethanol). In these experiments, 3 ml of
successive dilutions from saturated cultures (OD660 0.05,
0.01 and 0.002) were spotted on the plates. Growth was
monitored after two days of incubation at 288C. Tolerance
to H2O2 was carried out by liquid growth tests in YPD
media supplemented with 1 mM of H2O2 using 96-well ster-
ile plates. Growth was monitored at 630 nm after 16 h of
incubation at 28 8C using an iEMS Reader MF (Labsys-
tems). For each tested condition a score (1–7) was given
as a function of the growth observed and the intensity of
the stress, following previously reported procedures (Bar-
reto et al., 2011), so a tolerance similar to the ptc1 mutant
received a score of 1, whereas tolerances equivalent to
that of the wild type strain received a score of 6.
Immunodetection of phospho-Slt2, Slt2, and phospho-
Cdc28
For immunodetection of phosphorylated and total Slt2,
strains BY4741 and MAR143 (ptc1) were transformed with
the indicated plasmids and grown until saturation in SC
medium lacking uracil. Then, cells were diluted to an OD600
of 0.2 in the same medium and grown until OD600 of 0.8-1.
Cell cultures (10 ml) were centrifuged at 1500 xg for 3 min
at 48C, the pellet was washed once with cold water and
stored at 2808C. Cell pellets were resuspended in 100 ll of
lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 15%
glycerol, 0.5% Tween-20) containing phosphatase inhibitors
(10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenyl phos-
phate, 10 mM sodium pyrophosphate, and 10 mM b-glycer-
ophosphate), 1 mM phenylmethylsulfonyl fluoride (PMSF),
and EDTA-free protease inhibitor cocktail (Roche). One vol-
ume of glass beads was added, and cells were broken by
vigorous shaking in a Vibrax VXR basic orbital shaker (IKA)
for 20 min at 2500 rpm at 48C. Cell debris was removed by
centrifugation at 500 xg for 3 min at 48C. The protein con-
centration of the lysate was determined by the method of
Bradford (Bio-Rad). Twenty microgram of protein were frac-
tionated by SDS-PAGE in 10% polyacrylamide gels and
transferred to Protran BA85 nitrocellulose membranes (GE
Healthcare). Membranes were incubated overnight with
anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (New
England Biolabs), at 1:2000 dilution or anti-GST-Slt2 anti-
body (Martin et al., 1993) at 1:3000 dilution. Secondary
peroxidase-conjugated anti-rabbit antibody was used at
dilution 1:15,000. Immunocomplexes were visualized using
Super signal West Pico Chemiluminiscent substrate
(Thermo Scientific) and chemiluminescence was detected
using CL-Xposure films (Thermo Scientific).
14 L. Tatjer et al. 䊏

For detection of phospho-Cdc28 the same procedure 2004). Evaluation of DNA content by flow cytometry in pro-
was used except that the primary antibody was an anti- pidium iodine-stained cells was carried out as in (Tatjer
phospho-cdc2 (Tyr15) antibody (Cell Signaling), at 1:1000 et al., 2016).
dilution. Actin levels were monitored as loading and transfer
control for immunoblots by mean of an anti-actin antibody
(MAB1501, Millipore) at 1:5000 dilution. Acknowledgements
We acknowledge Montserrat Robledo for excellent technical
assistance and Amparo Ruiz and Raquel Serrano for their
Determination of Clb2-immunoprecipitated Cdk28
contribution to the initial steps of this work. We thank Ethel
activity
Queralt (IDIBELL, Barcelona, Spain) for her unconditional
Clb2-tagged yeast strains were grown in 125 ml cultures until support (advice and reagents) in developing the determina-
OD60050.6–0.8. Cells were harvested by centrifugation (1500 tion of Cdc28 activity. Supported by grants BFU2011-30197-
xg, 10 min at 48C), washed with water and transferred to C3-01 and BFU2014-54591-C2-1-P to JA (Ministerio de
screw cap Eppendorf tubes. Cells were spun down for 3 min Economıa y Competitividad, Spain and FEDER). JA was
at 2000 rpm, and the supernatant removed. Cells were resus- recipient of an ‘Ajut 2014SGR-4’ (Generalitat de Catalunya)
pended in 450 ll of EBX buffer (50 mM HEPES/KOH pH 7.5, and an ICREA Academia 2009 Award (Generalitat de Catalu-
100 mM KCl, 2.5 mM MgCl2, 0.25% Triton X-100, 1 mM nya). L. Tatjer was supported by a predoctoral fellowship from
DTT) supplemented with protease inhibitors (Protease inhibi- the Ministry of Science and Innovation, Spain.
tor cocktail set III (Calbiochem), Complete EDTA-free tablet The authors have no conflicts of interest to declare.
(Roche), plus 1 mM PMSF) and phosphatase inhibitors
(50 mM NaF, 10 mM b-glicerophosphate, PhosStop tablets
(Roche)). Three-hundred ml of zirconia beads was then
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