The Yeast Sks1p Kinase Signaling Network Regulates Pseudohyphal Growth and Glucose Response
et al. (2014) The Yeast Sks1p Kinase Signaling Network Regulates Pseudohyphal Growth
and Glucose Response. PLoS Genet 10(3): e1004183. doi:10.1371/journal.pgen.1004183
The Yeast Sks1p Kinase Signaling Network Regulates Pseudohyphal Growth and Glucose Response
Cole Johnson 0
Hye Kyong Kweon 0
Daniel Sheidy 0
Christian A. Shively 0
Dattatreya Mellacheruvu 0
Alexey I. Nesvizhskii 0
Philip C. Andrews 0
Anuj Kumar 0
Scott Erdman, Syracuse, United States of America
0 1 Department of Molecular, Cellular, and Developmental Biology, University of Michigan , Ann Arbor , Michigan, United States of America, 2 Department of Biological Chemistry, University of Michigan Medical School , Ann Arbor , Michigan, United States of America, 3 Department of Pathology, University of Michigan Medical School , Ann Arbor , Michigan, United States of America, 4 Department of Computational Medicine and Bioinformatics, University of Michigan Medical School , Ann Arbor , Michigan, United States of America, 5 Department of Chemistry, University of Michigan , Ann Arbor, Michigan , United States of America
The yeast Saccharomyces cerevisiae undergoes a dramatic growth transition from its unicellular form to a filamentous state, marked by the formation of pseudohyphal filaments of elongated and connected cells. Yeast pseudohyphal growth is regulated by signaling pathways responsive to reductions in the availability of nitrogen and glucose, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. Here, we identify the glucoseresponsive Sks1p kinase as a signaling protein required for pseudohyphal growth induced by nitrogen limitation and coupled nitrogen/glucose limitation. To identify the Sks1p signaling network, we applied mass spectrometry-based quantitative phosphoproteomics, profiling over 900 phosphosites for phosphorylation changes dependent upon Sks1p kinase activity. From this analysis, we report a set of novel phosphorylation sites and highlight Sks1p-dependent phosphorylation in Bud6p, Itr1p, Lrg1p, Npr3p, and Pda1p. In particular, we analyzed the Y309 and S313 phosphosites in the pyruvate dehydrogenase subunit Pda1p; these residues are required for pseudohyphal growth, and Y309A mutants exhibit phenotypes indicative of impaired aerobic respiration and decreased mitochondrial number. Epistasis studies place SKS1 downstream of the G-protein coupled receptor GPR1 and the G-protein RAS2 but upstream of or at the level of cAMPdependent PKA. The pseudohyphal growth and glucose signaling transcription factors Flo8p, Mss11p, and Rgt1p are required to achieve wild-type SKS1 transcript levels. SKS1 is conserved, and deletion of the SKS1 ortholog SHA3 in the pathogenic fungus Candida albicans results in abnormal colony morphology. Collectively, these results identify Sks1p as an important regulator of filamentation and glucose signaling, with additional relevance towards understanding stressresponsive signaling in C. albicans.
Funding: This work was supported by grants 1R01-A1098450-01A1 from the National Institutes of Health and 1-FY11-403 from the March of Dimes (to AK), grant
P41-RR18627 from the National Institutes of Health National Research for Proteomics and Pathways (to PCA), and grant R01-GM094231 from the National
Institutes of Health (to AIN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Multiple fungal species exhibit complex morphological changes
in response to environmental conditions, generating multicellular
forms or structures critical to the respective life cycles of these
organisms . These morphological transitions have been
linked to virulence in several human and plant fungal pathogens,
including Candida albicans, Cryptococcus neoformans, Aspergillus fumigates,
and Ustilago maydis . In particular, several lines of study have
established that the formation of hyphal filaments is required for
virulence in the opportunistic human fungal pathogen C. albicans
. The budding yeast Saccharomyces cerevisiae also exhibits a
morphogenetic transition from its typical form to a filamentous
state , and the study of this dimorphism in S. cerevisiae has
contributed considerably to our understanding of important cell
signaling mechanisms, while also providing insight into the
molecular basis of fungal pathogenicity .
The morphological transition in S. cerevisiae is pronounced: yeast
cells undergoing pseudohyphal growth are elongated and remain
connected after cytokinesis, forming multicellular chains, or
filaments . These filaments of connected cells can spread
out along the surface of a solid growth substrate as well as invade
the substrate  and are referred to as pseudohyphae, since they
resemble the hyphae of other fungal species but lack the structure
of a true hyphal tube . Strains of S. cerevisiae competent to
undergo pseudohyphal growth (e.g., the S1278b strain used here)
initiate this transition in response to conditions of nitrogen
limitation and/or glucose limitation [11,17,18]. Consequently,
pseudohyphal growth is considered to be an adaptive mechanism,
enabling non-motile yeast cells to forage for nutrients when local
resources become limited . The morphological changes
associated with pseudohyphal growth are driven by a host of
altered developmental processes, including a delay in the G2/M
cell-cycle transition that produces the elongated cell morphology
, a switch to a unipolar budding pattern [11,20], and
increased cell-cell adhesion .
At least 700 single gene deletions in the filamentous S1278b
strain of S. cerevisiae result in pseudohyphal growth phenotypes
Eukaryotic cells respond to nutritional and environmental
stress through complex regulatory programs controlling
cell metabolism, growth, and morphology. In the budding
yeast Saccharomyces cerevisiae, conditions of limited
nitrogen and/or glucose can initiate a dramatic growth
transition wherein the yeast cells form extended
multicellular filaments resembling the true hyphal tubes of
filamentous fungi. The formation of these pseudohyphal
filaments is governed by core regulatory pathways that
have been studied for decades; however, the mechanism
by which these signaling systems are integrated is less well
understood. We find that the protein kinase Sks1p
contributes to the integration of signals for nitrogen
and/or glucose limitation, resulting in pseudohyphal
growth. We implemented a mass spectrometry-based
approach to profile phosphorylation events across the
proteome dependent upon Sks1p kinase activity and
identified phosphorylation sites important for
mitochondrial function and pseudohyphal growth. Our studies place
Sks1p in the regulatory context of a well-known
pseudohyphal growth signaling pathway. We further find that
SKS1 is conserved and required for stress-responsive
colony morphology in the principal opportunistic human
fungal pathogen Candida albicans. Thus, Sks1p is part of
the mechanism integrating glucose-responsive cell
signaling and pseudohyphal growth, and its function is required
for colony morphology linked with virulence in C. albicans.
[23,24], and classic studies have established three well-studied
signaling pathways as regulators of pseudohyphal differentiation:
the mitogen-activated protein kinase (MAPK) pathway, the
cAMP-dependent protein kinase A (PKA) pathway, and the
sucrose non-fermentable (SNF) pathway. The yeast pseudohyphal
growth MAPK pathway consists of the MAPKKK Ste11p, the
MAPKK Ste7p, and the MAPK Kss1p [12,13,2527]. Ste11p is
phosphorylated by the p21-activated kinase Ste20p , and
Kss1p phosphorylates the key heterodimeric transcription factor
Ste12p/Tec1p [29,30]. In S. cerevisiae, PKA consists of the
regulatory subunit Bcy1p and one of three catalytic subunits,
Tpk1p, Tpk2p, and Tpk3p [31,32]. Deletion of TPK2 results in a
loss of pseudohyphal growth, and Tpk2p has been implicated most
extensively in filamentation and the response to nitrogen stress
. Tpk2p phosphorylates the transcription factor Flo8p, which
is required for pseudohyphal growth [32,33]. Snf1p is a member of
the AMP-activated kinase family and regulates transcriptional
changes associated with glucose derepression [34,35]. Snf1p
regulates the key pseudohyphal growth effector FLO11 through
repression of the negative regulators Nrg1p and Nrg2p . The
Kss1p MAPK pathway and PKA also activate FLO11
transcription through Ste12p/Tec1p and Flo8p, respectively .
Notably, each pathway above is involved in the cellular response
to nutrient availability [18,39,40]. In particular, glucose, the
preferred carbon source of budding yeast, effects changes in
transcription principally through the Ras/PKA pathway , and
glucose limitation activates the heterotrimeric Snf1p kinase
complex through phosphorylation of T210 in Snf1p [42,43].
The mechanisms by which these signals are then propagated and
executed to elicit pseudohyphal differentiation, however, are still
under investigation. Studies from Bisson and colleagues 
identified the SKS1 gene, encoding a Ser/Thr kinase, as a
multicopy suppressor of snf3D; mutants deleted for SNF3 are
defective in high-affinity glucose transport and cannot grow by
fermentation on low-glucose medium. Yang and Bisson also
demonstrated that Sks1p kinase activity was required for
phenotypic suppression of snf3D. Recent work in our lab indicated
that Sks1p undergoes a localization shift to the nucleus during
butanol-induced pseudohyphal growth and that its kinase activity
is required for wild-type localization of Ksp1p, a stress
granuleassociated protein with pseudohyphal growth deletion phenotypes
. Thus, SKS1 may regulate both glucose-responsive signaling
and pseudohyphal development, with the potential to serve as an
integrator between these interrelated signaling processes.
Sks1p kinase activity is required for pseudohyphal
We initially assessed the role of Sks1p in pseudohyphal growth
through a series of phenotypic studies analyzing pseudohyphal
filamentation in SKS1 mutants under conditions of nitrogen
limitation. For this work, we constructed homozygous diploid
sks1D/D, and sks1-K39R/sks1-K39R mutants in the filamentous
S1278b genetic background, with the latter strain containing a
site-directed mutation yielding greatly diminished kinase activity.
On low-nitrogen (SLAD) media, loss of either the gene or its kinase
activity resulted in decreased surface-spread filamentation relative
to an isogenic wild-type strain (Figure 1A). The introduction of a
centromeric plasmid bearing wild-type SKS1 under transcriptional
control of its native promoter was able to rescue the loss of
pseudohyphal growth exhibited by the sks1D/D mutant, while
introduction of a similar plasmid bearing the kinase-dead variant
of SKS1 (sks1-K39R) was unable to restore wild-type filamentation
(Figure 1B). Overexpression of SKS1 from a high-copy 2m plasmid
induced hyper-filamentation under conditions of nitrogen
limitation (Figure 1C).
Identification of the Sks1p signaling network
To determine the molecular basis of Sks1p kinase regulation of
peudohyphal growth, we analyzed the Sks1p kinase signaling
network by quantitative phosphoproteomics. Our approach was
straightforward; we implemented a mass spectrometry-based
method utilizing stable isotope labeling of amino acids in cell
culture (SILAC) to identify proteins differentially phosphorylated
upon loss of Sks1p kinase activity [46,47]. As outlined in Figure 2A,
a strain that was wild-type with respect to SKS1 and an otherwise
isogenic strain carrying the sks1-K39R allele in the filamentous
S1278b background were made auxotrophic for arginine and lysine;
the wild-type and sks1kinase-dead strains were subsequently grown
in triplicate for five cell doublings in media containing unlabeled or
labeled arginine and lysine, respectively, in the presence of butanol
to induce pseudohyphal growth. Prepared proteins from both sets of
samples were enriched for phosphopeptides, and differences in
phosphopeptide abundance between the wild-type and kinase-dead
samples were determined by mass spectrometry.
By this approach, we profiled 903 phosphorylation sites across
the yeast proteome, identifying 114 phosphopeptides differentially
abundant upon enrichment from the sks1 kinase-dead strain
relative to wild-type (Figure 2B). These peptides correspond to 91
proteins in total, encompassing phosphorylation events directly
and indirectly dependent upon the presence of Sks1p kinase
activity. Interestingly, by comparing the phosphorylation sites
determined in this study against S. cerevisiae phosphorylation sites
reported in public databases, we identified 39 new
phosphorylation sites in the yeast proteome. Table 1 presents novel
phosphorylation sites in peptides differentially abundant upon
phosphopeptide enrichment from the sks1-K39R mutant relative to
wild type. A listing of phosphopeptides is presented in Dataset S1,
and the full mass spectrometry dataset can be accessed at
ProteomeXchange (dataset identifier PXD000414).
Sks1p signaling network connectivity
The set of Sks1p-dependent phosphoproteins identified in this
study is statistically enriched (p-value of 1022) for the cellular
pathway enabling glycolysis and gluconeogenesis (Figure 3A), as
defined in the KEGG database; the glycolysis and gluconeogenesis
pathway is annotated in KEGG as sce00010. To gain a better
understanding of the means through which Sks1p-dependent
glucose signaling impacts additional cell processes and pathways,
we used the glycolysis/gluconeogenesis pathway as a starting point
for the construction of an interaction network. In brief, reported
genetic and physical interactions with components of the KEGG
glycolysis/gluconeogenesis pathway were incorporated and
expanded until Sks1p was included in the network as well as MAPK
signaling and cell cycle pathways known to be required for wild-type
pseudohyphal growth (Figure 3B and C). The resulting interaction
network structure indicates two points. First, the clusters of genes
enriched in MAPK signaling and cell cycle control exhibit a greater
number of genetic and physical interactions between each other
than with the cluster of genes enriched for
glycolysis/gluconeogenesis; this is evident visually from the dark blue lines in Figure 3A
indicating increased interaction density connecting the MAPK- and
cell cycle-enriched clusters. Second, the network distance of Sks1p
to proteins exhibiting Sks1p-dependent phosphorylation in the
glycolysis/gluconeogenesis-enriched cluster is typically small and
establishes a stronger interconnection between Sks1p and this
cluster than with clusters enriched for MAPK signaling and cell
cycle regulation. This result is consistent with the observed
enrichment for the KEGG glycolysis/gluconeogenesis pathway in
the set of proteins exhibiting Sks1p-dependent phosphorylation.
The interactions used to construct this network are presented in
Figure 2. Quantitative phosphoproteomic analysis of the Sks1p signaling network in the filamentous S1278b genetic background.
A) Schematic overview of major steps in the SILAC-based mass spectrometric analysis of Sks1p signaling; strain auxotrophies are indicated in the
control and kinase-dead strains. B) A listing of proteins exhibiting decreased or increased phosphopeptide abundance after enrichment from a strain
carrying the sks1-K39R kinase-dead allele relative to a strain carrying wild-type SKS1.
aPhosphosites indicating a localization probability of p.0.75.
bPhosphopeptide abundance as sks1-K39R/WT ratio.
Phenotypic analysis of Sks1p-dependent
As a first step towards identifying the phosphorylation events
responsible for the filamentation defect observed in sks1-K39R, we
screened a panel of yeast proteins exhibiting Sks1p-dependent
phosphorylation for phenotypes similar to those of genes whose
deletion affects pseudohyphal growth. Genes were selected by
crossreferencing the list of phosphoproteins identified by our mass
spectrometry study with genes identified as having pseudohyphal
growth phenotypes [23,24]. For this analysis, we prioritized genes
with a role in glucose signaling. Homozygous diploid gene deletions
were generated and screened for surface-spread filamentation under
conditions of nitrogen limitation (SLAD medium) and nitrogen/
glucose limitation (SLALD medium). Wild-type S. cerevisiae of the
S1278b genetic background exhibits surface-spread filamentation on
both SLAD and SLALD medium, and the homozygous sks1D/D
mutant displays a loss of filamentation on both media. Figure 4A
indicates deletion mutants exhibiting pseudohyphal growth
phenotypes under these conditions. Strains deleted for BUD6, ITR1,
MDS3, NPR3, and PDA1 displayed significantly decreased
pseudohyphal growth on both SLAD and SLALD medium; lrg1D/D
mutants exhibited decreased pseudohyphal growth under conditions
of nitrogen/glucose limitation. These genes contribute to pathways
and cell processes required for pseudohyphal growth. MDS3 and
NPR3 are TOR pathway components [48,49], and LRG1 encodes a
putative GTPase-activating protein involved in the Pkc1p signaling
pathway controlling cell wall integrity . Bud6p is a polarity
protein required for budding , and Itr1p is a myo-inositol
transporter ; Pda1p is a subunit of the pyruvate dehydrogenase
complex and will be discussed below. As indicated in Figure 4A,
deletion of the HXT1 gene encoding a low-affinity glucose
transporter yielded hyperactive pseudohyphal growth on SLAD
and SLALD media. Deletion of RCK2 encoding a kinase responsive
to oxidative and osmotic stress resulted in increased pseudohyphal
growth; a similar phenotype has been observed upon decreased
activity of the osmo-responsive Hog1p MAPK pathway .
To assess the functional significance of Sks1p-dependent
phosphorylation sites, we constructed homozygous diploid strains
containing chromosomal point mutations in BUD6, ITR1, LRG1,
NPR3, and PDA1, substituting a non-phosphorylatable residue for
the Sks1p-dependent phosphorylation site (Figure 4B).
Corresponding phosphopeptides for each phosphorylation site exhibited
decreased abundance upon zirconium dioxide enrichment in the
kinase-dead sks1-K39R mutant. These integrated point mutants
were assayed for fitness in SLAD and SLALD media, and each
mutant exhibited a fitness defect relative to wild-type (Figure 4C
and D), indicating that the mutated residues are necessary for
optimal response to nitrogen and nitrogen/glucose limitation.
Growth and growth rates for these strains in synthetic complete
(SC) media are provided in Tables S5 and S6.
Pda1p residues Y309 and S313 are necessary for
pseudohyphal differentiation and respiratory growth
Of the point mutants assayed above, strains with mutations in
PDA1 exhibited pseudohyphal growth defects on low-nitrogen
medium, with a much less acute growth defect in SC media. As
indicated in Figure 5A and B, two distinct point mutations in
PDA1, the Y309A and S313A alleles, yield a dramatic fitness defect
and loss of pseudohyphal differentiation in nitrogen-limiting
conditions. Pda1p is a subunit of the mitochondrial pyruvate
dehydrogenase complex involved in the conversion of pyruvate to
acetyl-CoA . Cells lacking PDA1 demonstrate diminished
growth on glucose from a respiratory deficiency due to
mitochondrial DNA loss . We found no noticeable difference in protein
levels for either single point-mutant (Y309A and S313A) or the
double mutant (Y309A-S313A) relative to a wild-type strain
(Figure 5C), indicating that the observed phenotypes were not due
to instability of Pda1p. Interestingly, when each mutant was
screened on glycerol-containing media that forced respiratory
growth, the S313A mutant grew as well as wild-type, while the
Y309A mutant exhibited a phenotype analogous to the pda1D/D
mutant  (Figure 5D). The double Y309A-S313A mutant
shared the respiratory deficiency of the Y309A mutant. We also
investigated whether the mutation of these residues altered
mitochondrial structure or mitochondrial DNA. By live-cell DAPI
staining, no abnormal mitochondrial DNA phenotypes were
observed between the wild-type strain, the pda1D/D mutant, or
any of the site-directed mutants (Figure 5E, lower). However,
staining with the membrane-potential-dependent dye MitoTracker
illustrates dramatic differences in mitochondrial membrane
potential or structure between these mutants and wild-type
(Figure 5E, middle). The wild-type and pda1-S313A strains exhibit
similar mitochondrial staining, while the pda1D, pda1-Y309A, and
pda1-Y309A-S313A mutants all demonstrate a mitochondrial
membrane phenotype. Collectively, both the Y309A and S313A
mutations result in the abolishment of pseudohyphal growth in
conditions of low nitrogen, and the Y309A mutation also yields
phenotypes indicative of impaired respiration.
Epistasis analysis of Sks1p with respect to glucose
signaling and pseudohyphal growth
The studies presented here support a role for Sks1p in enabling
wild-type glucose signaling and pseudohyphal growth; however,
the molecular context and genetic relationships of SKS1 with
respect to the corresponding signaling pathways is unclear.
Consequently, we performed epistasis experiments examining
the phenotypic consequences of over-expressing SKS1 in diploid S.
cerevisiae strains deficient for components of both glucose signaling
pathways and pseudohyphal signaling networks (Figure 6A and B).
Here, we examined whether SKS1 could act as a high-copy
suppressor of mutations in cAMP signaling (gpr1D/D, ras2D/D,
and tpk2D/D), MAPK signaling (ste20D/D), or Snf1p signaling
(snf1D/D). Each of these mutations generates a yeast strain
deficient in pseudohyphal differentiation under conditions of
limiting nitrogen. We found that over-expression of SKS1 was able
to suppress the gpr1D/D phenotype (Figure 6C). Interestingly, a
ras2D/D mutant also demonstrated a moderate phenotypic rescue
from overexpression of SKS1; however, SKS1 overexpression did
not restore pseudohyphal growth in a tpk2D/D mutant, indicating
that SKS1 acts downstream of GPR1 and RAS2 but upstream of or
at the level of TPK2 (Figure 6D). Overexpression of SKS1 in the
gpr1D/D and ras2D/D backgrounds did not result in filamentation
on media with normal levels of nitrogen. SKS1 did not suppress
mutations in STE20 or SNF1. Consistent with this STE20 result,
the sks1D/D mutant exhibited no loss of pseudohyphal MAPK
signaling under conditions of nitrogen limitation (data not shown),
as assessed using a PFRE(TEC1)-lacZ reporter system that is
specifically responsive to the MAPK signaling components
required for filamentous growth . We also tested whether
deletion of SKS1 could affect the hyper-filamentous phenotype of
strains deleted for PDE1, which encodes a phosphodiesterase that
degrades cAMP , and strains deleted of GPB1, which encodes
a Gb-subunit of the Gpa2p heterotrimeric G protein . In each
case, the double deletion mutants remained hyperfilamentous,
indicating no clear genetic relationship between SKS1 and these
Mss11p and Rgt1p are required for wild-type SKS1
In complement to our analysis of Sks1p kinase activity, we also
investigated whether known transcriptional regulators of
pseudohyphal development influenced the expression of SKS1. Analysis of
SKS1 transcription via quantitative real-time (qRT) PCR identified
several results, as follows. First, SKS1 mRNA levels were
responsive to nitrogen and glucose limitation in wild-type S.
cerevisiae of the filamentous S1278b background. SKS1 transcript
levels increased by nearly 180% under conditions of nitrogen
limitation coupled with glucose limitation (SLALD medium)
(Figure 7A). A comparison of SKS1 transcript levels between
mutants deleted for known transcriptional regulators of
pseudohyphal differentiation (flo8D/D, mfg1D/D, mga1D/D, mss11D/D,
phd1D/D, phd1D/D, and tec1D/D) and wild-type S. cerevisiae found
that the transcription factor Mss11p, involved in glucose signaling,
invasive growth, and starch degradation, as well as Flo8p, the
wellknown filamentous growth transcriptional activator, exhibited
minor decreases in SKS1 mRNA levels under the indicated
extracellular conditions (Figure 7B). The flo8D/D mutant
displayed an approximate 30% reduction in SKS1 transcript levels
relative to a wild-type strain in both standard and low nitrogen/
glucose SLALD media. Mss11p demonstrated a reduction in SKS1
mRNA levels of nearly 65%, but only in standard media
promoting vegetative growth. We also examined the SKS1
transcriptional response in a diploid strain deleted for RGT1,
encoding a glucose-responsive transcriptional regulator known to
repress the expression of many HXT genes [58,59]. Compared to
the wild-type control, the rgt1D/D mutant demonstrated a marked
increase in SKS1 transcript abundance under conditions of low
nitrogen coupled with glucose limitation (Figure 7B).
The SKS1 ortholog SHA3 is required for wild-type colony
morphology in Candida albicans
Candida albicans is both a successful commensal and pathogen
of humans, sharing with S. cerevisiae the ability to undergo
morphological transitions in response to appropriate
environmental cues [7,6062]. The importance of this morphological
differentiation is underscored by the fact that hyphal development
is required for virulence in C. albicans. The S. cerevisiae SKS1 gene is
conserved in C. albicans, and considering the strong conservation of
pathway structure between these organisms, we hypothesized that
the SKS1 ortholog in C. albicans may serve a similar function in
integrating environmental cues to regulate fungal morphology. To
test this hypothesis, we generated a heterozygous deletion of the
SKS1 ortholog SHA3 in the C. albicans strain BWP17. SHA3 shares
approximately 33% sequence identity with SKS1 and also encodes
a kinase involved in glucose transport and glucose-responsive
signaling  (Figure 8A). On Spider growth medium in which
mannitol is the carbon source, the C. albicans SHA3 heterozygous
mutant displayed a decrease in colony wrinkling relative to an
isogenic wild-type strain (Figure 8B). Consistent with this result,
Uhl et al.  found that a heterozygous mutant containing a
transposon insertion upstream of SHA3 in its promoter region
exhibited decreased hyphal growth on Spider medium. In liquid
culture, cell morphology is wild type in the SHA3 heterozygous
deletion mutant (Figure 8C), but the mutant does exhibit a
statistically significant decrease in biofilm formation on Spider
medium (Figure 8D). The ratio of biofilm formation on Spider
media versus control RPMI buffer also indicates an approximately
three-fold decrease in the sha3D/SHA3 strain relative to wild type.
Since the heterozygous sha3 mutant exhibits a phenotype on
Spider medium, we examined if colony morphology was affected
on media containing other carbon sources. As indicated in
Figure 8E, the sha3D/SHA3 mutant exhibits a colony morphology
distinct from wild type on medium with glucose as the carbon
source and a slight phenotype on media containing sucrose.
Cellular adaptation to nitrogen or carbon deprivation in S.
cerevisiae requires the remodeling of cellular metabolism and the
precisely coordinated restructuring of cellular morphology. Here,
we identify the glucose-responsive Sks1p kinase as a signaling
protein required for pseudohyphal growth induced by nitrogen
limitation and nitrogen limitation coupled with glucose limitation.
Ninety-one proteins undergo Sks1p-dependent phosphorylation,
and the functional scope of these phosphoproteins identifies Sks1p
contribution to glucose signaling as well as additional processes
and pathways required for pseudohyphal growth, including
mitochondrial function. Epistasis studies indicate that SKS1 acts
downstream of GPR1 and RAS2, consistent with Sks1p regulation
of or by glucose-responsive cAMP signaling. SKS1 transcript levels
are dependent upon Mss11p and Rgt1p. SKS1 is conserved, and
the SKS1 ortholog SHA3 in C. albicans is required for wild-type
colony morphology on glucose-containing medium and on Spider
medium with mannitol as a carbon source. Collectively, these
results are consistent with a function for Sks1p kinase activity in
the integration of glucose-responsive signaling and filamentous
development an example of signaling crosstalk that has not been
extensively studied or well understood.
In this study, we utilized a SILAC-based mass spectrometry
approach to identify phosphorylation events dependent upon Sks1p
kinase activity. In S. cerevisiae, several phosphoproteomic strategies
have been utilized recently to profile differential phosphorylation
. In particular, Bodenmiller et al.  implemented a
label-free mass-spectrometry approach to investigate the global
phosphoproteomic response of S. cerevisiae to the systematic deletion
of protein kinases and phosphatases. Trade-offs exist in considering
the relative advantages of both label-free and labeling strategies.
Label-free methods have been shown to identify a larger number of
proteins than label-based methods; however, SILAC-based
strategies typically enable better quantification and identification of
differentially abundant proteins, while also providing greater
reproducibility across samples . It is important to bear in mind
that both label-free and SILAC-based interventional
phosphoproteomic methods identify direct and indirect phosphorylation events;
consequently, the studies here are intended to identify the broad
scope of cell processes and pathways encompassed within the Sks1p
Notably, the study by Bodenmiller and colleagues did address
the Sks1p signaling network in a non-filamentous strain under
vegetative growth conditions, and approximately 30% of the
proteins detected in this analysis were also identified by label-free
methods in that work. Further, the overlap between the datasets is
striking, in that nine proteins exhibiting Sks1p-dependent
phosphorylation in a non-filamentous strain under vegetative
conditions were also identified as being differentially phosphorylated in
our analysis of sks1-K39R in a filamentous strain under conditions
inducing pseudohyphal growth; these phosphoproteins include
Cdc37p, Crp1p, Fyv8p, Hxt1p, Mrh1p, Mtc1p, Pda1p, Pil1p,
Ptr2p, Rck2p, Zuo1p, and Ymr196w. The proteins Hxt1p, Rck2p,
and Pil1p are stress-responsive, and Mrh1p, Pil1p, and Pda1p
have been reported to localize to mitochondria, highlighting
important processes and functions required for wild-type glucose
signaling and pseudohyphal growth.
The dependence of Pda1p phosphorylation upon Sks1p and
phenotypic analysis of Pda1 Y309A and S313A mutants
underscores that wild-type mitochondrial membrane structure and
function is interconnected with Sks1p kinase signaling.
Interestingly, signaling pathways that regulate filamentation, cAMP-PKA
and Snf1p, have also been shown to target mitochondria ,
and genetic screens of pseudohyphal deficient mutants have
identified genes required for mitochondrial function [17,23,74].
The S313 residue of Pda1p is a known phosphorylation site, and
phosphorylation of S313 inhibits Pda1p activity in vitro [75,76].
Further, Oliveira et al.  report that the S313A mutant exhibits
increased flux through pyruvate dehydrogenase during growth on
glucose in a non-filamentous strain. As reported here, a
filamentous strain containing the S313A mutation is able to grow
on medium with glycerol as the sole carbon source. Interestingly,
however, both the Pda1p S313 and Y309 residues are required for
pseudohyphal growth. Sks1p is a Ser/Thr kinase; consequently,
the Y309 residue in Pda1p is not expected to be a direct
phosphorylation target of Sks1p. Further, Pda1p is a
mitochondrial protein, and our previous analysis of Sks1p-YFP subcellular
distribution did not identify mitochondrial localization .
Rather, we expect that Sks1p is required for wild-type
phosphorylation of Pda1p Y309 because it functions in a signaling network
that results in this phosphorylation event. Ongoing investigations
are directed towards identifying the kinase that phosphorylates
Pda1p Y309 and the mechanism by which the Y309 and S313
residues contribute to pseudohyphal growth.
Our results indicate that SKS1 mRNA levels are
glucoseregulated in the filamentous S1278b strain and that this regulation
in SLALD medium is carried out in part by Rgt1p. Two lines of
evidence support this result. First, analysis of the yeast
transcriptional response to glucose by Wang et al.  indicated that SKS1
mRNA levels increase more than two-fold when cells are switched
from galactose to glucose-containing media. Second, the snf3D
phenotype is subject to high copy number suppression by the SKS1
promoter sequence, which titrates Rgt1p [78,79]. In addition to
possessing binding sites for Rgt1p, the SKS1 promoter is reportedly
bound by Mss11p and Flo8p [24,80], although we observe that the
relative individual contributions of these transcription factors to
the establishment of SKS1 mRNA levels is modest under
conditions of nitrogen limitation and nitrogen/glucose limitation.
Considered collectively, transcriptional regulation of SKS1 likely
results from the combinatorial contributions of numerous
transcription factors. Under conditions of glucose limitation,
Rgt1p actively binds target promoters to repress transcription of
glucose-induced genes, and the observed increase in SKS1
transcript levels upon RGT1 deletion under low-glucose conditions
is consistent with this observation. However, SKS1 mRNA levels
increase upon growth in SLALD media, indicating that Rgt1p
cannot be predominantly responsible for the establishment of
overall SKS1 transcript levels. Additional factors, including Mss11p
and Flo8p, must contribute to this transcriptional control as
well, presenting a more complex picture of SKS1 transcriptional
Coupling findings from this study with previous work, we
suggest that Sks1p mediates cellular response to glucose limitation
and nitrogen limitation by signaling through Gpr1p and the
cAMP-PKA pathway. In this study, we demonstrate that SKS1 is a
high-copy suppressor of pseudohyphal-deficient gpr1D/D and
ras2D/D mutants. Both Gpr1p and Ras2 are components of the
cAMP-dependent PKA pathway. Gpr1p is a nutrient sensor that
activates cAMP in response to low-levels of extracellular glucose
 and regulates pseudohyphal differentiation in S. cerevisiae .
Overexpression of SKS1 failed to restore pseudohyphal growth in a
strain deleted for TPK2. Tk2p is one of three catalytic subunits of
PKA; TPK2 is required for pseudohyphal growth, and its function
is required for the phosphorylation of Flo8p and additional key
signaling events necessary for pseudohyphal differentiation
[32,83]. Thus, Sks1p may contribute to the regulation of Tpk2p
or may be regulated indirectly by Tpk1p or Tpk3p. Sks1p has not
been identified as a phosphoprotein, and any such mechanisms of
Sks1p regulation have not been identified to date.
Pseudohyphal growth in S. cerevisiae is an excellent model of
related processes of filamentous development in the principal
opportunistic human fungal pathogen Candida albicans. In C.
albicans, a variety of culture conditions, including growth on Spider
medium with mannitol as a carbon source, results in the
development of pseudohyphae and true hyphal tubes .
Orthologs of many S. cerevisiae pseudohyphal growth genes play
similarly important roles in C. albicans hyphal development, and we
find that the SKS1 ortholog SHA3 is required for wild-type colony
morphology in C. albicans on Spider medium. The cAMP-PKA
pathway is required for hyphal development and virulence in C.
albicans, exhibiting structural similarity to the orthologous pathway
in S. cerevisiae. Notably, GPR1 is conserved, and Ras1p in C. albicans
contributes to the production of cAMP through adenylate cyclase
in response to various stimuli . The PKA catalytic subunits
Tpk1p and Tpk2p have been identified in C. albicans, and it will be
interesting to determine if the functional relationship between
SHA3 and this cAMP-PKA pathway is similar to that which we
observe in S. cerevisiae.
Materials and Methods
Yeast strains, plasmids, and growth conditions
The strains used in this study are listed in Table S1 and are
isogenic derivatives of the S1278b strain [11,85]. All strains were
generated from the MATa haploid strain Y825 (ura3-52 leu2D)
and the MATa haploid strain HLY337 (ura3-52 trp1-1).
Standard yeast media and microbiological techniques were used
. Briefly, standard growth media consisted of YPD (1% yeast
extract, 2% peptone, 2% glucose) or Synthetic Complete (SC)
(0.67% yeast nitrogen base (YNB) without amino acids, 2%
glucose, and 0.2% of the appropriate amino acid drop-out mix).
Nitrogen deprivation and filamentous phenotypes were assayed
using Synthetic Low Ammonium Dextrose (SLAD) medium
(0.17% YNB without amino acids, 2% glucose, 50 mM ammonium
sulfate and supplemented with appropriate amino acids) and
Synthetic Low Ammonium Low Dextrose (SLALD) medium
(0.17% YNB without amino acids, 0.05% glucose, 50 mM
ammonium sulfate and supplemented with appropriate amino
acids) [11,87,88]. Respiratory competency was assayed using YPG
(1% yeast extract, 2% peptone, 3% glycerol). For plates,
autoclaved 2% agar was added to the media. To promote C.
albicans vegetative growth, 80 mg/mL of uridine was added to all
media unless otherwise noted. Hyphal growth was induced in C.
albicans via growth in Spider medium and/or 10%
serumcontaining medium for the indicated times at 37uC .
Plasmids used in this study are listed in Table S2. Plasmids
pFRE-lacZ and pSKS1-K39R-vYFP were constructed as
described [26,45]. To overexpress SKS1, the SKS1 open reading
frame was amplified from genomic DNA and cloned into
XmaIXhoI-digested p426GPD . The GPD promoter was then
replaced with the ADH1 promoter amplified from genomic DNA
(21464 to 0) and cloned into SacI-XmaI-digested p426-GPD-SKS1
to generate plasmid pCK020.
Yeast gene deletions and site-directed mutagenesis
Gene deletion mutants were constructed in strains Y825 and
HLY337 using a one-step PCR-based gene-disruption strategy
[91,92] with the G418 resistance cassette from plasmid
pFA6aKanMX6 . Integrated point mutations were generated using
the one-step site-directed mutagenesis strategy described in Zheng
et al. . After confirming the haploid mutants via PCR and
sitedirected mutants via sequencing, the strains were allowed to mate
on YPD+G418 plates for approximately 20 hours at 30uC. Mated
cells were then streaked on SC-Trp-Leu plates to select for
Y8256HLY337 diploids. All yeast transformations were
performed according to standard lithium acetate-mediated protocols
Surface-spread filamentation assays
Defects in surface spread filamentation were assessed as
described . In brief, yeast strains were grown overnight and
were subsequently diluted to an OD600 of approximately 0.2 in
fresh media. Cells were grown for at least two doublings, to an
OD600 of approximately 0.61.0. Approximately 1 mL of each cell
culture was transferred to a microcentrifuge tube, where the
cultures were washed twice with sterile water before suspending in
1 mL sterile water and serially diluting such that the density of
plating was approximately102103 cells per plate; high-density
plating has been shown to decrease the rate at which cells
transition to the filamentous form . Diluted cultures were then
spread on SLAD and/or SLALD plates supplemented with
appropriate amino acids and incubated at 30uC for 3 or more
days. Cells were imaged using a Photometrics CoolSnapES2
digital camera mounted on a Nikon Eclipse 80i upright
microscope. Colony morphology was imaged using a 46objective,
while cellular morphology was imaged with a 1006 oil-immersion
Peptide sample preparation and phosphopeptide
S. cerevisiae Y825 control and sks1-K39R mutant cells were
isotopically labeled with medium (Lys-4/Arg-8) amino acids
during cell culture (SILAC). Cell cultures were lysed by bead
beating in lysis buffer; the lysis buffer was composed of 50 mM tris
buffer (pH 8.2), 8 M urea, and protease inhibitors (Roche) and
phosphatase inhibitors (50 mM NaF, 50 mM
beta-glycerophosphate, 1 mM sodium vanadate, 10 mM sodium pyrophosphate,
1 mM phenylmethylsulfonyl fluoride). Frozen cells were
suspended in 400 ml lysis buffer and were lysed by applying three cycles of
bead beating (for one minute each) with a 2-minute rest on ice
between cycles. Supernatants containing protein extract were
recovered by centrifugation at 14,000 g for 10 minutes, and
protein concentrations were measured by Bradford assay. Equal
amounts of protein from three SILAC-labeled cells were
combined, treated for disulfide reduction and alkylation, and
digested with TMPK-treated trypsin (Worthington Biochemical
Corp., Lakewood, NJ) at a trypsin:protein ratio of 1:10 at 37uC
Peptide mixtures were desalted with C18 (Waters) and
separated into 12 strong cation exchange (SCX) fractions on a
PolySulfoethyl A column (PolyLC, 15064 mm) over a 48 minute
salt gradient with two mobile phases: 100% solvent A (5 mM
KH2PO4, 30% acetonitrile, pH 2.7) for 5 minutes, a linear
gradient of 040% solvent B (250 mM KH2PO4, 30% acetonitrile,
pH 2.7) in the following 35 min, a stiff increase of 40100% B in
3 min, and flushing with 100% B for 5 min. Collected SCX
fractions were desalted with C18 (Waters) and subjected to
selective phosphopeptide enrichment using ZrO2 (Glygen, 50 mm
i.d. resin) under acidic conditions in the presence of 2,5-dihydroxy
benzoic acid [100,101]. Phosphopeptides selectively bound on
ZrO2 were eluted with 4% NH4OH. The ZrO2 eluate of enriched
phosphopeptides and the flow-through of each SCX fraction were
analyzed by nanoLC-tandem mass spectrometry (MSMS).
Mass spectrometric analysis and SILAC quantification
NanoLC-MSMS experiments were performed on a hybrid type
mass spectrometer (Thermo, LTQ-Orbitrap XL) coupled to a
nanoLC system (Eksigent, 2D nanoLC). Samples were separated
on a custom capillary column (150 mm675 mm, 3 mm Sepax
HPC18) using a 120 min linear aqueous gradient (990% acetonitrile,
0.01% formic acid) delivered at 250 nL/min. The eluent was
introduced on-line to the LTQ-Orbitrap via an electrospray
device (Advion, TriVersa NanoMate) in positive ion mode.
The LTQ-Orbitrap was operated in a data-dependent mode
alternating a full MS scan (3001700 m/z at 60,000 resolution
power at 400 m/z) in the Orbitrap analyzer and collision-induced
dissociation scans (CID-MSMS) for the 7 most abundant ions with
signal intensity above 500 from the previous MS scan in LTQ.
Recurring precursor ions were dynamically excluded for 30 sec by
applying charge-state monitoring, ions with 1 or unassigned
charge states were rejected to increase the fraction of ions
producing useful fragmentation. Lock mass ([(Si(CH3)2O)6]1+, m/
z = 445.120029) was used for internal calibration. Each sample
was analyzed by two LC-MS experiments. Raw LC-MS data file
sets were processed, database searched, and quantified using
MaxQuant (ver 188.8.131.52)  and the Mascot search engine
together. Mascot database searches were performed against a
composite database of forward and reverse sequences of verified
yeast open reading frames from the Saccharomyces Genome
Database. Variable modifications were allowed for oxidation (M)
and phosphorylations (STY), as well as a fixed modification of
carbamidomethylation (C). Peptide, protein, and phosphorylation
site identifications were filtered at a false discover rate of 5%. The
MaxQuant normalized M/L (medium/light) ratios with
significance B scores less than 0.05 were considered statistically
significant. 1068 peptides were identified, corresponding to 552
The mass spectrometry proteomics data have been deposited to
the ProteomeXchange Consortium (http://proteomecentral.
proteomeexchange.org) via the PRIDE partner repository 
with the dataset identifier PXD000414.
Identification of previously unreported phosphorylation
sites and network analysis
A network scaffold was constructed was constructed using
interactions from the publicly available Kyoto Encyclopedia of
Genes and Genomes (KEGG), GeneMania and BioGrid databases
. KEGG xml files for the glycolysis/gluconeogenesis
(accession sce00010), cell cycle (accession sce04111), meiosis
(accession sce04113) and MAPK signalling pathways (accession
sce04011) were downloaded and parsed using an in-house
program to create a network. The genes in the resulting network
were then uploaded to GeneMania in order to retrieve additional
genetic and physical interactions. Finally, the interactions for
SKS1 were downloaded from BioGrid and appended to the
Differentially phosphorylated proteins, identified by their
differentially abundant phosphopeptides upon enrichment, were
first filtered using the significance of the medium/light isotope
ratios; we implemented a significance (A) cut off at or below 0.05.
The resulting protein list was mapped on to the network scaffold
using Cytoscape . The network was clustered by node
attributes to reflect the pathways from which the genes originated.
As expected, the network consisted of three groups/sub-networks
(glycolysis/gluconeogenesis, cell cycle/meiosis, and MAPK
Assays for fitness and respiratory deficiency
Yeast strains were inoculated in 5 mL SC and incubated
overnight at 30uC with constant shaking (250 rpm). Cell cultures
were subsequently diluted in 6 mL of SC, SLAD, and SLALD to
an OD600 of approximately 0.1 and incubated at 30uC with
constant shaking (250 rpm) for approximately 15 hours. OD600
measurements were collected approximately every 3 hours from
the time of dilution. Full growth curve datasets for the analysis of
mutants in SLAD and SLALD media are provided in Tables S3
and S4, respectively. Assays for respiratory deficiency were
implemented as follows. Single colonies were inoculated in 5 mL
YPD media and incubated with continuous agitation overnight.
Cell cultures were diluted to an OD600 of approximately 0.3 in
fresh YPD media and grown at 30uC with shaking for at least two
doublings, to an OD600 of approximately 1.0. Each yeast cell
culture was then adjusted to an identical OD600 and serially
diluted 1021, 1022, 1023, and 1024, respectively. Subsequently,
5 mL of each diluted yeast culture was spotted onto YPD and YPG
plates and incubated at 30uC for three to five days.
RNA preparation and qRT-PCR analysis
Yeast strains were inoculated in 5 mL SC and incubated
overnight at 30uC with constant shaking (250 rpm). Cell cultures
were diluted to an OD600 of approximately 0.3 in fresh SC, SLAD,
and SLALD media and grown at 30uC with shaking for 4 hours.
Afterward, cell cultures were collected by centrifugation at 3000 g
for 5 minutes; the supernatant was removed, and cell pellets were
flash frozen in a dry ice/ethanol bath. Total RNA was extracted
using the RiboPure Yeast Kit (Ambion) following the
manufacturers protocol. First-strand cDNA synthesis was performed using
the Superscript II Reverse Transcriptase Kit (Invitrogen) with 2 mg
of total RNA as template and Oligo d(T)1218 as primers according
to the manufacturers protocol. Quantitative real-time assays were
performed in triplicate with a Mastercycler EP Realplex4 S
(Eppendorf) using SYBR Green I dye-based detection (Life
Technologies). Each reaction contained 10 mL SYBR Green
PCR Master Mix (Life Technologies), 0.2 mM of the appropriate
primers, and 120 ng of cDNA template in a total volume of 20 mL.
The real time PCR reactions were performed at 95uC for
5 minutes followed by 40 cycles of 30 seconds at 95uC, 30 seconds
at 60uC, and a final step at 72uC for 30 seconds. Relative
differences in RNA levels were normalized against ACT1 levels
using the delta delta CT method .
Yeast strains were analyzed by Western blotting according to
standard protocols . For Western analysis, 10 mL of protein
sample were separated via SDS-PAGE and transferred to
ImmunBlot PVDF (Bio-Rad) using standard methods. Protein detection
was carried out using antibodies against Hemagglutinin (HA)
(1:2000; Abcam) in TBS+0.1% Tween20 and 5% milk. After
immunodetection of Hemagglutinin, the membrane was stripped
using Stripping Buffer (62.5 mM Tris pH 6.8, 100 mM
bmercaptoethanol, and 2% SDS) at 65uC for 30 minutes with
occasional agitation. Normalization of loading was achieved by
probing the original membrane with antibodies against yeast
3phosphoglycerate kinase Pgk1p (1:5000; Invitrogen) in the buffer
conditions used previously.
Observation of mitochondrial morphology
Mitochondrial morphology was scored using MitoTracker
CMXR (Molecular Probes) for labeling the mitochondrial
membrane and 49,6-diamidino-2-phenylindole (DAPI) for labeling
mitochondrial DNA. MitoTracker was added to 1 mL aliquots of
each cell culture to a final concentration of 0.5 mM, and
the samples were incubated at 30uC for 30 minutes similar to
Nunnari et al. . 3 mL of stained culture was then mixed
with 3 mL of DAPI mounting media (9.25 mM
p-Phenylenediamine (Sigma), 0.18 mM DAPI (Sigma), in glycerol) on a glass
slide . The cell suspension was then covered with a
glass coverslip and imaged using a Photometrics CoolSnapES2
digital camera mounted on a Nikon Eclipse 80i upright
Phenotypic analysis of C. albicans sha3D/SHA3
Construction of the heterozygous C. albicans sha3::CdHIS1/SHA3
and homozygous sha3::CdHIS1/sha3::ARG4 deletion mutants was
performed using the transformation methods described in Walther
et al. . Wild-type and mutant colonies were grown overnight
at 30uC in 3 ml YPD or SC media minus the appropriate amino
acids and supplemented with uridine. To assess colony
morphology, cell cultures were diluted to an OD600 of approximately 0.25
in fresh YPD+uri and grown at 30uC with shaking for at least two
doublings, to an OD600 of approximately 0.61.0. 5 ml of each
culture was spotted onto YPD+uri, YPD+uri+10% serum, and
Spider plates. After drying on the bench, YPD+uri plates were
incubated at 30uC and YPD+uri+10% serum and Spider plates
were incubated at 37uC for 35 days.
For the study and measurement of C. albicans biofilm
development, we used a metabolic
2,3-bis(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction
assay as described in Pierce et al.  with slight modifications.
Briefly, triplicate cell cultures were grown overnight at 30uC in SC
media. The cultures were centrifuged, washed twice with 16 PBS
and then resuspended in pre-warmed (37uC) medium (RPMI
1640-MOPS or Spider) at a final concentration of OD520 = 0.38, a
cell concentration that was demonstrated to correlate with
optimum biofilm formation . Subsequently, 100 mL of each
culture was added in triplicate to a 96-well plate. The plate was
then incubated at 37uC with shaking (100 rpm) for 24 hours. After
24 hours, the wells were washed 3 times with 16 PBS, and100 mL
of pre-warmed SC media was added to each well followed by
shaking (100 rpm) at 37uC for an additional 8 hours. Post
incubation, the media was removed and the XTT assay performed
as described .
Dataset S1 Listing of phosphopeptides identified in this study by
SILAC-based quantitative phosphoproteomics. Each
phosphopeptide sequence is presented; ph indicates the predicted site of
phosphorylation. The corresponding protein for each peptide is
presented, with standard and systematic yeast names. The
normalized ratio of M/L isotope is indicated along with the
significance. Phosphopeptides identified multiple times are
presented as such with their respective M/L ratios and significance
Dataset S2 Listing of genetic and physical interactions used to
construct the Sks1p signaling connectivity network. Each binary
interaction is represented in a row; the source of the interaction
data (e.g., KEGG, GeneMania, BioGrid) is indicated. Standard
protein names are used as available.
Strains used in this study.
Plasmids used in this study.
Table S4 Growth curve datasets for the analysis of S. cerevisiae
strains in low-nitrogen/low-glucose (SLALD) media. Cell growth
is approximated by optical density readings at a wavelength of
660 nm. Optical density measurements are presented as the
average of triplicate experiments.
We thank Mark Johnston, Kobi Simpson, Jason E. Gestwicki, and Daniel J.
Klionsky for reagents and/or helpful discussions regarding this manuscript.
Conceived and designed the experiments: CJ HKK DM AIN PCA AK.
Performed the experiments: CJ HKK DS CAS DM. Analyzed the data: CJ
HKK DS CAS DM AIN PCA AK. Contributed reagents/materials/
analysis tools: CAS DS. Wrote the paper: CJ DM HKK PCA AK.
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