BACKGROUND
cation homeostasis is an indispensable process for every living cell.
the intracellular concentration of h+, k+ and na+ cations must be tightly regulated because h+ and k+ are involved in important processes and activities of many cellular systems.
on the other hand, extreme na+ concentrations are toxic for cells.
aberrant cellular cation homeostasis is related to human neurological diseases like alzheimer’s disease  <cit>  and epilepsy  <cit> .
furthermore, cation homeostasis is critically important for apoptosis  <cit>  and aberrant serum potassium levels are related to diseases like hypo- and hyperkalemia and crohn's disease.
properties of ion homeostasis in plants determine their ability to grow in environments with very low or high concentrations of salts .
the yeast saccharomyces cerevisiae is an excellent model organism to study cation homeostasis .
in this yeast intracellular potassium concentration is relatively stable at  <dig> – 300 mm.
on the other hand, the intracellular na+ concentration is an order of magnitude lower, around 20 mm.
maintenance of high intracellular concentrations of potassium is facilitated by the generation of a membrane potential by the proton atpase pma <dig>  <cit>  and two high affinity potassium transporters trk <dig> and trk <dig>  <cit> .
sodium ions are mainly extruded by the ena1- <dig> atp-dependent sodium-potassium transporters  <cit> .
potassium is extruded from the cells by the nha <dig> and tok <dig> transporters  <cit> .
the activity of these transporters is highly regulated by several protein kinases and protein phosphatases and by protein turnover.
regulation of ion homeostasis at the transcriptional level is less well understood.
genome-wide studies mainly addressed the transcriptional response to high sodium- or potassium levels rather than the response to cation starvation conditions .
to further understand cation homeostasis in s. cerevisiae a number of european research groups including ours collaborate in the translucent consortium  <cit> .
to allow studies on the response of yeast to different concentrations of potassium, a new ynb medium containing negligible concentrations of alkali metal cations has been developed by the consortium  <cit> .
in a recent study this medium was used to study the short-term response to potassium starvation at the transcriptional level using dna microarrays  <cit> .
this study showed that the lack of potassium drastically activates sulfur metabolism , triggers an oxidative stress response and activate the retrograde pathway.
the expression of genes encoding ribosomal proteins and proteins involved in the cell cycle was considerably lowered.
in this study, we investigated the transcriptional response to potassium starvation using serial analysis of gene expression -tag sequencing.
the expression of genes involved in several cellular processes was shown to be affected during potassium starvation including genes encoding proteins involved in stress response and phosphate metabolism.
the effect of potassium starvation on the expression of pho <dig> encoding an high affinity phosphate transporter was shown to be caused by an activation of the pho <dig> promoter through the pho <dig> transcription factor as well as at the level of rna turnover.
RESULTS
transcriptome analysis by sage tag sequencing
to further understand the mechanisms of ion homeostasis we analyzed the effects of potassium starvation on the genome-wide transcription in s. cerevisiae.
in our studies we used the translucent ynb medium containing very low concentrations of alkali metal cations, allowing controlled addition of potassium.
exponential growing cells in four independent cultures were transferred to medium containing 50 mm kcl or lacking kcl and grown for 60 min.
rna was isolated for transcriptome analysis.
recent technological developments made rna sequencing the method of choice over analysis using dna microarrays for such studies.
we used sage tag sequencing allowing accurate quantification of rna levels.
the sage tags were aligned to a virtual tag library corresponding to all s. cerevisiae annotated open reading frames  including 200 bp downstream sequences.
in this way, transcriptional information was obtained for  <dig> loci .
comparison of transcript levels isolated from cells grown in media without potassium with those isolated from cells grown under standard potassium concentrations showed that the mrna levels of  <dig> genes were significantly  up-regulated more than  <dig> -fold during potassium starvation and the mrna levels of  <dig> genes significantly down-regulated.
the  <dig> most up-regulated genes are shown in table  <dig>  the  <dig> most down-regulated genes in table  <dig>  classification according to go-term biological process using funspec  <cit>  of the genes up-regulated at least 2-fold  showed that the up-regulated genes belong to many different categories and that especially genes involved in phosphate metabolism, cytogamy and stress response are enriched in this set of genes .
in addition, genes involved carbohydrate metabolism are affected and genes subject to glucose repression like hxk <dig> are induced.
classification of the genes down-regulated at least 2-fold  by potassium starvation shows a very clear enrichment of genes involved in cell cycle-related processes.
in addition genes involved in ammonia assimilation and amino acid transport are enriched in this set of genes .table  <dig>
the  <dig> most up-regulated genes after 60 min of potassium starvation
0 mm
ssn8
yer138w-a
cit3
hsp82
pdc5
pho84
ybr085c-a
ygr161w-c
yil082w-a
spl2
mcm10
hsp104
ies5
phm6
ynl146w
hsp42
ecl1
sti1
are2
hxk1
fus1
ssa3
esc2
met28
ybl005w-b
yil171w-a
rer1
clb6
tos6
pol30
pcl1
tos4
kcc4
ymr230w-a
yox1
hta1
cdc45
swe1
cdc21
alg14
yig1
mcd1
rpl18b
lsm4
gap1
yer088c-a
ylr413w
tos2
pmi40
kip1
<cit>
of genes up-regulated at least 2-fold  during potassium starvation
a
a
vtc <dig> vtc <dig> pho84
fus <dig> fig  <dig> ste2
ssa <dig> hsp <dig> aad <dig> hsp <dig> mdj <dig> pog <dig> hsp <dig> sti <dig> hsp82
rom <dig> nap <dig> reh1
glc <dig> hxk <dig> pgm <dig> glc <dig> zwf <dig> sol1
mdj <dig> hsp82
eds <dig> ybr053c ybr085c-a pcs <dig> rtn <dig> phm <dig> ies <dig> yer138w-a tmt <dig> ygr127w ecl <dig> ygr161w-c hua <dig> aim <dig> yil102c-a iks <dig> yjl127c-b ykl023w tti <dig> ykr075c ylr149c ylr271w cmc <dig> pgm <dig> ynl058c ynl146w ynr014w aim <dig> yor289w uip4
idp <dig> zwf1
fig  <dig> sag1
hxk <dig> pgm <dig> zwf1
bat <dig> pdc5
far <dig> pog1
swf <dig> vtc <dig> vtc4
edc <dig> dcs2
vtc <dig> vtc4
a, k: number of genes from the input cluster in given category; f: number of genes total in given category.
functional analysis using funspec
<cit>
of genes down-regulated at least 2-fold  during potassium starvation
a
a
lte <dig> kip <dig> hsl <dig> kcc <dig> mcd <dig> ubc <dig> pds <dig> spc <dig> clb <dig> clb <dig> swe <dig> pcl <dig> sgo <dig> nud <dig> cln <dig> clb <dig> kar3
clb <dig> clb <dig> swe <dig> pcl <dig> cln <dig> clb5
lte <dig> kip <dig> hsl <dig> kcc <dig> mcd <dig> psa <dig> ubc <dig> pds <dig> spc <dig> clb <dig> clb <dig> swe <dig> sld <dig> cdc <dig> yox <dig> tof <dig> pcl <dig> sgo <dig> nud <dig> cln <dig> clb <dig> kar3
lte <dig> kip <dig> hsl <dig> mcd <dig> ubc <dig> pds <dig> spc <dig> clb <dig> swe <dig> sgo <dig> nud <dig> kar3
gap <dig> put <dig> gdh1
gnp <dig> hnm <dig> gap <dig> bio <dig> put4
rdh <dig> sld <dig> sld <dig> cdc <dig> ctf4
pol <dig> pol <dig> sld <dig> sld <dig> cdc <dig> tof <dig> rfa <dig> ctf4
hsl <dig> ubc <dig> clb <dig> swe <dig> clb5
clb <dig> clb5
psa <dig> pmi40
pol <dig> msh <dig> pms1
kcc <dig> gin <dig> tos <dig> dfg5
ade <dig> ade <dig> ade2
spc <dig> spc <dig> stu <dig> spc98
gap <dig> put4
clb <dig> clb5
pol <dig> tof <dig> ctf <dig> kar3
psa <dig> pmi <dig> ktr1
shm <dig> gcv2
kcc <dig> gin4
gnp <dig> hnm <dig> gap <dig> bio <dig> put4
rdh <dig> pol <dig> mcd <dig> dun <dig> msh <dig> hta <dig> srs <dig> tos <dig> ogg <dig> pms <dig> tof <dig> smc5
est <dig> rap <dig> rfa2
rdh <dig> pol <dig> msh <dig> hta <dig> srs <dig> ogg <dig> pms <dig> tof <dig> rfa <dig> smc <dig> ctf4
htb <dig> hta1
smi <dig> yox1
clb <dig> clb5
dun <dig> tof1
a, k: number of genes from the input cluster in given category; f: number of genes total in given category.
our sage-tag sequencing showed that the stress response genes hsp <dig>  tpo <dig> and yro <dig> were highly up-regulated upon potassium starvation, but due to the variation in expression in the individual cultures the effect did not meet our criteria for significance, i.e.
p <  <dig> .
by real time qrt-pcr we verified the effects of potassium starvation on these genes, using act <dig> as control.
as shown in table  <dig> qrt-pcr clearly showed that hsp <dig>  tpo <dig> and yro <dig> were strongly up-regulated upon potassium starvation in agreement with the rnaseq data.
as an additional control we verified the expression of gap <dig> and alg <dig> by qrt-pcr.
both by rnaseq and qrt-pcr we showed that gap <dig> was strongly down-regulated whereas the control gene alg <dig> <cit>  was only slightly affected by potassium starvation .table  <dig>
analysis of the effect of potassium starvation by qrt-pcr
tpo2
hsp30
yro2
gap1
alg9
a, down-regulation is indicated with ‘-‘.
growth of selected deletion strains at low potassium concentrations
an obvious question is whether the up-regulated genes are required for growth at low potassium concentrations.
to this end strains deleted for the most up-regulated genes or deleted for pho <dig>  were spotted on plates with  <dig> or  <dig>  mm kcl.
the trk1trk <dig> double disruptant, lacking the high affinity potassium transporters trk <dig> and trk <dig>  was used as a control.
none of the selected disruptants  showed an aberrant growth at  <dig>  mm kcl, whereas the trk1trk <dig> mutant  did not grow at this kcl concentration .
these results indicate that the most up-regulated genes are not by themselves required for growth at low potassium concentrations.
to study the survival of the tested disruption strains at low potassium conditions, these strains were spotted on a plate lacking potassium and after six days the cells were transferred to plates with 50 mm kcl.
no significant differences in growth compared to the wild type strain were observed, except for the trk <dig> trk <dig> strain .figure  <dig>
growth at  <dig> and  <dig>  mm kcl of deletion strains lacking genes highly up-regulated during potassium starvation or lacking
pho4
.
ten-fold serial dilutions of the indicated strains are spotted on translucent ynb plates supplemented with histidine, methionine, leucine and uracil containing  <dig> or  <dig>  mm kcl.
plates were incubated for 3 days at 30°c.
promoter analysis
using the information available in the yeastract database  it became clear that many different transcription factors are associated with the up-regulated genes.
this suggests that the transcriptional response to potassium starvation is not linked to the activation of a single transcription factor.
two of the up-regulated genes related to stress-response, i.e.
yro <dig> and tpo <dig>  are also up-regulated upon weak acid stress  <cit> .
the latter up-regulation is dependent on the transcription factor haa <dig>  however, in a Δhaa <dig> mutant yro <dig> is still highly up-regulated upon potassium starvation  making a role of haa <dig> in the response to potassium starvation less likely.
to further analyze the regulation of yro <dig> and tpo <dig> the promoter sequences were placed upstream of the bacterial ß-galactosidase gene in plasmid prul <dig> and introduced in yeast strain by <dig>  the resulting strains were cultivated in the presence of 50 mm kcl till mid-exponential growth, transferred to media with 50 mm kcl or to media lacking kcl and grown for 60 min.
then, cells were isolated and ß-galactosidase activity was determined.
using 510 bp of sequences of the tpo <dig> promoter or 2141 bp of sequences of the yro <dig> promoter we found an only  <dig> -fold or  <dig> -fold, respectively, increase in β-galactosidase activity upon potassium starvation, whereas rnaseq showed  <dig> -fold  and 23-fold , respectively, increase in mrna level, suggesting that also other mechanisms than activation of the promoter are involved in the expression of yro <dig> and tpo <dig> during potassium starvation.
regulation of pho <dig> by the transcription factor pho4
our rnaseq data reveal that genes involved in phosphate metabolism are well represented in the set of genes that were significantly up-regulated.
many of these genes, including pho <dig>  are strongly regulated in response to external phosphate concentrations .
the transcription factor pho <dig> translocates into the nucleus at low phosphate concentrations and plays a prominent role in activation of pho <dig> expression  <cit> .
to investigate the involvement of pho <dig> in the activation of the pho <dig> promoter during potassium starvation we inserted 600 bp promoter sequences upstream of the ß-galactosidase gene in plasmid prul <dig> and introduced the resulting plasmid in by <dig> and the Δpho <dig> disruptant in the by <dig> background .
the resulting strains were cultivated as described above to determine the effect of potassium starvation on the pho <dig> promoter.
as shown in table  <dig>  in the wild type background  there is a clear activation of the pho <dig> promoter, whereas in the Δpho <dig> strain hardly any activity can be detected, both after cultivation at 50 mm and 0 mm kcl.
introduction of an intact copy of pho <dig>  in the wild type background gives a stimulation of the pho <dig> promoter, both at  <dig> and 0 mm kcl.
introduction of an intact copy of pho <dig>  in the Δpho <dig> strain restores and activates the pho <dig> promoter.
these results indicate that the pho <dig> transcription factor is required for the expression of pho <dig> at low potassium concentrations.table  <dig>
role of pho <dig> in the transcriptional regulation of
pho84
during potassium starvation
results of a typical experiment are given.
in each experiment two independent transformants were analyzed, the average is shown.
promoter replacement
to further investigate the regulation of gene expression during potassium starvation we replaced the promoter sequences of a number of genes  by the cyc <dig> promoter.
this promoter is most likely not influenced by potassium starvation as the levels of cyc <dig> rna were hardly changed .
the resulting strains were cultivated as above and rna was isolated.
gene expression was determined by qrt-pcr using act <dig> as a reference gene.
in this way we found the following effects of potassium starvation on the rna levels: hsp <dig>   <dig> - and  <dig> -fold increase in two independent experiments; tpo <dig>   <dig> - and  <dig> -fold increase; yro <dig>   <dig> - and  <dig> -fold decrease; pho <dig>   <dig> - and  <dig> --fold increase; alg <dig> ,  <dig> - and  <dig> -fold increase.
these observations show that especially for pho <dig> other regulatory aspects are involved besides activation of the promoter.
surprisingly, the levels of yro <dig> rna are reduced at 0 mm kcl.
antisense transcription
several studies have shown that pho <dig> is also transcribed in the opposite direction yielding antisense rna  <cit> .
sage tag sequencing is able to identify antisense transcripts  <cit> .
this is illustrated for pho <dig> in figure 2a.
alignment of the sequenced tags to the complementary strand of the s. cerevisiae open reading frames  reveals potential antisense transcripts corresponding to  <dig> loci .
the total number of tags aligning to the complementary strand is  <dig> to  <dig> percent of the total number of tags aligning to the coding strand.
comparison of the antisense transcript levels in cells grown in medium without potassium with the levels in cells grown under standard potassium concentrations shows that antisense transcription of  <dig> genes was significantly  up-regulated more than 2-fold, whereas antisense transcripts of  <dig> genes were significantly down-regulated .
several tags aligned to the complementary strand of pho <dig>  almost all tags were found at a higher level using rna isolated from cells grown at 0 mm kcl compared to cells grown at 50 mm kcl .
the total number of tags corresponding to antisense transcripts of the pho <dig> gene is  <dig> -fold  increased after potassium starvation.figure  <dig>
antisense transcription of
pho84
.
a, scheme showing the position of sage tags corresponding to sense and antisense transcripts.
b, primers used to detect pho <dig> antisense transcripts.
c. detection of antisense transcripts using strand specific cdna synthesis and pcr.
rna was isolated from cells grown for 60 min in translucent ynb medium lacking potassium.
arrow indicates the position of the expected pcr fragment.
antisense pho <dig> transcripts were further analyzed by rt-pcr using strand specific primers.
cdna synthesis using a primer specific for antisense rna  yields cdna dependent on antisense transcripts.
using primers as-pcr-fw and as-pcr-rv  this cdna can be detected by pcr.
as shown in figure 2c  the expected pcr fragment was found.
this band was not found when primer se-cdna  was used as primer for cdna synthesis, which generates cdna corresponding to sense transcripts.
these results indicate that antisense pho <dig> rna can be detected rt-pcr, in agreement with our rnaseq experiments and previous studies.
discussion and
CONCLUSIONS
the yeast s. cerevisiae is an excellent organism to study fundamental aspects of cation homeostasis.
although many components involved in the maintenance of cation homeostasis in this yeast have already been revealed, still many aspects remain unclear.
understanding the response of yeast cells to potassium starvation is essential for understanding the mechanisms involved in cation homeostasis.
therefore, the translucent consortium developed a new medium, based on the widely used ynb-medium, containing negligible amounts of alkali metal cations  <cit> .
this medium allowed potassium starvation studies.
studies have been conducted to address effects at the transcriptional  <cit>  and proteomic level  <cit> .
using dna microarrays it was shown that the lack of potassium affects the expression of more than one thousand genes including a strong activation of genes involved in sulfur metabolism, oxidative stress response and the retrograde pathway and a reduced expression of genes involved in ribosome biogenesis and cell cycle control.
at the protein level a general decrease in protein content and an increased level of stress-related proteins were found.
in this study we used sage-tag sequencing to analyze the transcriptional response of s. cerevisiae to potassium starvation.
we used four independent biological replicates and for reasons unknown the variation between these replicates is somewhat larger than expected.
despite this variation, similarly to the study with dna microarrays we found a very pronounced effect of potassium starvation on gene expression and the expression of genes belonging to many functional categories was affected.
the power of sage-tag sequencing is that also lowly expressed genes can be analyzed.
therefore, we were able to obtain information on the expression of  <dig> different loci, much higher than what can be achieved by dna microarrays.
both in this study and in the study with dna microarrays a strong up-regulation of genes involved in stress response and a strong down-regulation of genes involved in various aspects of cell cycle control were found.
although the overall trend found in this and the previous study is very similar, also quantitative differences were found.
one of the examples is the effect on the expression of genes involved in sulfur metabolism.
in the previous study after 60 min of potassium starvation sul <dig>  mup <dig>  met <dig>  str <dig>  met <dig>  met <dig>  ser <dig> and met <dig> were all up-regulated more than 4-fold.
for these genes we also found up-regulation, although much less pronounced.
the effect of potassium starvation on the s. cerevisiae transcriptome has also been investigated using chemostat cultures  <cit> .
in that study, only a limited number of genes were strongly affected by potassium limitation, and the great majority of these were genes known to be involved in nitrogen metabolism.
genes encoding ammonium ion and amino acid transporters  were down-regulated 30-fold and virtually all other nitrogen catabolite-repressed genes were strongly down-regulated as well.
sps-regulated amino acid permeases were strongly up-regulated.
in our study we also found a strong down-regulation of gap <dig> and mep <dig> .
on the other hand, in our study genes encoding amino acid permeases were down-regulated instead of up-regulated .
in this respect it has to be mentioned that the physiological state of yeast cells in chemostat cultures can be greatly different from that of cells in shake flask cultures and that different media were used in the two studies.
potassium homeostasis is regulated by a complex interplay between transporters and regulatory proteins.
many proteins have been identified that bind to the various s. cerevisiae ion transports  <cit>  .
the function of most of these interactions is still unknown and a role in the regulation of the ion transporters is imaginable.
in figure  <dig> a number of s. cerevisiae ion transporters and their binding partners is shown and the effect of potassium starvation on their transcript levels is indicated in a color scale, with down-regulation in green and up-regulation in red.
it is clear that the rna levels of some binding partners are increased and of some binding partners decreased.
for example, hsp <dig>  encoding a nha <dig> binding partner, and arr <dig>  encoding an ena <dig> binding partners are strongly up-regulated.
the rna levels of many, but not all, of the binding partners of the nhx <dig> transporter are decreased.
however, the significance of these observations still has to be shown.figure  <dig>
the effect of potassium starvation on the rna level of a number of
s. cerevisiae
ion transporters and their binding partners.
protein binding partners  were taken from http://www.yeastgenome.org.
squares: ion transporters; circles: binding partners.
fold changes in rna levels are indicated in a color scale, down-regulation in green and up-regulation in red; pink, rnaseq data not available.
protein-protein interactions are shown with blue lines.
the figure was made using cytoscape  <cit> .
identification of transcription factors involved in the regulation of gene expression during potassium starvation is very complex.
using the information on transcription factor associations in the yeastract database  many different transcription factors were found associated with the up- or down-regulated genes.
the transcription factor pho <dig> may participate in gene regulation during potassium starvation.
this transcription factor is found to be associated with seven of the  <dig> most up-regulated genes, i.e.
are <dig>  fus <dig>  hsp <dig>  phm <dig>  pho <dig>  spl <dig> and ynl146w .
deletion of pho <dig> strongly affected the activity of pho <dig> promoter.
however, other transcription factors are most likely be involved as well.
in addition, rna turnover is important.
after replacement of the pho <dig> promoter by the cyc <dig> promoter pho <dig> rna levels are still approximately five-fold increased.
in this study as well as in the study using dna microarrays  <cit>  a strong induction if genes involved in phosphate metabolism, including pho <dig>  was found.
thus, despite the presence of phosphate in the medium, these genes are not repressed, when potassium is absent.
the transcription factor pho <dig> plays a key role in the regulation of these genes.
the subcellular localization of this transcription factor is regulated via phosphorylation by the pho80-pho <dig> cyclin-kinase complex  <cit> .
when phosphate is available pho <dig> is retained in the cytoplasm, whereas under phosphate limiting conditions it translocates into the nucleus, resulting in expression of genes involved in phosphate uptake.
during potassium starvation we observed an increase in nuclear pho4-gfp .
the expression of pho <dig>  pho <dig> and pho <dig> is only slightly and not significantly affected by potassium starvation  , suggesting that transcriptional regulation of these genes does not play a role.
however, the mechanism of the regulation of pho <dig> during potassium starvation still has to be revealed.
several studies have shown that pho <dig> is also transcribed in the opposite direction yielding antisense rna  <cit> .
this antisense rna induces transcriptional silencing of pho <dig> via histone deacetylation of the pho <dig> promoter when phosphate is available.
our sage tag sequencing revealed many tags corresponding to antisense transcripts corresponding to  <dig> loci  and the levels of many of these tags were affected by potassium starvation, including those of pho <dig> .
by applying sage tag sequencing, tags corresponding to antisense transcripts were also found in other studies  <cit> .
however, it has been suggested that spurious synthesis of second-strand cdna during reverse transcription reactions triggers antisense artifacts  <cit> .
in our studies we performed cdna synthesis on-the-bead making the formation of artifacts less likely  <cit> .
furthermore, several studies showed the widespread occurrence of antisense transcription in s. cerevisiae .
by rt-pcr using antisense-specific primers during cdna synthesis we were able to provide additional evidence for antisense transcription of pho <dig> .
however, the role of antisense transcription in the regulation of pho <dig> during potassium starvation still has to be disclosed.

 cation homeostasis is an indispensable process for every living cell. the intracellular concentration of h+, k+ and na+ cations must be tightly regulated because h+ and k+ are involved in important processes and activities of many cellular systems. on the other hand, extreme na+ concentrations are toxic for cells. aberrant cellular cation homeostasis is related to human neurological diseases like alzheimer’s disease and epilepsy. furthermore, cation homeostasis is critically important for apoptosis and aberrant serum potassium levels are related to diseases like hypo- and hyperkalemia and crohn's disease. properties of ion homeostasis in plants determine their ability to grow in environments with very low or high concentrations of salts. the yeast saccharomyces cerevisiae is an excellent model organism to study cation homeostasis. in this yeast intracellular potassium concentration is relatively stable at – 300 mm. on the other hand, the intracellular na+ concentration is an order of magnitude lower, around 20 mm. maintenance of high intracellular concentrations of potassium is facilitated by the generation of a membrane potential by the proton atpase pma and two high affinity potassium transporters trk and trk. sodium ions are mainly extruded by the ena1- atp-dependent sodium-potassium transporters. potassium is extruded from the cells by the nha and tok transporters. the activity of these transporters is highly regulated by several protein kinases and protein phosphatases and by protein turnover. regulation of ion homeostasis at the transcriptional level is less well understood. genome-wide studies mainly addressed the transcriptional response to high sodium- or potassium levels rather than the response to cation starvation conditions. to further understand cation homeostasis in s. cerevisiae a number of european research groups including ours collaborate in the translucent consortium. to allow studies on the response of yeast to different concentrations of potassium, a new ynb medium containing negligible concentrations of alkali metal cations has been developed by the consortium. in a recent study this medium was used to study the short-term response to potassium starvation at the transcriptional level using dna microarrays. this study showed that the lack of potassium drastically activates sulfur metabolism, triggers an oxidative stress response and activate the retrograde pathway. the expression of genes encoding ribosomal proteins and proteins involved in the cell cycle was considerably lowered. in this study, we investigated the transcriptional response to potassium starvation using serial analysis of gene expression -tag sequencing. the expression of genes involved in several cellular processes was shown to be affected during potassium starvation including genes encoding proteins involved in stress response and phosphate metabolism. the effect of potassium starvation on the expression of pho encoding an high affinity phosphate transporter was shown to be caused by an activation of the pho promoter through the pho transcription factor as well as at the level of rna turnover.  transcriptome analysis by sage tag sequencing to further understand the mechanisms of ion homeostasis we analyzed the effects of potassium starvation on the genome-wide transcription in s. cerevisiae. in our studies we used the translucent ynb medium containing very low concentrations of alkali metal cations, allowing controlled addition of potassium. exponential growing cells in four independent cultures were transferred to medium containing 50 mm kcl or lacking kcl and grown for 60 min. rna was isolated for transcriptome analysis. recent technological developments made rna sequencing the method of choice over analysis using dna microarrays for such studies. we used sage tag sequencing allowing accurate quantification of rna levels. the sage tags were aligned to a virtual tag library corresponding to all s. cerevisiae annotated open reading frames including 200 bp downstream sequences. in this way, transcriptional information was obtained for loci. comparison of transcript levels isolated from cells grown in media without potassium with those isolated from cells grown under standard potassium concentrations showed that the mrna levels of genes were significantly up-regulated more than -fold during potassium starvation and the mrna levels of genes significantly down-regulated. the most up-regulated genes are shown in table  the most down-regulated genes in table  classification according to go-term biological process using funspec of the genes up-regulated at least 2-fold showed that the up-regulated genes belong to many different categories and that especially genes involved in phosphate metabolism, cytogamy and stress response are enriched in this set of genes. in addition, genes involved carbohydrate metabolism are affected and genes subject to glucose repression like hxk are induced. classification of the genes down-regulated at least 2-fold by potassium starvation shows a very clear enrichment of genes involved in cell cycle-related processes. in addition genes involved in ammonia assimilation and amino acid transport are enriched in this set of genes.table the most up-regulated genes after 60 min of potassium starvation 0 mm ssn8 yer138w-a cit3 hsp82 pdc5 pho84 ybr085c-a ygr161w-c yil082w-a spl2 mcm10 hsp104 ies5 phm6 ynl146w hsp42 ecl1 sti1 are2 hxk1 fus1 ssa3 esc2 met28 ybl005w-b yil171w-a rer1 clb6 tos6 pol30 pcl1 tos4 kcc4 ymr230w-a yox1 hta1 cdc45 swe1 cdc21 alg14 yig1 mcd1 rpl18b lsm4 gap1 yer088c-a ylr413w tos2 pmi40 kip1 of genes up-regulated at least 2-fold during potassium starvation a a vtc vtc pho84 fus fig ste2 ssa hsp aad hsp mdj pog hsp sti hsp82 rom nap reh1 glc hxk pgm glc zwf sol1 mdj hsp82 eds ybr053c ybr085c-a pcs rtn phm ies yer138w-a tmt ygr127w ecl ygr161w-c hua aim yil102c-a iks yjl127c-b ykl023w tti ykr075c ylr149c ylr271w cmc pgm ynl058c ynl146w ynr014w aim yor289w uip4 idp zwf1 fig sag1 hxk pgm zwf1 bat pdc5 far pog1 swf vtc vtc4 edc dcs2 vtc vtc4 a, k: number of genes from the input cluster in given category; f: number of genes total in given category. functional analysis using funspec of genes down-regulated at least 2-fold during potassium starvation a a lte kip hsl kcc mcd ubc pds spc clb clb swe pcl sgo nud cln clb kar3 clb clb swe pcl cln clb5 lte kip hsl kcc mcd psa ubc pds spc clb clb swe sld cdc yox tof pcl sgo nud cln clb kar3 lte kip hsl mcd ubc pds spc clb swe sgo nud kar3 gap put gdh1 gnp hnm gap bio put4 rdh sld sld cdc ctf4 pol pol sld sld cdc tof rfa ctf4 hsl ubc clb swe clb5 clb clb5 psa pmi40 pol msh pms1 kcc gin tos dfg5 ade ade ade2 spc spc stu spc98 gap put4 clb clb5 pol tof ctf kar3 psa pmi ktr1 shm gcv2 kcc gin4 gnp hnm gap bio put4 rdh pol mcd dun msh hta srs tos ogg pms tof smc5 est rap rfa2 rdh pol msh hta srs ogg pms tof rfa smc ctf4 htb hta1 smi yox1 clb clb5 dun tof1 a, k: number of genes from the input cluster in given category; f: number of genes total in given category. our sage-tag sequencing showed that the stress response genes hsp tpo and yro were highly up-regulated upon potassium starvation, but due to the variation in expression in the individual cultures the effect did not meet our criteria for significance, i.e. p < . by real time qrt-pcr we verified the effects of potassium starvation on these genes, using act as control. as shown in table  qrt-pcr clearly showed that hsp tpo and yro were strongly up-regulated upon potassium starvation in agreement with the rnaseq data. as an additional control we verified the expression of gap and alg by qrt-pcr. both by rnaseq and qrt-pcr we showed that gap was strongly down-regulated whereas the control gene alg was only slightly affected by potassium starvation.table analysis of the effect of potassium starvation by qrt-pcr tpo2 hsp30 yro2 gap1 alg9 a, down-regulation is indicated with ‘-‘. growth of selected deletion strains at low potassium concentrations an obvious question is whether the up-regulated genes are required for growth at low potassium concentrations. to this end strains deleted for the most up-regulated genes or deleted for pho were spotted on plates with or  mm kcl. the trk1trk double disruptant, lacking the high affinity potassium transporters trk and trk was used as a control. none of the selected disruptants showed an aberrant growth at  mm kcl, whereas the trk1trk mutant did not grow at this kcl concentration. these results indicate that the most up-regulated genes are not by themselves required for growth at low potassium concentrations. to study the survival of the tested disruption strains at low potassium conditions, these strains were spotted on a plate lacking potassium and after six days the cells were transferred to plates with 50 mm kcl. no significant differences in growth compared to the wild type strain were observed, except for the trk trk strain.figure growth at and  mm kcl of deletion strains lacking genes highly up-regulated during potassium starvation or lacking pho4. ten-fold serial dilutions of the indicated strains are spotted on translucent ynb plates supplemented with histidine, methionine, leucine and uracil containing or  mm kcl. plates were incubated for 3 days at 30°c. promoter analysis using the information available in the yeastract database it became clear that many different transcription factors are associated with the up-regulated genes. this suggests that the transcriptional response to potassium starvation is not linked to the activation of a single transcription factor. two of the up-regulated genes related to stress-response, i.e. yro and tpo are also up-regulated upon weak acid stress. the latter up-regulation is dependent on the transcription factor haa however, in a Δhaa mutant yro is still highly up-regulated upon potassium starvation making a role of haa in the response to potassium starvation less likely. to further analyze the regulation of yro and tpo the promoter sequences were placed upstream of the bacterial ß-galactosidase gene in plasmid prul and introduced in yeast strain by the resulting strains were cultivated in the presence of 50 mm kcl till mid-exponential growth, transferred to media with 50 mm kcl or to media lacking kcl and grown for 60 min. then, cells were isolated and ß-galactosidase activity was determined. using 510 bp of sequences of the tpo promoter or 2141 bp of sequences of the yro promoter we found an only -fold or -fold, respectively, increase in β-galactosidase activity upon potassium starvation, whereas rnaseq showed -fold and 23-fold, respectively, increase in mrna level, suggesting that also other mechanisms than activation of the promoter are involved in the expression of yro and tpo during potassium starvation. regulation of pho by the transcription factor pho4 our rnaseq data reveal that genes involved in phosphate metabolism are well represented in the set of genes that were significantly up-regulated. many of these genes, including pho are strongly regulated in response to external phosphate concentrations. the transcription factor pho translocates into the nucleus at low phosphate concentrations and plays a prominent role in activation of pho expression. to investigate the involvement of pho in the activation of the pho promoter during potassium starvation we inserted 600 bp promoter sequences upstream of the ß-galactosidase gene in plasmid prul and introduced the resulting plasmid in by and the Δpho disruptant in the by background. the resulting strains were cultivated as described above to determine the effect of potassium starvation on the pho promoter. as shown in table  in the wild type background there is a clear activation of the pho promoter, whereas in the Δpho strain hardly any activity can be detected, both after cultivation at 50 mm and 0 mm kcl. introduction of an intact copy of pho in the wild type background gives a stimulation of the pho promoter, both at and 0 mm kcl. introduction of an intact copy of pho in the Δpho strain restores and activates the pho promoter. these results indicate that the pho transcription factor is required for the expression of pho at low potassium concentrations.table role of pho in the transcriptional regulation of pho84 during potassium starvation results of a typical experiment are given. in each experiment two independent transformants were analyzed, the average is shown. promoter replacement to further investigate the regulation of gene expression during potassium starvation we replaced the promoter sequences of a number of genes by the cyc promoter. this promoter is most likely not influenced by potassium starvation as the levels of cyc rna were hardly changed. the resulting strains were cultivated as above and rna was isolated. gene expression was determined by qrt-pcr using act as a reference gene. in this way we found the following effects of potassium starvation on the rna levels: hsp - and -fold increase in two independent experiments; tpo - and -fold increase; yro - and -fold decrease; pho - and --fold increase; alg, - and -fold increase. these observations show that especially for pho other regulatory aspects are involved besides activation of the promoter. surprisingly, the levels of yro rna are reduced at 0 mm kcl. antisense transcription several studies have shown that pho is also transcribed in the opposite direction yielding antisense rna. sage tag sequencing is able to identify antisense transcripts. this is illustrated for pho in figure 2a. alignment of the sequenced tags to the complementary strand of the s. cerevisiae open reading frames reveals potential antisense transcripts corresponding to loci. the total number of tags aligning to the complementary strand is to percent of the total number of tags aligning to the coding strand. comparison of the antisense transcript levels in cells grown in medium without potassium with the levels in cells grown under standard potassium concentrations shows that antisense transcription of genes was significantly up-regulated more than 2-fold, whereas antisense transcripts of genes were significantly down-regulated. several tags aligned to the complementary strand of pho almost all tags were found at a higher level using rna isolated from cells grown at 0 mm kcl compared to cells grown at 50 mm kcl. the total number of tags corresponding to antisense transcripts of the pho gene is -fold increased after potassium starvation.figure antisense transcription of pho84. a, scheme showing the position of sage tags corresponding to sense and antisense transcripts. b, primers used to detect pho antisense transcripts. c. detection of antisense transcripts using strand specific cdna synthesis and pcr. rna was isolated from cells grown for 60 min in translucent ynb medium lacking potassium. arrow indicates the position of the expected pcr fragment. antisense pho transcripts were further analyzed by rt-pcr using strand specific primers. cdna synthesis using a primer specific for antisense rna yields cdna dependent on antisense transcripts. using primers as-pcr-fw and as-pcr-rv this cdna can be detected by pcr. as shown in figure 2c the expected pcr fragment was found. this band was not found when primer se-cdna was used as primer for cdna synthesis, which generates cdna corresponding to sense transcripts. these results indicate that antisense pho rna can be detected rt-pcr, in agreement with our rnaseq experiments and previous studies. discussion and  the yeast s. cerevisiae is an excellent organism to study fundamental aspects of cation homeostasis. although many components involved in the maintenance of cation homeostasis in this yeast have already been revealed, still many aspects remain unclear. understanding the response of yeast cells to potassium starvation is essential for understanding the mechanisms involved in cation homeostasis. therefore, the translucent consortium developed a new medium, based on the widely used ynb-medium, containing negligible amounts of alkali metal cations. this medium allowed potassium starvation studies. studies have been conducted to address effects at the transcriptional and proteomic level. using dna microarrays it was shown that the lack of potassium affects the expression of more than one thousand genes including a strong activation of genes involved in sulfur metabolism, oxidative stress response and the retrograde pathway and a reduced expression of genes involved in ribosome biogenesis and cell cycle control. at the protein level a general decrease in protein content and an increased level of stress-related proteins were found. in this study we used sage-tag sequencing to analyze the transcriptional response of s. cerevisiae to potassium starvation. we used four independent biological replicates and for reasons unknown the variation between these replicates is somewhat larger than expected. despite this variation, similarly to the study with dna microarrays we found a very pronounced effect of potassium starvation on gene expression and the expression of genes belonging to many functional categories was affected. the power of sage-tag sequencing is that also lowly expressed genes can be analyzed. therefore, we were able to obtain information on the expression of different loci, much higher than what can be achieved by dna microarrays. both in this study and in the study with dna microarrays a strong up-regulation of genes involved in stress response and a strong down-regulation of genes involved in various aspects of cell cycle control were found. although the overall trend found in this and the previous study is very similar, also quantitative differences were found. one of the examples is the effect on the expression of genes involved in sulfur metabolism. in the previous study after 60 min of potassium starvation sul mup met str met met ser and met were all up-regulated more than 4-fold. for these genes we also found up-regulation, although much less pronounced. the effect of potassium starvation on the s. cerevisiae transcriptome has also been investigated using chemostat cultures. in that study, only a limited number of genes were strongly affected by potassium limitation, and the great majority of these were genes known to be involved in nitrogen metabolism. genes encoding ammonium ion and amino acid transporters were down-regulated 30-fold and virtually all other nitrogen catabolite-repressed genes were strongly down-regulated as well. sps-regulated amino acid permeases were strongly up-regulated. in our study we also found a strong down-regulation of gap and mep. on the other hand, in our study genes encoding amino acid permeases were down-regulated instead of up-regulated. in this respect it has to be mentioned that the physiological state of yeast cells in chemostat cultures can be greatly different from that of cells in shake flask cultures and that different media were used in the two studies. potassium homeostasis is regulated by a complex interplay between transporters and regulatory proteins. many proteins have been identified that bind to the various s. cerevisiae ion transports. the function of most of these interactions is still unknown and a role in the regulation of the ion transporters is imaginable. in figure  a number of s. cerevisiae ion transporters and their binding partners is shown and the effect of potassium starvation on their transcript levels is indicated in a color scale, with down-regulation in green and up-regulation in red. it is clear that the rna levels of some binding partners are increased and of some binding partners decreased. for example, hsp encoding a nha binding partner, and arr encoding an ena binding partners are strongly up-regulated. the rna levels of many, but not all, of the binding partners of the nhx transporter are decreased. however, the significance of these observations still has to be shown.figure the effect of potassium starvation on the rna level of a number of s. cerevisiae ion transporters and their binding partners. protein binding partners were taken from http://www.yeastgenome.org. squares: ion transporters; circles: binding partners. fold changes in rna levels are indicated in a color scale, down-regulation in green and up-regulation in red; pink, rnaseq data not available. protein-protein interactions are shown with blue lines. the figure was made using cytoscape. identification of transcription factors involved in the regulation of gene expression during potassium starvation is very complex. using the information on transcription factor associations in the yeastract database many different transcription factors were found associated with the up- or down-regulated genes. the transcription factor pho may participate in gene regulation during potassium starvation. this transcription factor is found to be associated with seven of the most up-regulated genes, i.e. are fus hsp phm pho spl and ynl146w. deletion of pho strongly affected the activity of pho promoter. however, other transcription factors are most likely be involved as well. in addition, rna turnover is important. after replacement of the pho promoter by the cyc promoter pho rna levels are still approximately five-fold increased. in this study as well as in the study using dna microarrays a strong induction if genes involved in phosphate metabolism, including pho was found. thus, despite the presence of phosphate in the medium, these genes are not repressed, when potassium is absent. the transcription factor pho plays a key role in the regulation of these genes. the subcellular localization of this transcription factor is regulated via phosphorylation by the pho80-pho cyclin-kinase complex. when phosphate is available pho is retained in the cytoplasm, whereas under phosphate limiting conditions it translocates into the nucleus, resulting in expression of genes involved in phosphate uptake. during potassium starvation we observed an increase in nuclear pho4-gfp. the expression of pho pho and pho is only slightly and not significantly affected by potassium starvation, suggesting that transcriptional regulation of these genes does not play a role. however, the mechanism of the regulation of pho during potassium starvation still has to be revealed. several studies have shown that pho is also transcribed in the opposite direction yielding antisense rna. this antisense rna induces transcriptional silencing of pho via histone deacetylation of the pho promoter when phosphate is available. our sage tag sequencing revealed many tags corresponding to antisense transcripts corresponding to loci and the levels of many of these tags were affected by potassium starvation, including those of pho. by applying sage tag sequencing, tags corresponding to antisense transcripts were also found in other studies. however, it has been suggested that spurious synthesis of second-strand cdna during reverse transcription reactions triggers antisense artifacts. in our studies we performed cdna synthesis on-the-bead making the formation of artifacts less likely. furthermore, several studies showed the widespread occurrence of antisense transcription in s. cerevisiae. by rt-pcr using antisense-specific primers during cdna synthesis we were able to provide additional evidence for antisense transcription of pho. however, the role of antisense transcription in the regulation of pho during potassium starvation still has to be disclosed. 
comparison of transcript levels in cells grown for 60 min in media without potassium with those in cells grown under standard potassium concentrations showed that the mrna levels of genes were significantly up-regulated more than -fold during potassium starvation and the mrna levels of genes significantly down-regulated. our study showed that yeast cells respond to potassium starvation in a complex way and reveals a direct link between potassium homeostasis and phosphate metabolism. the yeast saccharomyces cerevisiae is an excellent organism to study fundamental aspects of cation homeostasis. ion homeostasis is essential for every cell and aberrant cation homeostasis is related to diseases like alzheimer’s disease and epilepsy. the mechanisms responsible for cation homeostasis are only partly understood. saccharomyces cerevisiaecation homeostasissage tag sequencingrnaseqpho84antisense rnapotassium starvationissue-copyright-statement© the author 2014 in this study we investigated the transcriptional response of this yeast to potassium starvation by using serial analysis of gene expression -tag sequencing. many genes involved in cell cycle control, and to a lesser extent genes involved in amino acid transport, were strongly down-regulated. analysis of the promoter of pho encoding a high affinity phosphate transporter, revealed that increased pho rna levels are caused by both increased pho4-dependent transcription and decreased rna turnover. genes involved in stress response including hsp yro and tpo and phosphate metabolism including pho pho and spl were highly up-regulated. these genes belong to several functional categories.
 ion homeostasis is essential for every cell and aberrant cation homeostasis is related to diseases like alzheimer’s disease and epilepsy. the mechanisms responsible for cation homeostasis are only partly understood. the yeast saccharomyces cerevisiae is an excellent organism to study fundamental aspects of cation homeostasis. in this study we investigated the transcriptional response of this yeast to potassium starvation by using serial analysis of gene expression -tag sequencing.  comparison of transcript levels in cells grown for 60 min in media without potassium with those in cells grown under standard potassium concentrations showed that the mrna levels of genes were significantly up-regulated more than -fold during potassium starvation and the mrna levels of genes significantly down-regulated. these genes belong to several functional categories. genes involved in stress response including hsp yro and tpo and phosphate metabolism including pho pho and spl were highly up-regulated. analysis of the promoter of pho encoding a high affinity phosphate transporter, revealed that increased pho rna levels are caused by both increased pho4-dependent transcription and decreased rna turnover. in the latter process antisense transcription may be involved. many genes involved in cell cycle control, and to a lesser extent genes involved in amino acid transport, were strongly down-regulated.  our study showed that yeast cells respond to potassium starvation in a complex way and reveals a direct link between potassium homeostasis and phosphate metabolism. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords saccharomyces cerevisiaecation homeostasissage tag sequencingrnaseqpho84antisense rnapotassium starvationissue-copyright-statement© the author 2014 