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Phylogenetic origin and transcriptional

regulation at the post-diauxic phase of SPI1, in
Saccharomyces cerevisiae
ARTICLE in CELLULAR & MOLECULAR BIOLOGY LETTERS MAY 2012
Impact Factor: 1.59 DOI: 10.2478/s11658-012-0017-4 Source: PubMed

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PHYLOGENETIC ORIGIN AND TRANSCRIPTIONAL REGULATION

AT THE POST-DIAUXIC PHASE OF SPI1 IN Saccharomyces cerevisiae
FERNANDO CARDONA1,2, MARCEL.L DEL OLMO2
and AGUSTN ARANDA1
1Departamento

de Biotecnologa, Instituto de Agroqumica y Tecnologa

de Alimentos, CSIC, Paterna, Spain, 2Departament de Bioqumica i Biologia
Molecular, Universitat de Valncia, Burjassot, Spain

Abstract: The gene SPI1 of Saccharomyces cerevisiae encodes a cell wall

protein that is induced in several stress conditions, particularly in the
postdiauxic
and stationary phases of growth. It has a paralogue, SED1, which shows
some common features in expression regulation and in the null mutant
phenotype. In this work we have identified homologues in other species of
yeasts and filamentous fungi, and we have also elucidated some aspects of the
origin of SPI1 by duplication and diversification of SED1. In terms of
regulation, we have found that the expression in the post-diauxic phase is
regulated by genes related to the PKA pathway and stress response (MSN2/4,
YAK1, POP2, SOK2, PHD1 and PHO84) and by genes involved in the PKC
pathway (WSC2, PKC1 and MPK1).
Key words: SPI1, Phylogenetic origin,
diauxic,Nutrient starvation, PKA, PKC

Transcriptional

regulation,

Post-

INTRODUCTION
There is a clear relationship between the nutritional state of the cell and
its ability to resist stress conditions. When nutrients are available, a high
activity of the Ras/adenylate cyclase/protein kinase A (PKA) pathway
determines growth stimulation and cell division, as well as repression of
stress, respiration, the protein kinase C (PKC) pathway and autophagy related
genes [1]. Depletion of any essential nutrient stops the cell cycle,
activates the stress response, and cells enter the stationary phase; when all
the essential nutrients are available again, cells exit this phase [2]. These
changes are regulated, at least partially, by PKA pathway and cyclindependent kinases Pho80/85p, which control the entry into the stationary
phase, activating the transcription of genes characteristic of this phase via
several transcription factors, such as Msn2/4p [3].
S. cerevisiae gene SPI1 is a good example of a stress-response gene. It encodes
a
serine/threonine-rich
protein
anchored
to
the
cell
wall
by
glycophosphatidylinositol (GPI) [4]. It has been shown that it is important
in resistance to herbicides, wall lytic enzymes, food preservatives and weak
lipophilic acids [5, 6]; besides, its overexpression causes pseudohyphal
growth [7]. Its expression is induced in several adverse conditions,
particularly under oxidative, heat, ethanol,acetaldehyde and hyperosmotic
stresses, lack of nitrogen and amino acids, and acidic or basic pH [8, 9],
being particularly high during diauxic change, the stationary phase and

nutrient starvation in laboratory and wine-making conditions [10, 11]. Its

strong (and almost exclusive) expression under stress conditions has been
used successfully to express stress response genes in the later stages of
wine-making [12].
Some data on the transcriptional regulation of this gene are known. The
induction by glucose starvation is partially dependent on Msn2p/Msn4p [10].
Expression in the post-diauxic phase is regulated by Sok2p and the ubiquitin
ligase Rsp5p [11], as well as the phosphatidylinositol-4-phosphate kinase
Mss4p and the flavodoxin-like proteins Rfs1p and Ycp4p [13].
The Spi1p homologous protein with the highest degree of similarity in
S. cerevisiae is Sed1p. It is also a serine/threonine-rich protein anchored by
GPI to the cell wall, and has important functions in cell wall structure and
biogenesis [14]. It is also induced in nutrient starvation and the stationary
phase,being the major protein of the cell wall in these conditions. The
sed1
mutant is sensitive to lytic enzymes and oxidative stress and the gene
is induced under stress conditions in a PKC-dependent manner [14].
In this work we have studied the phylogenetic origin of Spi1p and the role
some transduction pathways in its expression, concluding that SPI1 was
originated by duplication and diversification of SED1. In terms
regulation,expression in the post-diauxic phase of SPI1 is controlled
genes related to the PKA pathway and stress response and by genes involved
the PKC pathway.

of
of
by
in

MATERIALS AND METHODS

Yeast strains, plasmids and growth conditions
The yeast strains and plasmids used in this work are listed in Supplementary
Tables 1A and B respectively in Supplementary material at http://dx.doi.org/
10.2478/s11658-012-0017-4. For yeast growth the following media were used:
yeast extract peptone dextrose (YPD) medium (1% (w/v) yeast extract, 2% (w/v)
peptone, 2% (w/v) glucose); synthetic defined (SD) medium (0.17% (w/v) yeast
nitrogen base without amino acids and ammonium sulphate, 0.5% (w/v)
ammonium sulphate, 2% (w/v) glucose) supplemented with the required amino
acids; S (SD without glucose). Cultures were incubated at 30C with shaking,
unless a different temperature is indicated. Solid plates contained in
addition 2% (w/v) agar.
Gene expression analysis
The SPI1p/lacZ fusion expression was determined as
-galactosidase activity
in liquid medium via the method of permeabilized cells, using orthonitrophenyl- - galactoside (ONPG) as substrate, as described [11]. RNA
isolation, quantification and analysis by Northern blot analysis were
performed as previously described [11].
Phylogenetic studies
To search for homologous sequences the protein-protein BLAST tool of NCBI

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) with default settings was employed.

The sequences obtained were aligned using Clustal 2.0 [15] and Probcons [16].
The alignments obtained are shown in Supplementary Figs 1 and 2
(http://dx.doi.org/10.2478/s11658-012-0017-4). For the phylogenetic analysis,
we used the alignment obtained with Clustal 2.0 (Suppl. Fig. 1) edited with
Gblocks 0.91b [17]. Preliminary phylogenetic trees were obtained using MEGA 4
[18]. The final tree was obtained using MrBayes 3.1.2 [19], using the
parameters detailed in the supplementary material.
Identification of domains, motifs and repetitions
For these analyses we used the resources available at ExPASy
(http://expasy.org/tools/)
and
Conserved
Domains
(CDD)
of
NCBI
(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).
The
search
for
structural
homologues
was
made
using
Phyre
0.2
(http://www.sbg.bio.ic.ac.uk/~phyre/)
and
3D-BLAST
(http://3dblast.life.nctu.edu.tw/dbsas.php).
Bioinformatics analysis
For structural modelling we used the resources available at ExPASy. We
obtained similar results with several tools for secondary structure (only
results obtained from PSIPRED are shown). For homology modelling, no adequate
structural templates were found. Threading structural modelling with
acceptable
results
was
performed
using
the
LOMETS
meta-server
(http://zhanglab.ccmb.med.umich.edu/LOMETS/). The structures generated were
evaluated using the tools available in SWISSMODEL.
The models and the quality assessment data were deposited in the
Protein Model Database (http://mi.caspur.it/PMDB/) to make them publicly
available (PMDB IDs: Spi1p PM0077324; Sed1p PM0077325).
Statistical analysis of the results
The statistical significance of the numerical results was performed using a
onetailed Student s t test with 2 degrees of freedom (3 samples) available in
Excel (Microsoft Office package). Data were considered significant if the pvalue was 0.05 or lower, and the statistic t does not exceed the limits for
considering the test as correct.
RESULTS
Phylogenetic study
To perform the phylogenetic study, we searched for homologous proteins by
means of the BLAST tool of NCBI using the reference sequence database. To
root the tree we used species belonging to the genera Pichia and Yarrowia as
outgroups; they are frequently considered in this way because they are the
most distant from S. cerevisiae among the yeast species commonly studied [20].

Fig. 1. Phylogenetic relationships between Spi1p and homologues in fungi. A

Phylogenetic tree of Sed1p and Spi1p obtained using MrBayes. In the tree are shown
the posterior probabilities obtained by Bayesian statistics (MrBayes) and the
bootstrap values (500 replicates) obtained using the Neighbour Joining method with a
JTT matrix (Mega 4.0) for each branch. Also protein ID (NCBI) and the abbreviated
name of the species are shown (G: Gibberella, N: Neurospora, Ss: Sclerotinia, M:
Magnaporthe, B: Botryotinia, C: Candida, Sc: Saccharomyces, A: Ashbya and K:
Kluyveromyces). B
Edited alignment used for the performance of the phylogenetic
tree shown in A.

According to the phylogenetic tree obtained (Fig. 1A) by means of the

sequence alignment performed (Fig. 1B and Suppl. Fig. 1), there are Spi1p
orthologues in at least three species of yeast: Kluyveromyces lactis, Candida
glabrata and Ashbya gossypii; and five filamentous fungi: Sclerotinia sclerotiorum,
Botryotinia fuckeliana, Magnaporthe grisea, Neurospora crassa and Gibberella
zeae, among the sequenced organisms. Moreover, as expected [4], Spi1p has one
paralogue in S. cerevisiae, Sed1p. Also C. glabrata presents two paralogues of
the protein.
Bioinformatics analysis of Spi1p and Sed1p domains and structure
Although we did not find conserved functional domains with acceptable
probabilities, small similarity (e-value
3) was found in the conserved
sequence (amino acids 17 to 52 in Fig. 1B) with PRK08383 (E subunit of
H/monovalent
cation
antiporter),
PRK11512
(putrescine-2-oxoglutarate
aminotransferase) and DM13 (protein of unknown function present in Drosophila
melanogaster and Caenorhabditis elegans). The prediction of potential sites of
post-translational modifications is shown on the alignment of Sed1p and Spi1p
(Suppl. Fig. 5).
The N-terminal glycosylation has already been tested [14], as well as the
phosphorylation [29], and there are several motifs that can potentially be
a substrate of these post-translational modifications. There are also some

Fig. 2. Tertiary structure prediction obtained by threading using LOMETS meta-server.

A
Three-dimensional model obtained for Spi1p. The structure shows the
-sheet
barrels forming Ig-like domains. -sheets and coiled-coil regions are dark-coloured,
whereas
-loops are light-coloured. Three views of the protein are shown, referring
the turn of the axis to the first view. B
Three-dimensional model obtained for
Sed1p. The structure shows a threelobular structure formed by Ig-like domains.
Helices are shown in yellow and coiled-coil regions in green. The results show that
it is also composed of three lobes of -sheet barrels like those that form Spi1p.

interesting protein-binding motifs. There is one for AP (adaptor protein),

typical of the endocytic route, very common in membrane or cell wall
proteins, especially those involved in transport. The structural models
obtained for Spi1p (Fig. 2A and Suppl. Fig. 6) show that the conserved
sequences shown in Fig. 1B (TTFVT and TTLTITNCP) form
-sheets that are part
of a domain similar to the eight-stranded anti-parallel
-sheet barrel (Iglike domain), typical of porins, adhesins and other cell wall or outermembrane proteins that bind hydrophobic ligands. Sed1p (Fig. 2B) is composed
of three lobes of
-sheet barrels like those that form Spi1p, showing that
these proteins also have related three-dimensional structure. The search for
structural homologues shows that there are similar
-sheet barrels in solved
structures, such as 2nnc (sulphur carrier protein from sulphur bacterium),
1st8 (fructan 1-exohydrolase from plants) and 3por (calcium porin from nonsulphur photosynthetic bacterium). These results point to a channel and/or
enzymatic function of this protein in the yeast cell wall. The search for
structural homologues for Sed1p also points to bacterial adhesins and

surface antigens as the closest structures. It is also remarkable that the

alignment of Spi1p and Sed1p (Suppl. Fig. 5) shows that cysteines forming
disulfide bonds that maintain the Ig-like domain (see models deposited in
PMDB) are often conserved in both proteins.
Study of the expression of SPI1 during growth and its involvement in the response to
nutrient starvation
To gain a global view on SPI1 transcriptional regulation we performed a
search for transcription factors for which a direct or indirect role in the
regulation of SPI1 has been described (Suppl. Table 2A). Notably, using
transcription factors listed in this table, over-represented gene ontology
terms were found for stimulus and nutrient response. Suppl. Table 2B shows
the transcription factors which have binding consensus sequences in the
promoter of SPI1. Although for some of them there is evidence indicating that
they participate in the regulation of SPI1 in other conditions (underlined;
Crz1p, Gis1p, Hsf1p, Mcm1p, Msn2/4p, Sko1p, Ste12p and Yap1p), no studies
have been carried out in post-diauxic conditions until now. Besides, in most
cases there are no data about its involvement in the transcriptional
regulation of this gene, so it remains to be demonstrated that the putative
binding sequence is functional. In this case, many of the transcription
factors included in this table are involved in filamentous growth and the
stress response. It is remarkable that in both cases (Suppl. Tables 1 and 2),
the most over-represented classes (adjusted p-value < 0.001) were the same
(nitrogen compound metabolic process, ion-binding and nucleic acid
metabolism),indicating the importance of these transcription factors in these
processes.
The expression of SPI1 during growth was studied by Northern blot in the
strain BY4742 (Fig. 3A). The expression is increased up to 3 days, its
maximum level being found between 1 and 1.5 days (coinciding with the diauxic
shift), then decreased and maintained for 10 days. No expression was detected
with the method used after 10 days.
The spi1
mutant presents a sensitive phenotype in starvation conditions. As
shown in Fig. 3B, it has significantly lower viability than the wild type
strain at 20 days in nutrient deficient medium (transferring the cells from
YPD medium in exponential growth to S medium with no glucose), although these
differences could also be explained by previously described variations in
viability in rich medium after the diauxic shift [11].

Fig. 3. Study of SPI1 expression during the growth and its role in the response to
nutrient starvation. A
SPI1 expression in BY4742 was analysed by Northern blot. We
present the quantification and the standard deviation of the data obtained with three
different cultures. B
Loss of viability in starvation medium (S medium) in wild
type strain (represented by squares and continuous line) and spi1 (diamonds and
discontinuous line). Counts were made by plate count in YPD plates and are presented
as N/No [(cfu/ml)/ initial (cfu/ml)] to cancel the effects due to different initial
cfu/mL number. C
Comparison of
-galactosidase activity of SPI1p/lacZ fusion
(squares) and Northern blot of SPI1 mRNA (diamonds, discontinuous line) during growth
in the strain BY4742. D
Same as C but for YPH499 strain. The data were multiplied
by the factor needed to be represented in the same scale. For Northern blot analyses
shown in this figure, data were obtained by normalization to rRNA. Statistically
significant differences are marked with asterisks ( p-value < 0.05, p-value <
0.005).

Verification of the appropriateness of SPI1p/lacZ fusion and -galactosidase activity as a

simple method to analyse expression of SPI1
In order to use the
-galactosidase activity generated by the SPI1p/lacZ
fusion as a reporter of SPI1 expression, YPH499 and BY4742 strains were
transformed with the fusion and the expression was measured in parallel by
-galactosidase activity and Northern blot during growth (Figs. 3C and D).
It is noted that in early stages, until 32-48 hours depending on the strain,
the results obtained using the SPI1p/lacZ fusion are comparable to those
obtained by Northern blot. In contrast, for longer times, the expression
detected by Northern blot decays, while -galactosidase activity remains.
However, no significant differences were found between mRNA levels of the
lacZ gene expressed under the control of SPI1 promoter and mRNA levels of SPI1
(Suppl. Fig. 3A). For both strains, the maximum -galactosidase activity is
approximately at 80 hours, while the highest mRNA level occurs at about 40
hours. This could be explained by enzyme accumulation due to its higher

stability compared to SPI1 mRNA, while differences in SPI1 mRNA between

strains could be due to strain-specific differences in transcriptional
regulation. These differences among the strains could explain different
vitality, so BY4742, which shows higher expression of SPI1 at late phases,
also grows to reach higher OD and during a longer time than YPH499 (Suppl.
Fig. 3B).
Searching for transduction pathways that regulate SPI1 transcription in post-diauxic
phase
We studied the role of different transduction pathways and transcription
factors in the regulation of SPI1 transcription during the post-diauxic
phase. These analyses were made using some mutant strains, including some
genes that were described as regulators of SPI1 by direct or indirect
evidence in other conditions, as well as genes that encode transcription
factors with binding consensus sequences in the SPI1 promoter, according to
the data shown in Suppl. Table 2. The results indicate that some of the
selected transcription factors, such as Pho4p, Crz1p, Mig1p, Yap1p, Msn1p and
Sko1p, have no relevant effect on the expression of SPI1 at the post-diauxic
phase (Suppl. Fig. 4), although they have been related to the regulation of
SPI1 expression in other conditions (Suppl. Table 2A). We also studied the
effect of mutants in some genes that encode proteins involved in signal
transduction and other functions related to gene expression, but no effect
was detected in the deletion strains of PHO85, CNB1, SST2, MSN5, PUF5 and
HOG1 (Suppl. Fig. 4). However, an effect was found in some deletion or
thermosensitive mutants in genes related to PKA/stress and PKC pathways, as
is shown below.
The PKA/stress pathway has a role in the regulation of SPI1 transcription at the post-diauxic
phase
The expression of SPI1 at the post-diauxic phase decreases in msn2msn4
strain compared to the wild type (Fig. 4A). According to this, the
transcription factors Msn2/4p are involved in the transcriptional regulation
of this gene, as expected according to previous studies [10].
A regulatory effect on transcription of SPI1 was found for Yak1p (Fig. 4A)
and the related protein Pop2p (Fig. 4B), involved in the PKA pathway and
response to glucose depletion [20]. Also pho84 (Fig. 4C) mutation has an
effect on this expression, suggesting a role of phosphate starvation as a
sensor for the induction of the gene by this pathway.

Fig. 4. Effect of several mutants in the expression of SPI1 during exponential [C]
and postdiauxic phase [PD]. A
Study by Northern blot in deletion strains in the
MSN2/MSN4 and YAK1 genes, in W303-1a strain grown in YPD. B
Study by Northern
blot of the effect of POP2 gene deletion in BY4742 strain in YPD. C
Study of the
effect of deletions in WSC2 and PHO84 in BY4741 strain in SC-URA using SPI1p/lacZ
fusion. D
Study of the effect of deletions in SOK2 and PHD1 in MLY40 strain in
SC-URA using SPI1p/lacZ fusion. E
Effect of mutations in PKC pathway genes in SPI1
expression. Northern blot after 0, 6 and 9 hours at 37C in W303-1a and the

thermosensitive mutant pkc1ts and its quantification are shown. F

Effect of the
overexpression of PKC pathway genes in SPI1 expression. Quantification in W303-1a
containing multicopy plasmids with indicated genes and Northern blot spots are shown.
Statistically significant differences are marked with asterisks ( p-value < 0.05,
p-value < 0.005).

The transcriptional factor Sok2p and its antagonist Phd1p, also related to
the PKA pathway, regulate the expression of SPI1 in these conditions (Fig.
4D). In the mutant sok2, induced (post-diauxic) and basal (exponential
phase) expression is lower, as we previously described [11], whereas in the
mutant phd1 the mRNA levels are higher than in the wild type strain, under
both control and post-diauxic conditions (Fig. 4D).
The PKC pathway has a role in the regulation of SPI1 transcription at the postdiauxic phase
Since Spi1p is a cell wall protein, we also studied the involvement of the
PKC pathway in the expression of SPI1. In the thermosensitive mutant pkc1ts,
SPI1 expression decreases in post-diauxic phase cultures changed from 30 to
37C in rich medium (Fig. 4E), although the differences are not
statistically significant.
Also wsc2 shows significantly lower induction of SPI1 in post-diauxic
conditions (Fig. 4C), pointing to activation via the canonical transduction
pathway of PKC, which starts in this sensor protein. However, in mpk1 the
induction was not affected in the post-diauxic phase (Suppl. Fig. 4),
supporting the results of other authors who proposed that there is a bypass
on the pathway at this level [22]. We also studied the expression in yeast
transformed with multicopy plasmid containing MPK1 and PKC1. The
overexpression of both genes increases the SPI1 expression in a statistically
significant manner, both under control and post-diauxic conditions (Fig. 4D).
DISCUSSION
The results obtained from the phylogenetic analysis suggest that Sed1p
duplication and subsequent differentiation of one copy could be the origin of
Spi1p. Homologues of Sed1p and Spi1p are present in yeasts only in the
subphylum Saccharomycotina/class Saccharomycetes/order Saccharomycetales,
whereas in filamentous fungi they are present in the subphylum Pezizomycotina,
classes Leotiomycetes (B. fuckeliana and S. sclerotiorum) and Sordariomycetes
(G. zeae, N. crassa and M. grisea), but they are not found in Eurotiomycetes
class (including other sequenced model organisms belonging to Aspergillus
genus) or throughout the subphylum Taphrinomycotina (which includes other
sequenced model organisms such as Schizosaccharomyces pombe and Pneumocystis
carini). Based on the phylogenetic relationship of Ascomycota proposed by
Scannell et al. [23], these results suggest that Sed1p and orthologous genes
appear in the evolution of the subphylum Pezizomycotina when Eurotiomycetes
diverge from Sordariomycetes and Leotiomycetes, and are maintained later on
(Leotiomycetes, Sordariomycetes and Saccharomycetales) or duplicate (Saccharomyces
sensu stricto), explaining the lack of these genes in Eurotiomycetes and

Taphrinomycotina. It is interesting to note the presence of a conserved

sequence of amino acids (TLTITDCPC, amino acids in positions 235-244 in the
alignment) in these proteins (Fig. 1B). The protein of A. gossypii and Sed1p
have two repetitions of this sequence (Suppl. Fig. 1, amino acids in
positions 332-341 and 483-492 in the alignment), which could indicate that
these proteins have a common ancestor with Spi1p that would have originated
by duplication and specialization in S. cerevisiae, after losing one copy of
this sequence. The conservation of these proteins (even duplicated) in the
mentioned species points to an important function, for instance in stress
resistance, as has been demonstrated [11, 14]. In S. cerevisiae, gene expression
changes at the diauxic shift and the stationary phase have been studied by
genome-wide analyses [24, 25]. These works clearly showed the importance of
protein biosynthesis, as well as carbohydrate and amino acid metabolism, in
these conditions. In this sense, our group has reported the relation of Spi1p
regulation with these pathways [11, 13]. In other yeasts, the homologue
proteins have also been related to nutrient limitation. In N. crassa,
Spi1p/Sed1p homologue (XP960686, named ccg6) is light-activated and
development-related [26], and the expression of the gene is induced in
glucose-limitation conditions, in medium with acetate as a carbon source
[27]. In K. lactis it would be interesting to analyse the expression of the
hypothetical protein XP453996 in post-diauxic conditions, to investigate the
relationship of this protein with the changes that occur in this shift.
Sed1p duplication could have happened during the total genome duplication
proposed by Wolfe in 1997 [28], so it would be present only in S. cerevisiae and
C. glabrata, and the subsequent gene loss or specialization would have not been
equal in both species, so the divergence may be different. Subsequent
specialization of one copy probably originated Spi1p in S. cerevisiae, which may
explain the partially coincident phenotypes of null mutants and regulation
pathways in Sed1p and Spi1p, even more important considering that the
conserved domain described in this work is present in both proteins, Sed1p
(repeated) and Spi1p (single) (Suppl. Fig. 5).
There is controversy over what yeast genus (Candida or Kluyveromyces) is
phylogenetically closer to Saccharomyces [20, 23, 30]. The tree obtained for
Spi1p supports the theory proposed by Wolfe in 1997 [24], given that
C. glabrata presents two sequences homologous to Spi1p and Sed1p, while
C. albicans and K. lactis have only one. Thus, the results of this phylogenetic
study point to a Sed1p gene duplication and diversification as the origin of
Spi1p. This diversification would imply the loss of part of the sequence of
Sed1p, including one repetition of the conserved motif TLTITDCPC, which is
not lost in A. gossypii.
In this work we have analysed the involvement of several transcription
factors in the regulation of the SPI1 gene at the post-diauxic phase. Sok2p
and Pdh1p, two transcription factors involved in pseudohyphal growth
regulation, are involved in SPI1 transcriptional regulation. In the mutant
sok2, -galactosidase activity is decreased in the post-diauxic phase,
whereas in phd1 it is increased (Fig. 4D).
This result fits with the model proposed by Pan and Heitman [31] that
suggests antagonist effects of these factors; Sok2p inhibits PHD1

transcription, which induces FLO11 (a glycoprotein anchored by GPI) and

triggers pseudohyphal invasive growth. In the case of SPI1, Phd1p would act
as a transcriptional repressor to fit this model. In addition, SOK2 is
strongly induced under heat shock, osmotic stress and the stationary phase,
and PHD1 is activated under osmotic stress and nitrogen starvation [32],
pointing to the involvement of these transcription factors in the stress
response. Although pho85 mutation has no effect in SPI1 induction in these
conditions (Suppl. Fig. 4), pho84 has an evident effect (Fig. 4C),
suggesting a role of inorganic phosphate signalling in the induction of the
gene. This phosphate transporter has also been related to PKA pathway
activation [33], suggesting an explanation for its role in SPI1 induction.
Bioinformatics analysis of transcription factors which regulate SPI1
transcription by gene ontology (Suppl. Table 2) shows the following as
enriched terms: nitrogen compound metabolic process, ion binding, nucleic
acid metabolism, stimulus response, glucose metabolism regulation, nutrient
response, filamentous growth and stress transcriptional response. These
results are also consistent with the phenotype observed (stress and nutrient
starvation sensitivity) and together with the putative function proposed by
the bioinformatics analysis open new fields that could be explored with these
proteins, as transport or signal transduction.
In this work, a connection has been established between the expression of
SPI1 in the post-diauxic phase and the PKC pathway, confirming the
relationship between this gene and the cell integrity pathway. Plasmids
containing MPK1 and PKC1 show greater SPI1 induction (Fig. 4F), and the
WSC2 (sensor where the pathway begins) deletion has significantly smaller
induction (Fig. 4C). However, in the mpk1 strain (last kinase in the
pathway) no altered expression was detected (Suppl. Fig. 4), so other
proteins at the end of the pathway may be responsible for this
transcriptional activation. Some proteins that have been described with a
partially redundant function with Mpk1p are Rpi1p [34] and Mlp1p [35].
According to these results, SPI1 expression is regulated by two major cell
pathways: PKA, which regulates the stress response and cell growth, and PKC,
which regulates cell integrity and cell wall metabolism and biogenesis. These
pathways are the key to control of global cellular physiology, regulating the
expression of a large number of genes in different conditions, and are
probably controlled together by TOR kinase, also involved in the response to
nutrient and environmental conditions. Thus, it seems clear that the
transcription of this gene is regulated in a manner dependent on the
availability of nutrients and cell growth, consistent with its expression
pattern and the phenotype of the null mutant in starvation conditions (Fig.
3B).
Acknowledgements. The authors thank Vicente Herranz-Prez for the critical
review of this manuscript. This work has been supported by grants AGL200500508 and AGL2008-00060 from the
Ministerio de Educacin y Ciencia
to
M.O. and A.A. respectively and GRUPOS03/012 from the
Generalitat
Valenciana
to both; F.C. was a F.P.U. fellow of the
Ministerio de
Educacin .

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SUPPLEMENTARY METHODS

Identification of domains, motives and repetitions

For

these

analyses

(http://expasy.org/tools/)

we

and

used

the

Conserved

resources

available

Domains

(CDD)

at

ExPASy

of

NCBI

(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Structural homologues search

was made using Phyre 0.2 (http://www.sbg.bio.ic.ac.uk/~phyre/) and 3D-BLAST
(http://3d-blast.life.nctu.edu.tw/dbsas.php). We did not found conserved functional
domains with acceptable probabilities using CDD, but small similarity (e-value 3, data
not shown) was found in conserved sequence (amino acids form 15 to 52 in Figure 1b)
with PRK08383 (E subunit of H+/monovalent cation antiporter), PRK11512
(putresceine-2-oxoglutarate aminotransferase) and DM13 (protein of unknown function
present in Drosophila melanogaster and Caenorhabditis elegans).

Structural predictions
For structural modelling we used the resources available at ExPASy. We
obtained similar results with several tools for secondary structure (only results obtained
from PSIPRED are showed). For homology modelling, no adequate structural templates
found.
Threading structural modelling with acceptable results was made using
LOMETS meta-server (http://zhanglab.ccmb.med.umich.edu/LOMETS/). The
structures generated were evaluated using the tools available in SWISS-MODEL. The
models and the quality assessment data were deposited in Protein Model Database
(http://mi.caspur.it/PMDB/) to make them publicly available (PMDB IDs: Spi1p
PM0077324; Sed1p PM0077325).

Details of the phylogenetic analysis with MrBayes

Supp. Fig. 1a. Unedited alignment of amino acid sequences used for the phylogenetic study. The alignment was made
using ClustalX 2.0. The conserved motif repeated twice in A. gossypii protein and in Sed1p is squared.

Supp. Fig. 1b. Unedited alignment of amino acid sequences using Probcons. The conserved motif is squared. Note
that the results are similar to those obtained used with Clustal 2.0, at least for the sequence used for the phylogenetic
analysis (Fig. 1).

mRNA relative expression

(a)

40
35
30
25
SPI1

20

lacZ

15
10
5
0
0

10

20

30

40

50

60

70

t (h)

(b)
30
25

OD600

20

BY4742_SD
YPH499_SD

15

BY4742_YPD
YPH499_YPD

10
5
0
0

20

40

60

80

100

120

t (h)
Supp. Fig. 2. (a) Comparison of RNAm expression by northern blot of SPI1 and lacZ controlled by SPI1 promoter in
the strain YPH499. (b) Comparison of the growth (measured as 600 nm optical density) of BY4742 and YPH499
strains in rich (YPD) and minimal media (SD). Strains and media are indicated in the legend.

30,00

20,00
15,00
10,00
5,00

W303-1a

MCY829

hog1

sko1

pho4

pho85

sst2

WT

mig1

msn5

msn1

WT

yap1

puf5

mpk1

crz1

cnb1

0,00

WT

SPI1 mRNA

25,00

BY4742

Supp. Fig. 3. Mutant strains tested without effect in SPI1 induction in post-diauxic conditions. SPI1 expression
analysed by northern blot is showed after 8h growing in YPD from DO600= 0.3.

MLSNAKLLLSLAMASTALGLVSNSSSSVIVVPSSDATIAGNDTATPAPEPSSAAPIFYNSTATAT
QYEVVSEFTTYCPEPTTFVTNGATFTVTAPTTLTITNCPCTIEKPTSETSVSSTHDVETNSNAAN
ARAIPGALGLAGAVMMLL
b
Spi1p

Sed1p

WW4

USP7

AP
FHA

FHA

FHA

Supp. Fig. 4. (a) Amino acid sequence of Spi1p and domains found by SMART. Domains are squared by the
following colours: Red, signal peptide; pink, low complexity areas; blue, intrinsic disorder; RPT (blue), internal
repeats. (b) Distribution of domains found by SMART in Sed1p and Spi1p. The same colour code than in (a) is used.
(c) Alignment of the amino acid sequence of Spi1p and its homolog Sed1p. It is shown in pink the serine-threonine
rich domain, characteristic of these proteins and potential substrate of serine-threonine kinases. For Sed1p it is shown
in blue the repeated domain, present only once in Spi1p (RPT in B). It is also shown squared the motifs recognized as
domains (indicated by the name) or substrate of post-translational modifications. Black continued: N-miristoilation;
red dashed: phosphorylation; green dashed: N-glycosylation; FHA: phosphothreonine union; WW4: phosphorylationdependent interaction domain; USP7: binding domain; AP: interaction with AP (Adaptor Protein; typical of the
endocytic route, very common in membrane or cell wall proteins, especially those involved in transport).

Supp. Fig. 5. Secondary structure prediction of Spi1p. c-helices are shown as green cylinders (H above the amino
acid sequence) and d-sheets as yellow arrows (E above the amino acid sequence). The probability is shown as blue
bars over the prediction. The results show that in Spi1p there are 9 putative regions adopting a d-sheet structure, four
of them dubious due to their small size and/or their low probability. The first one corresponds to the signal
peptide, the fourth includes one conserved region (VVSeFTTYCP; Figure 1b), fifth and sixth are also within one
conserved domain (TTFVT-TFTVT; Figure 1b). The seventh is the most conserved (TTLTITNCP), and probably is
important in the function of the protein. Probably these motifs, due to its evolutionary conservation, are part of the
secondary structure and play a central role in the function of protein. An g-helix is also predicted at the C-terminus of
the protein, although with low probability.

y=90

x=90

Supp. Fig. 6. Tertiary structure prediction obtained for Spi1p by threading using LOMETs meta-server. The structure
shows the -sheet barrels forming Ig-like domains. -sheets and coiled coil regions are dark-coloured, whereas gloops are light-coloured. Three views of the protein are showed, referring the turn of the axis to the first view. The
results show that the protein folds in a domain similar to the eight-stranded anti-parallel d-sheets barrel (Ig-like
domain), typical of porins, adhesins and other cell wall or outer-membrane proteins that bind hydrophobic ligands.
The search of structural homologues using 3D-BLAST tool shows that there are similar d-sheets barrels in solved
structures, as 2nnc (sulphur carrier protein from sulphur bacterium; identity = 40.7%), 1st8 (fructan 1-exohydrolase
from plants; identity = 34.3%) and 3por (calcium porin from non-sulphur photosynthetic bacterium, identity =
31.6%). These results point to a channel and/or enzymatic function of this protein in the yeast cell wall.

x=90

y=90

Supp. Fig. 7. Tertiary structure prediction obtained for Sed1p by threading using LOMETs meta-server [3]. The
structure shows a three-lobular structure formed by Ig-like domains. Helixes are shown in yellow and coiled-coil
regions in green. Three views of the protein are showed. The turn of the axis shown is referred to the first panel. The
results shows that it is also composed by three lobes of d-sheets barrels as those that form Spi1p, showing that also
these proteins has related three-dimensional structure. 3D-BLAST points to bacterial adhesins and surface antigens as
structures close to Sed1p. It is also remarkably that the alignment of Spi1p and Sed1p (Supplementary figure 2c)
shows that cysteines forming disulfide bonds that maintain the Ig-like domain (see models deposited at PMDB) are
often conserved in both proteins.

Suppl. Tab. 1
(a) S. cerevisiae strains used in this work

Strain
W303-1a
W303-1a msn2Fmsn4F
W303-1a cnb1F
W303-1a crz1F
W303-1a mpk1F
W303-1a pkc1ts

Genotype
MATa, ade 2-1, ura 3-1, leu 2-3, his 3-1, trp1-1
W303-1a msn2F3::HIS3, msn4-1::TRP1
W303-1a , cnb1::LEU2
W303-1a , crz1::G418
W303-1a mpk1::TRP1
W303-1a pkc1ts-X

W303-1a puf5F

W303-1a uth4::KanMX4

W303-1a yak1F

W303-1a yak1::KanMX4

W303-1a yap1F
MCY829
MCY msn1F
MCY msn5F
MCY mig1F
BY4741
BY4741 pho84F
BY4741 wsc2F
BY4742
BY4742 hog1F
BY4742 pho85F
BY4742 pop2F
BY4742 pho4F
BY4742 sko1F
BY4742 sst2F
MLY40
MLY40 phd1F
MLY40 sok2F

W303-1a yap1::KanMX4
MATa his3-F200 lys2-801 ura3-52
MCY829 msn1-F1::URA3
MCY829 msn5-F2::HIS3
MCY829 leu2::HIS3 mig1-F2::LEU2
MAT a, his3-1, leu2-0, met15-0, ura3-0
BY4741 pho84::KanMX4
BY4741 wsc2::KanMX4
MATc, his3-1, leu2-0, lys2-0, ura3-0
BY4742 hog1::KanMX4
BY4742 pho85::KanMX4
BY4742 pop2::KanMX4
BY4742 pho4::KanMX4
BY4742 sko1::KanMX4
BY4742 sst2::KanMX4
MATc, ura 3-52
MLY40 phd1::G418
MLY40 sok2::hygB

Reference
[1]
[2]
[3]
[4]
[5]
[6]
P. Carrasco,
doctoral thesis
P. Carrasco,
doctoral thesis
F. Rndez-Gil
[7]
[7]
[8]
[9]
Euroscarf
Euroscarf
Euroscarf
Euroscarf
Euroscarf
Euroscarf
Euroscarf
Euroscarf
Euroscarf
Euroscarf
[10]
[10]
[10]

(b) Plasmids used in this work

Plasmid
YEp352
YEp352-PKC1
YEp352-MPK1
YEp357
YEp357SPI1p/lacZ

Description
Reference
Multicopy shuttle vector. URA3 marker.
[11]
Multicopy shuttle vector. URA3 marker. PKC1 in PstI/BglII
[12]
Multicopy shuttle vector. URA3 marker. MPK1 in SphI/Ncori.
[12]
Multicopy shuttle vector. URA3 marker. lacZ.
[13]
Multicopy shuttle vector. URA3 marker. lacZ expression controlled by
[14]
SPI1 promoter.

Suppl. Tab. 2.
(a) Analysis of transcription factors (TF), using YEASTRACT tool (http://www.yeastract.com/), which regulate SPI1
expression documented by direct or indirect evidences. Abbreviations: northern blot (NB); expression microarrays
(ARR); chromatin inmunoprecipitation (ChIP); ChIP-on-CHIP (ChIP-CH), mutant (mt), wild-type (WT).

TF

Reference

Adr1p

[15]

Indirect: ARR (WT/TFmt)

Aft1p

[16]

Indirect: ARR (WT/TFmt)

Cat8p

[15]

Indirect: ARR (WT/TFmt)

Cin5p

[17]

Direct: ChIP

[15]

Indirect: ARR (WT/TFmt)

[18]

Indirect: ARR (WT/TFmt)

Cst6p

[15]

Indirect: ARR (WT/TFmt)

Gat4p

Indirect: ARR (WT/TFmt)

Gcr2p

[15]
[19]

Indirect: ARR (WT/TFmt)

Gis1p

[15]

Indirect: ARR (WT/TFmt)

Gzf3p

Indirect: ARR (WT/TFmt)

Haa1p

[15]
[20]

Hap4p

[21]

Indirect: ARR (WT/TFmt)

Hot1p

[22]

Direct: ChIP

[15]

Indirect: ARR (WT/TFmt)

[23]

Indirect: RT-PCR (WT/TFmt)

[24]

Indirect: RT-PCR (WT/TFmt)

[25]

Indirect: RT-PCR (WT/TFmt)

Ino2p

[26]

Indirect: ARR (WT/TFmt)

Ino4p

Indirect: ARR (WT/TFmt)

Mbp1p

[26]
[15]

Indirect: ARR (WT/TFmt)

Mcm1p

[27]

Indirect: NB (WT/TFmt)

Met31p

[15]

Indirect: ARR (WT/TFmt)

Met4p

[28]

Indirect: ARR (WT/TFmt)

Mga1p

[15]

Indirect: ARR (WT/TFmt)

Mig1p

[15]
[15]

Indirect: ARR (WT/TFmt)

Indirect: ARR (WT/TFmt)

[29]

Indirect: ARR (WT/TFmt)

[30]

Indirect: NB (WT/TFmt)

[31]

Indirect: NB (WT/TFmt)

[32]

Indirect: NB (WT/TFmt)

Pdr3p

[33]

Indirect: ARR (WT/TFmt)

Put3p

[15]

Indirect: ARR (WT/TFmt)

Crz1p

Hsf1p

Msn2p/Msn4p

Evidence

Indirect: NB (WT/TFmt)

TF

Reference

Rds2p

[34]

Direct: ChIP-CH

Rfx1p

[15]

Indirect: ARR (WT/TFmt)

Rgm1p

[15]

Indirect: ARR (WT/TFmt)

Rme1p

[15]
[15]

Indirect: ARR (WT/TFmt)

Indirect: ARR (WT/TFmt)

[35]

Indirect: ARR (WT/TFmt)

Rpn4p

[36]

Indirect: ARR (WT/TFmt)

Sfl1p

[37]

Indirect: ARR (WT/TFmt)

[38]

Direct: ChIP-CH

[15]

Indirect: ARR (WT/TFmt)

[22]

Direct: ChIP

[39]

Direct: ChIP

[40]

Direct: ChIP-CH

[41]

Indirect: ARR (WT/TFmt)

[15]

Indirect: ARR (WT/TFmt)

[42]

Indirect: ARR (WT/TFmt)

Ste12p

[41]

Direct: ChIP-CH

Stp2p

[15]

Indirect: ARR (WT/TFmt)

Tec1p

[41]

Direct: ChIP-CH

[15]

Indirect: ARR (WT/TFmt)

[28]

Indirect: ARR (WT/TFmt)

Rox1p

Skn7p

Sko1p

Sok2p

Yap1p

Analysis

of

transcription

Evidence

factors

found

with

Funcassociate

2.0

(http://llama.mshri.on.ca/funcassociate/), shows as enriched GO terms (adjusted p-value

<0.05): nitrogen compounds metabolic process (<0.001), ion-binding (<0.001), nucleic
acids metabolism (<0.001), stimulus response (0.009), glucose metabolism regulation
(0.03) and nutrient response (0.035).

(b) Transcriptional factors (TF), using YEASTRACT, which presents consensus binding motif in the sequence of the
SPI1 promoter. The position in the sequence respect the transcription start (ATG) is indicated, as well as its
orientation (forward in black and reverse in red). TF which have been demonstrated as SPI1 regulators, by direct or
indirect evidences (see a) are underlined.

TF

Position and orientation

TF

Position and orientation

Ash1p

-296, -65, -909, -565, -14

Nrg1p

-872, -700, -264, -257, -155, -373

Azf1p

-197

Pho4p

-889

Cbf1p

-888

Pip2p

-366

Crz1p

-358

Rph1p

-373, -265, -258

Fkh1/2p

-561, -812, -766, -333

Rtg1/3p

-575

Gcr1p

-268, -150, -488, -212

Sko1p

-708

Gis1p

-368, -260, -253

Gln3p

-303

Hac1p

-134

Ste12p

-993

Hsf1p

-187, -177

Tec1p

-207

Mcm1p

-311, -290, -168, -278, -372

Xbp1p

-928, -868, -361, -184, -922

Mot3p

-306, -978

Yap1p

-9, -834

Msn2/4p

-373, -265, -258

Yap3

-9

Yrr1p

-561

Stb5p

-565, -497, -366, -105, -879, 547, -322

Analysis of transcription factors found with Funcassociate 2.0, shows as enriched GO

terms (adjusted p-value <0.05): nitrogen compounds metabolic process (<0.001), ionbinding (<0.001), nucleic acids metabolism (<0.001), filamentous growth (0.005) and
stress transcriptional response (0.008). Note that the tree more over-represented
categories (adjusted p-value <0.001) were found in both tables.

Supplementary References
1.

Thomas, B.J. and Rothstein, R.: The genetic control of direct-repeat

recombination in Saccharomyces: the effect of rad52 and rad1 on mitotic
recombination at GAL10, a transcriptionally regulated gene. Genetics
123 (1989) 725-738.

2.

Estruch, F. and Carlson, M.: Two homologous zinc finger genes identified
by multicopy suppression in a SNF1 protein kinase mutant of
Saccharomyces cerevisiae. Mol Cell Biol. 13 (1993) 3872-3881.

3.

Cunningham, K.W. and Fink, G.R.: Calcineurin-dependent growth control

in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of
plasma membrane Ca2+ ATPases. J Cell Biol. 124 (1994) 351-363.

4.

Matheos, D.P., Kingsbury, T.J., Ahsan, U.S. and Cunningham, K.W.:

Tcn1p/Crz1p, a calcineurin-dependent transcription factor that
differentially regulates gene expression in Saccharomyces cerevisiae.
Genes Dev. 11 (1997) 3445-3458.

5.

Queralt, E. and Igual, J.C.: Functional connection between the Clb5

cyclin, the protein kinase C pathway and the Swi4 transcription factor in
Saccharomyces cerevisiae. Genetics 171 (2005) 1485-1498.

6.

Igual, J.C., Johnson, A.L. and Johnston, L.H.: Coordinated regulation of

gene expression by the cell cycle transcription factor Swi4 and the
protein kinase C MAP kinase pathway for yeast cell integrity. Embo J. 15
(1996) 5001-5013.

7.

Estruch, F. and Carlson, M.: Increased dosage of the MSN1 gene

restores invertase expression in yeast mutants defective in the SNF1
protein kinase. Nucleic Acids Res. 18 (1990) 6959-6964.

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