FEMS Yeast Research, 15, 2015, fov007
doi: 10.1093/femsyr/fov007
Research Article
RESEARCH ARTICLE
Vcx1 and ESCRT components regulate intracellular
pH homeostasis in the response of yeast cells
to calcium stress
Klara Papouskova1,∗ , Linghuo Jiang2,3 and Hana Sychrova1
1
Department of Membrane Transport, Institute of Physiology Academy of Sciences of the Czech Republic, v.v.i.,
Videnska 1083, 142 20 Prague 4, Czech Republic, 2 The National Engineering Laboratory for Cereal Fermentation
Technology, School of Biotechnology, Jiangnan University, Wuxi 214122, China and 3 The State Key Laboratory
of Food Science and Technology, School of Biotechnology, Jiangnan University, Wuxi 214122, China
∗ Corresponding author: Department of Membrane Transport, Institute of Physiology Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083,
142 20 Prague 4, Czech Republic. Tel: +420 24106 2419; Fax: +420 24106 2488; E-mail: [email protected]
One sentence summary: The vacuolar Ca2+ /H+ antiporter Vcx1 and some components of the highly conserved ESCRT pathway regulate intracellular pH
of Saccharomyces cerevisiae cells in the response to calcium stress.
Editor: Dr. Ian Dawes
ABSTRACT
Endosomal sorting complexes required for transport (ESCRTs) are involved in the formation of multivesicular bodies and
sorting of targeted proteins to the yeast vacuole. The deletion of seven genes encoding components of the ESCRT
machinery render Saccharomyces cerevisiae cells sensitive to high extracellular CaCl2 concentrations as well as to low pH in
media. In this work, we focused on intracellular pH (pHin ) homeostasis of these mutants. None of the studied ESCRT
mutants exhibited an altered pHin level compared to the wild type under standard growth conditions. Nevertheless, 60 min
of CaCl2 treatment resulted in a more significant drop in pHin levels in these mutants than in the wild type, suggesting that
pHin homeostasis is affected in ESCRT mutants upon the addition of calcium. Similarly, CaCl2 treatment caused a bigger
pHin decrease in cells lacking the vacuolar Ca2+ /H+ antiporter Vcx1 which indicates a role for this protein in the
maintenance of proper pHin homeostasis when cells need to cope with a high CaCl2 concentration in media. Importantly,
ESCRT gene deletions in the vcx1 strain did not result in an increase in the CaCl2 -invoked drop in the pHin levels of cells,
which demonstrates a genetic interaction between VCX1 and studied ESCRT genes.
Key words: calcium; ESCRT; pHin ; Vcx1
INTRODUCTION
Calcium ions, ubiquitously used in eukaryotic cells as signalling
molecules, are essential for living organisms. In the yeast Saccharomyces cerevisiae, calcium signalling is involved in cell responses
to various stimuli, such as mating pheromones (Iida et al. 1990),
hypotonic or hypertonic shock (Batiza et al. 1996; Denis and Cyert
2002), alkaline pH (Viladevall et al. 2004) or endoplasmic reticulum stress (Bonilla and Cunningham 2003).
The optimal cytosolic Ca2+ concentration is tightly regulated
and maintained by a precise interplay of several transporters
(Cui et al. 2009b). Extracellular calcium can enter the cells via
unknown transporters designated as X and M (Cui et al. 2009a)
or the plasma membrane channel composed of the proteins
Cch1, Mid1 and Ecm7 (Iida et al. 1994; Fischer et al. 1997; Locke
et al. 2000; Martin et al. 2011). Ca2+ release from intracellular
stores in the vacuole is mediated by the Yvc1 channel (Denis and
Cyert 2002). The increase in cytosolic calcium concentration is
Received: 8 October 2014; Accepted: 6 February 2015
C FEMS 2015. All rights reserved. For Permissions, please e-mail: [email protected].
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FEMS Yeast Research, 2015, Vol. 15, No. 2
Table 1. list of strains used in this study.
Strain
Genotype
Source
BY4741
vps20
vps25
vps28
vps36
snf7
snf8
stp22
vcx1
snf7vcx1 (YYSC446)
vps20vcx1 (YYSC448)
vps36vcx1 (YYSC194)
MATa his31 leu20 met150 ura30
BY4741 vps20::kanMX4
BY4741 vps25::kanMX4
BY4741 vps28::kanMX4
BY4741 vps36::kanMX4
BY4741 snf7::kanMX4
BY4741 snf8::kanMX4
BY4741 stp22::kanMX4
BY4741 vcx1::kanMX4
BY4741 snf7::kanMX4vcx1::natR
BY4741 vps20::kanMX4vcx1::natR
BY4741 vps36::kanMX4vcx1::natR
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Dr. Y. Zhao
Dr. Y. Zhao
Dr. Y. Zhao
sensed by calmodulin molecules and leads to the activation of
effector proteins, such as the protein phosphatase calcineurin
(Cunningham 2011). Activated calcineurin dephosphorylates the
zinc-finger transcription factor Crz1, which leads to its nuclear
localization (Matheos et al. 1997; Stathopoulos and Cyert 1997).
The calcium/calcineurin signalling pathway positively regulates
the expression of the genes PMC1 and PMR1 (Cunningham and
Fink 1996; Stathopoulos and Cyert 1997) encoding Ca2+ -ATPases
required to prevent sustained calcium accumulation in the cytosol. Ca2+ ions are sequestrated into the vacuole via both Pmc1
Ca2+ -ATPase and the Ca2+ /H+ antiporter Vcx1 (Pittman 2011).
The other Ca2+ -ATPase, Pmr1, supplies calcium to the ER and
Golgi apparatus (Strayle et al. 1999; Cunningham 2011), and excessive calcium ions in the secretory pathway are extruded via
exocytosis. Recently, the existence of a Ca2+ /H+ antiporter in the
Golgi membranes, Gdt1, was suggested (Demaegd et al. 2013). As
most yeast calcium transporters have their functional counterparts in higher eukaryotes, the studies in S. cerevisiae can help to
understand the mechanisms regulating calcium homeostasis in
higher eukaryotes, plants and mammals.
Endosomal sorting complexes required for transport (ESCRTs) are well conserved in eukaryotic cells. They are involved
in the formation of multivesicular bodies and sorting of targeted proteins to the vacuole or its mammalian equivalent, the
lysosome (Katzmann et al. 2002). The ESCRT machinery mainly
consists of four protein complexes designated as ESCRT-0, −I,
−II and −III, respectively, and several accessory components
(Hurley 2010). The conservation of multivesicular body components from yeast to humans highlights the importance of ESCRT complexes in eukaryotic organisms. They are implicated
in a variety of processes, including plasma membrane receptor downregulation, the sorting of newly synthesized degradative enzymes to the vacuole/lysosome, cell division, autophagy
(Hurley 2010) or plasma membrane repair (Jimenez et al. 2014).
Most membrane-enveloped viruses make use of the host ESCRT
machinery to facilitate their budding from cells. Moreover, dysfunctions of ESCRT complexes are also connected to cancer or
the development of neurodegenerative disorders (Hurley 2010).
In S. cerevisiae, deletions of genes encoding seven ESCRT components (Snf7, Snf8, Stp22, Vps20, Vps25, Vps28 and Vps36) render cells sensitive to high extracellular calcium concentrations
(Bowers et al. 2004; Zhao et al. 2013a). Strains lacking these genes
exhibit increased levels of calcium accumulation in response to
calcium stress and elevated activation levels of calcineurin signalling (Zhao et al. 2013b). ESCRT mutants lack the proteolytic
activation of the Rim101 transcription factor (Xu et al. 2004). As
a consequence, PMR1 expression is reduced in these mutants
through the Rim101/Nrg1 pathway (Zhao et al. 2013b) and thus
the ability to pump Ca2+ into the ER and Golgi lumens might be
diminished.
All calcium-sensitive ESCRT deletion mutants were previously identified as vps (vacuolar protein sorting) class E mutants
(Raymond et al. 1992). The growth of these ESCRT mutants was
also found to be sensitive to low extracellular pH (Kallay et al.
2011), which suggests that intracellular pH (pHin ) homeostasis
might be affected in these mutants. The aim of this work was to
investigate if the calcium sensitivity of ESCRT mutants is connected to their pHin homeostasis.
MATERIALS AND METHODS
Strains, media and growth tests
The S. cerevisiae wild-type strain BY4741 (MATa his31 leu20
met150 ura30) and its isogenic mutants used in this work
are listed in Table 1. Strains were routinely maintained in nonbuffered YPD medium (pH 6.2, ForMediumTM , UK). After transformation, cells were kept in YNB medium supplemented with
2% glucose and the BSM mixture of auxotrophic supplements
without uracil (ForMediumTM , UK). For pHin measurements,
strains were grown in low-fluorescence YNB medium without
ammonium sulphate, folic acid and riboflavin (MP Biomedicals,
USA) supplemented with 0.4% ammonium sulphate, 2% glucose and the BSM mixture of auxotrophic supplements without
uracil (starting pH 4.0). Glucose-induced medium acidification
was measured in YNB medium without amino acids (DifcoTM ,
USA) containing 20 μg ml−1 of appropriate auxotrophic supplements (starting pH 4.5). 2% agar was used for solid YPD media.
Calcium and magnesium tolerance of strains was tested in a
drop test experiment. 10-fold serial dilutions of cell suspensions
were spotted on YPD plates supplemented as indicated. Growth
was recorded after two days.
Measurement of pHin
The pHin of the strains was monitored using pHluorin [a pH sensitive ratiometric GFP variant (Miesenbock et al. 1998)]. Cells were
transformed with plasmid pHl-U, the pVT100U plasmid containing the pHluorin gene (Maresova et al. 2010), and grown in the
low-fluorescence YNB medium to the logarithmic growth phase
(OD600 0.5–0.6). The fluorescence intensity ratios of growing cells
were measured using a SynergyHT microplate reader (BioTek,
Papouskova et al.
USA) with a 516/20 nm emission filter and 400/30 and 485/20 nm
excitation filters in 20 wells for each strain, and a mean value
was calculated. To determine the pHin values from the fluorescence ratios, a calibration curve was obtained as described previously (Brett et al. 2005; Maresova et al. 2010).
Since calcium precipitation was observed in minimal YNB
medium when a high CaCl2 (0.2 M) concentration was added,
we monitored pHin of cells in response to CaCl2 addition in YPD
medium. Therefore, cells were first grown in YNB medium (to
maintain the pHl-U plasmid) to the logarithmic growth phase
(OD600 0.5–0.6), collected by centrifugation, and then resuspended in YPD medium at a 50% higher cell concentration to
enhance the fluorescent signal/noise ratio of the samples. Cell
suspensions were divided into two parallels and incubated for
60 min to allow cells to recover from the change in media. Subsequently, CaCl2 was added to one of the two parallels of cells
to a final concentration of 0.2 M; the other parallel was not
treated with CaCl2 . In 5 and 60 min, the fluorescence intensity ratios of these two parallel samples were measured in a
microplate reader (see above). The same procedure was used
to monitor pHin of cells in response to MgCl2 addition in YPD
medium.
Data are presented as means ± SD from at least three independent experiments (16–20 aliquots of cells were measured for
each strain/condition in individual experiments).
Glucose-induced medium acidification
Glucose-induced medium acidification was measured using the
method described previously (Maresova and Sychrova 2007).
Briefly, cells from an overnight culture grown in YPD were
washed with YNB medium without glucose and suspended in
the same medium containing the pH indicator (0.01% bromocresol green sodium salt, Fluka, Sigma-Aldrich) to OD600 0.1.
After 60 min, medium acidification was started by the addition of glucose to a final concentration of 2%. Changes
in absorbance (595 nm) were recorded in an ELx808 Absorbance Microplate Reader (BioTek Instruments, USA). The
experiments were repeated three times with very similar
results.
3
Statistical analysis
Statistically significant differences were analysed by the Student
t-test using MS Office Excel (in case of comparing two groups
of data) or by ANOVA analysis followed by Dunnett’s multiple
comparison test using GraphPad Prism 6 if more than two groups
of data were compared.
RESULTS
Deletions of seven ESCRT genes do not affect pHin
of yeast cells growing under standard conditions
First, we examined the effect of deletions of seven ESCRT genes
on the pHin homeostasis of exponentially growing cells in YNB
medium. The pHin values of ESCRT mutants ranged from 6.902
± 0.037 for the snf8 mutant to 7.018 ± 0.038 for the stp22
mutant; these values were not significantly different from the
value obtained for BY4741 (6.957 ± 0.068, Fig. 1). This suggests
that the deletion of any of the seven studied ESCRT genes
does not affect the pHin homeostasis of yeast cells growing
in YNB.
Deletions of ESCRT genes affect the pHin homeostasis
of yeast cells in response to calcium stress
Previous studies have shown that all seven studied ESCRT mutants are sensitive to high extracellular concentrations of calcium (Bowers et al. 2004; Zhao et al. 2013a). Therefore, we examined the effect of calcium stress on the pHin homeostasis
of these mutants. The pHin values of cells in response to 0.2 M
CaCl2 treatment were measured in YPD medium, since the addition of CaCl2 to a final concentration of 0.2 M caused precipitation in YNB. The pHin values of cells treated with CaCl2 were always compared to the pHin levels of control cells to which CaCl2
was not added.
The transfer of cells into YPD resulted in a decrease in their
pHin values; the pHin of cells incubated in YPD was about 0.3–
0.4 pH units lower than the pHin values of cells growing in YNB
(compare Fig. 1 with Fig. 2A). This difference was similar for all
7.27,2
7.07
pHin
pHin
6.86,8
6.66,6
6.46,4
6.26,2
6.06
BY4741
vps20Δ
vps25Δ
vps28Δ
vps36Δ
snf7Δ
snf7Δ
vps36Δ
BY4741
vps28Δ
vps25Δ
vps20Δ
snf8Δ
snf8Δ
stp22Δ
stp22Δ
Figure 1. intracellular pH of calcium-sensitive ESCRT mutants. Cells were grown to exponential growth phase (OD600 0.5–0.6) in low-fluorescent YNB medium and their
pHin values were determined.
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FEMS Yeast Research, 2015, Vol. 15, No. 2
(A)
7.2
7,2
7.07
pHin
pHin
6.8
6,8
6,6
6.6
6,4
6.4
6,2
6.2
6.06
min
BY4741
BY4741
vps20
vps20Δ
vps25
vps25Δ
5
5
5
60
60
vps28
vps28Δ
60
5
vps36
vps36Δ
60
5
60
snf7
snf7Δ
5
60
snf8
snf8Δ
5
60
stp22
stp22Δ
5
60
(B)
7,2
7.2
7.07
pHin
pHin
6,8
6.8
6,6
6.6
6,4
6.4
6.2
6,2
6.06
min
BY4741
BY4741
vcx1
vcx1Δ
5
5
60
60
vps20vcx1
vps36vcx1
snf7vcx1
snf7Δvcx1Δ
vps20Δvcx1Δ
vps36Δvcx1Δ
5
60
5
60
5
60
Figure 2. pHin response of cells to 0.2 M CaCl2 treatment. A—pHin response of the wild type BY4741 and calcium-sensitive ESCRT mutants to CaCl2 treatment; B—pHin
response of BY4741, vcx1 and double-gene deletion mutants to CaCl2 treatment. Cells grown into exponential growth phase (OD600 0.5–0.6) in YNB medium were
collected, transferred into YPD, and their pHin responses to 0.2 M CaCl2 treatment were examined in 5 and 60 min. White bars—pHin of control cells (not treated with
0.2 M CaCl2 ), grey bars—pHin of cells treated with 0.2 M CaCl2 for 5 or 60 min, respectively.
ESCRT mutants and the wild type. The pHin was relatively stable
in all strains during the 60 min of measurement in YPD (Fig. 2A,
cells untreated with CaCl2 , white bars).
The addition of 0.2 M CaCl2 to the YPD medium resulted in
a decreased pHin level in all ESCRT mutants and the wild type
compared to the pHin levels of corresponding CaCl2 -untreated
cells (Fig. 2A, Table 2). Five minutes after the calcium addition,
the 0.2 M CaCl2 -invoked drop in the pHin levels was similar in
the wild type BY4741 and most of the studied ESCRT mutants
(approximately 0.1–0.15 pH units), but about twice as high in the
snf7 and snf8 strains (Table 2). 60 min after the calcium addition, the pHin drop in the wild type was partially recovered
from 0.104 to 0.062. However, the pHin drops in all ESCRT mutants still remained at least twice as high than in the wild type
(Table 2). Taken together, these data suggest that wild-type cells
cope more effectively with the pHin drop provoked by the addition of 0.2 M CaCl2 than the ESCRT mutants, and the deletion of
each of the studied ESCRT genes leads to a defect in the regulation of pHin homeostasis in the response of yeast cells to extracellular calcium stress.
5
Papouskova et al.
Table 2. drop of pHin upon 0.2 M CaCl2 treatment in BY4741 and ESCRT mutant cells. Cells grown to exponential growth phase (OD600
0.5–0.6) in YNB medium were collected, transferred into YPD and
their pHin responses to 0.2 M CaCl2 were examined after 5 or 60 min
of calcium treatment.
Table 3. drop of pHin upon 0.2 M CaCl2 treatment in BY4741,
vcx1 and double-gene deletion mutants. Cells grown to exponential
growth phase (OD600 0.5–0.6) in YNB medium were collected, transferred into YPD and their pHin responses to 0.2 M CaCl2 were examined after 5 or 60 min of calcium treatment.
pHin drop = pHin (control) – pHin (CaCl2 )
Strain
5 min
BY4741
vps20
vps25
vps28
vps36
snf7
snf8
stp22
0.104 ± 0.093
0.114 ± 0.080
0.135 ± 0.055
0.127 ± 0.072
0.135 ± 0.059
0.214 ± 0.057
0.254 ± 0.065
0.173 ± 0.055
pHin drop = pHin (control) – pHin (CaCl2 )
60 min
Strain
±
±
±
±
±
±
±
±
BY4741
vcx1
vps20vcx1
vps36vcx1
snf7vcx1
0.062
0.156
0.133
0.136
0.144
0.154
0.146
0.132
0.044
0.065∗
0.045
0.021
0.024∗
0.014∗
0.041∗
0.008
pHin (control)—intracellular pH of control cells not treated with 0.2 M CaCl2 .
pHin (CaCl2 )—intracellular pH of cells treated with 0.2 M CaCl2 .
∗
Indicates values significantly different from the value obtained for BY4741 (P <
0.05).
To verify that the observed bigger pHin drops in ESCRT mutants are specific to CaCl2 treatment of cells, we used the same
procedure to monitor pHin changes of cells upon 0.2 M MgCl2
treatment. In contrast to CaCl2 , 0.2 M MgCl2 does not seem to
inhibit the growth of any of the studied ESCRT mutant strains
(Fig. S1, Supporting Information). When MgCl2 was added to
cells, we observed an initial variable drop in pHin levels ranging
from 0.021 to 0.112 pH units in all tested strains. This pHin drop
was completely or almost completely recovered after 60 min of
MgCl2 treatment both in wild-type cells and in all ESCRT mutants (summarized in Table S1, Supporting Information). Therefore, MgCl2 treatment (in contrast to calcium treatment) does
not seem to affect pHin homeostasis in ESCRT mutants.
Vcx1 and ESCRT components regulate the pHin
homeostasis of yeast cells through a common pathway
The vacuolar Ca2+ /H+ antiporter Vcx1 is able to rapidly sequester Ca2+ into the vacuole following a high burst of cytosolic
Ca2+ (Miseta et al. 1999; Cui et al. 2009a). Moreover, as a protein
translocating protons, Vcx1 might be important for pHin homeostasis regulation both in the vacuolar lumen and in the cytosol.
Therefore, we examined the effect of VCX1 deletion on pHin
homeostasis. In YNB medium, there was no significant difference in the pHin level between the vcx1 mutant (6.977 ± 0.049)
and the wild type (6.957 ± 0.068), suggesting that VCX1 deletion
does not significantly affect the pHin homeostasis of cells growing in YNB medium.
We next investigated if the Vcx1 antiporter has any effect
on pHin regulation upon cell treatment with CaCl2 . Using the
same approach as for ESCRT mutants, we determined the pHin
response to calcium addition in cells lacking the VCX1 gene.
Similarly to the situation in BY4741 and ESCRT mutants, the
transfer of vcx1 cells grown in YNB medium into YPD resulted
in a decrease in the pHin level of cells (approximately 0.3 pH
units). However, the vcx1 mutant exhibited a slightly, but significantly (n = 4, P < 0.05), higher pHin level than the wild type
in YPD within the 60 min of incubation in this medium (Fig. 2B).
Compared to wild-type BY4741, calcium invoked a significantly higher drop of the pHin in the vcx1 mutant in 5 min,
which was only partially recovered in 60 min of the 0.2 M CaCl2
treatment (Fig. 2B, Table 3). These data suggest that the Vcx1
5 min
0.104
0.235
0.215
0.262
0.270
±
±
±
±
±
0.093#
0.039∗
0.078
0.067∗
0.045∗
60 min
0.062
0.175
0.169
0.166
0.166
±
±
±
±
±
0.044#
0.034∗
0.027∗
0.076
0.009∗
pHin (control)—intracellular pH of control cells not treated with 0.2 M CaCl2 .
pHin (CaCl2 )—intracellular pH of cells treated with 0.2 M CaCl2 .
∗
Indicates values significantly different from the value obtained for BY4741 (P <
0.05).
#
Indicates values significantly different from the value obtained for the vcx1
strain (P < 0.05).
antiporter plays a role in the proper maintenance of cell pHin
homeostasis in response to extracellular calcium stress.
To investigate if the Vcx1 antiporter is important for pHin regulation upon calcium addition in calcium-sensitive ESCRT mutants, we determined the pHin response to CaCl2 treatment in
cells lacking the VCX1 gene in addition to three ESCRT genes
(VPS20, VPS36 and SNF7). The vps20, vps36 and snf7 strains
were the ESCRT mutants which exhibited the highest degrees of
pHin drops upon 60 minutes of 0.2 M CaCl2 treatment (Table 2).
The addition of the VCX1 gene deletion in three tested ESCRT
mutants resulted in an increase in the pHin levels of cells incubated in YPD, which was most significant in the vps20vcx1
and vps36vcx1 mutants (compare the values obtained for single ESCRT and double-gene deletion mutants not treated with
CaCl2 , Fig. 2A and B). Nevertheless, the way how VCX1 deletion
affects pHin levels of cells upon their transfer into YPD still remains to be elucidated.
Importantly, in all three double-gene mutants, 0.2 M CaCl2
treatment invoked similar decreases in pHin levels as in the
vcx1 mutant (Table 3). Although the cytosolic H+ concentrations varied among the studied strains due to their different pHin
levels in YPD, 60 min of CaCl2 treatment resulted in 1.45 to 1.49
times increased cytosolic proton concentrations both in the single vcx1 mutant and double-gene deletion mutants, while H+
concentration was only 1.16 times increased in wild-type cells.
These findings suggest that pHin recovery upon calcium treatment is affected to a similar extent in all vcx1 mutants.
Taken together, our results suggest a role for Vcx1 in the
maintenance of pHin homeostasis upon CaCl2 treatment of
BY4741 cells and a genetic interaction between the VCX1 and
ESCRT genes, as ESCRT gene deletions in the vcx1 strain did
not result in an increase in the CaCl2 -invoked drops in the pHin
levels of cells. The VCX1 and ESCRT genes seem to regulate pHin
homeostasis upon calcium treatment through a common pathway in yeast cells.
Deletions of seven ESCRT genes affect glucose-induced
medium acidification
Our data obtained in pHin measurements suggest that the activity of H+ -ATPase(s) might be affected in studied mutant strains
(discussed below). Therefore, we monitored glucose-induced
medium acidification both in ESCRT and vcx1 mutants.
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FEMS Yeast Research, 2015, Vol. 15, No. 2
Table 4. medium acidification by ESCRT and vcx1 mutant strains.
Cells were suspended in YNB medium containing pH indicator and
medium acidification was recorded within 180 min upon glucose
addition.
Strain
Drop in pH of medium
BY4741
vps20
vps25
vps28
vps36
snf7
snf8
stp22
vcx1
0.511 ± 0.052
0.263 ± 0.014∗ ∗ ∗
0.241 ± 0.025∗ ∗ ∗
0.278 ± 0.024∗ ∗ ∗
0.257 ± 0.022∗ ∗ ∗
0.257 ± 0.035∗ ∗ ∗
0.253 ± 0.019∗ ∗ ∗
0.282 ± 0.021∗ ∗ ∗
0.481 ± 0.027
∗∗∗
Indicates values significantly different from the value obtained for BY4741 (P
< 0.001).
4,6
4.6
4,2
4.2
4.04
Extracellular pH
Extracellular pH
4,4
4.4
3,8
3.8
3,6
3.6
3,4
3.4
3,2
3.2
3.03
00
50
50
100
100
Time (min)
Time (min)
150
150
200
200
Figure 3. comparison of medium acidification by BY4741 (diamonds), vps20
(squares) and vcx1 (triangles) cells. Cells were suspended in YNB medium containing pH indicator and medium acidification was started by glucose addition
(time 0 min). Closed symbols indicate 2% glucose added to cell suspensions.
Open symbols indicate no glucose added.
During 180 min of measurement, the wild-type strain exhibited
a higher acidification rate than all tested ESCRT mutants (Table 4
and Fig. 3 with the vps20 mutant shown as a representative ESCRT mutant strain). Glucose-induced medium acidification did
not differ among various ESCRT deletion strains as these mutants caused similar drops in pH levels of the medium. The acidification rate of the vcx1 mutant was not significantly different
from that of the wild-type strain.
DISCUSSION
Intracellular ion homeostasis is crucial to all living organisms,
including yeasts, and ESCRT complexes seem to play a very
complex role in its regulation. As they are involved in the
formation of multivesicular bodies, ESCRT complexes are indispensable for sorting of ubiquitinated ion transporters into the
vacuole. Endocytic regulation of ion transporters by ubiquitination is important not only in the removal of damaged or misfolded proteins, but also in the remodelling of protein composition in the plasma membrane in response to environmental
changes, and several metal ion transporters are known to be regulated by ubiquitination (Mulet et al. 2013). Ubiquitination also
targets the Pma1–7 mutant of the plasma membrane H+ -ATPase
to the endosomal/vacuolar pathway for degradation, probably
due to the Golgi-based quality control of a misfolded isoform
(Pizzirusso and Chang 2004).
Nevertheless, ESCRT complexes play additional roles in the
maintenance of cell ion homeostasis. Deletions of ESCRT components result in cell sensitivity to the presence of high sodium
and lithium concentrations; a functional connection between
endosomal sorting functions and the plasma membrane Na+ ATPase Ena1 was suggested (Warringer et al. 2003; Bowers et al.
2004; Logg et al. 2008).
Furthermore, deletions of seven tested ESCRT components
have been found to render cells sensitive to the presence of high
calcium concentrations in the media (Bowers et al. 2004; Zhao
et al. 2013a). This calcium hypersensitivity is mainly due to a defect of ESCRT mutants in the proteolytic processing of the transcriptional repressor Rim101, leading to an increased expression
level of one of the Rim101 targets, another transcriptional repressor, Nrg1. Nrg1 in turn represses expression of the ER/Golgi
Ca2+ -ATPase, Pmr1, in these mutants (Xu et al. 2004, Rothfels
et al. 2005; Zhao et al. 2013b). On the other hand, the expression
of the PMC1 gene encoding the vacuolar Ca2+ -ATPase seems to
be not affected in these ESCRT mutants (Zhao et al. 2013b). In addition, the growths of mutants lacking each of the seven studied
ESCRT genes have also been found to be sensitive to low pH in
the media (Kallay et al. 2011). In this study, we focused on the
pHin homeostasis of ESCRT mutants upon CaCl2 treatment.
pHin determines the relative protonation state of all proteins
and weak-acid compounds of the cytosol and contributes to the
regulation of many processes in the cell, as most enzymes have
their own specific pH optima. While pHin is a very dynamic parameter in the normal life of yeast, genetically it is a tightly controlled cellular parameter (Orij et al. 2012). Although the pHin of
studied ESCRT mutants was not affected when cells were growing in YNB medium, we have shown in this study that ESCRT
mutants fail to maintain a proper pHin level upon 0.2 M CaCl2
treatment. This result suggests that the pHin homeostasis of
yeast cells is regulated by ESCRT components in response to high
extracellular calcium concentrations.
Interestingly, the calcium hypersensitivity of studied ESCRT
mutants is connected to the activation of the protein phosphatase calcineurin, but not its downstream target, the transcription factor Crz1 (Zhao et al. 2013b). In addition to regulating
the transcription factor Crz1, activated calcineurin inhibits the
activity of the vacuolar Ca2+ /H+ antiporter Vcx1, which is another key player in Ca2+ sequestration into the vacuole and thus
the regulation of cytosolic Ca2+ concentration (Cunningham and
Fink 1996). Consistently, the deletion of the VCX1 gene has no effect on the sensitivity of seven studied ESCRT mutants to high
CaCl2 concentrations (Jiang and Zhao, unpublished data). Therefore, we decided to investigate if the decreased activity of Vcx1
antiporter due to calcineurin upregulation might be the cause of
the bigger pHin drops observed in ESCRT mutants upon calcium
treatment. In the vcx1 mutant strain, we measured a more
significant CaCl2 -invoked pHin decrease than in BY4741, which
demonstrates that the Vcx1 antiporter is involved in the maintenance of proper pHin homeostasis upon the calcium treatment
of cells. Importantly, ESCRT gene deletions in the vcx1 strain
did not cause further increase in the pHin drops invoked by cell
treatment with 0.2 M CaCl2 , which shows a genetic interaction
between VCX1 and ESCRT genes. As calcineurin seems to inhibit
the Vcx1 antiporter in ESCRT mutants, we hypothesize that Vcx1
Papouskova et al.
inhibition might affect pHin homeostasis upon CaCl2 addition in
these mutants similarly to VCX1 deletion in the vcx1 strain.
Several lines of experimental evidence document that the
deletion of genes encoding intracellular cation/H+ antiporters
can affect pHin homeostasis in yeast. Acid-stressed cells lacking
the endosomal Na+ , K+ /H+ antiporter Nhx1 exhibited a lower
pHin level than wild-type cells (Brett et al. 2005). This observation was explained as an indirect consequence of excessive
acidification of the vacuole in the nhx1 strain and downregulation of plasma membrane and/or vacuolar H+ -ATPases in order to maintain appropriate pH gradients across these membranes. The deletion of the VNX1 gene encoding the vacuolar
Na+ , K+ /H+ antiporter Vnx1 further decreased the pHin level
of yeast cells lacking the Trk1 and Trk2 potassium importers
(Petrezselyova et al. 2013). In our experiments, the pHin homeostasis of cells lacking the Vcx1 Ca2+ /H+ antiporter was affected
when the cells were treated with a high CaCl2 concentration,
therefore under conditions when Vcx1 is thought to help the
cells to sequestrate calcium ions into the vacuole. We hypothesize that the loss of Vcx1’s function might have an impact on
the activity of H+ -ATPase(s), as was proposed for cells lacking
the Nhx1 antiporter. The perturbation of H+ -ATPase activities
at both vacuolar and plasma membranes in mutants lacking
vacuolar Ca2+ /H+ antiporters, suggesting a close interplay between the Ca2+ /H+ exchangers and H+ pumps, was shown in
the plant Arabidopsis thaliana (Barkla et al. 2008). The activation
of calcineurin phosphatase and the consequent calcineurinmediated Vcx1 inhibition in studied calcium-sensitive ESCRT
mutants might affect the activity of H+ -ATPase(s) and pHin
homeostasis of these mutants upon calcium treatment similarly
to the absence of Vcx1 in the strain lacking the gene encoding
this antiporter.
In yeast, the activity of both plasma membrane H+ -ATPase
Pma1 and the vacuolar V-ATPase seem to be coordinated. Proton export is slower in vma mutants lacking V-ATPase subunits,
and Pma1 is partially mislocalized to the vacuole and other compartments in these mutants (Martinez-Munoz and Kane 2008).
The activity of Pma1 is also reduced in the vph1 mutant lacking a subunit of the vacuolar proton pump (Tarsio et al. 2011).
Therefore, we monitored glucose-induced medium acidification
to compare the activity of H+ -ATPases in studied mutant strains.
These measurements clearly showed a decreased rate of proton extrusion in all ESCRT mutants, but not in vcx1 cells. The
lack of components of the trafficking machinery may affect the
functioning and proper localization of several H+ transporters
and pH regulators, which might explain why we observed lower
acidification rates in ESCRT mutants, but not in the vcx1 mutant. Indeed, the endosomal Na+ , K+ /H+ antiporter Nhx1 was
found to be mislocalized in the vps36 mutant, which was also
sensitive to low pH, hygromycin B and salt stress similar to the
nhx1 mutant (Brett et al. 2011). Therefore, in addition to their
other roles in the maintenance of cell ion and pH homeostasis,
ESCRT components might be necessary for proper functioning
of yeast H+ -ATPase(s).
SUPPLEMENTARY DATA
Supplementary data is available at FEMSYR online.
ACKNOWLEDGEMENTS
We gratefully thank Dr Yunying Zhao (The National Engineering Laboratory for Cereal Fermentation Technology, School of
7
Biotechnology, Jiangnan University, Wuxi 214122, China) for providing of the vps20vcx1, vps36vcx1 and snf7vcx1 strains.
FUNDING
This work was supported by projects P503/10/0307 from the GA
CR, Kontakt II LH14297 from MSMT, and BIOCEV (Biotechnology
and Biomedicine Centre of the Academy of Sciences and Charles
University - CZ.1.05/1.1.00/02.0109) from the European Regional
Development Fund. LJ is supported by the National Natural Science Foundation of China (No. 81371784) and the Jiangnan University key grant (No. JUSRP51313B).
Conflict of interest statement. None declared.
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