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Glucose repression over Saccharomyces cerevisiae glycerol/H+

symporter gene STL1 is overcome by high temperature

Celia Ferreira*, Candida Lucas

Centro de Biologia (CB-UM), Departamento de Biologia da Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal

Received 9 March 2007; accepted 14 March 2007

Available online 9 April 2007

Edited by Francesc Posas

Abstract High temperature promotes an improved activity ofthe Saccharomyces cerevisiae glycerol/H+ symporter encodedby STL1, which correlates well with Stl1p levels. This happensin both fermentable and respiratory metabolic growth conditions,though the induction in the latter is much higher. The relief ofglucose repression by high temperature at the level of proteinexpression and activity (Stl1p) is reported for the first time.We reason that the glycerol internal levels fine-tuning, under

heat-stress as in other physiological condition, can be achievedwith the contribution of the tight regulation of the symporter. 2007 Federation of European Biochemical Societies. Publishedby Elsevier B.V. All rights reserved.

Keywords: Glycerol levels; Heat; Glycerol H+/symporter;STL1; Saccharomyces cerevisiae

1. Introduction

The key enzymes of glycerol metabolism interfere in glyc-

erol transport. Glycerol 3P dehydrogenase, from glycerol

production pathway, is encoded by two isogenes GPD1 and

GPD2 [1]. The gpd1Dgpd2D mutant is unable to produce en-

ough amounts of glycerol to counteract osmotic/salt stress

[1]. When grown on glucose with high amounts of salt, this

mutant presents the higher activity of the transporter re-

ported so far [2], overcoming glucose repression [3]. Also,

the glycerol kinase (Gutlp) from glycerol consumption path-

way interferes in glycerol transport creating measurement

artefacts [2,4]. When this gene is deleted the transport Vmaxis highly reduced [2,4]. In addition, in the pkc1D mutant

(defective in the MAPKinase from PKC pathway), the trans-

port Vmax is also diminished, possibly, due to the inability

this mutant presents to derepress GUT1 under induction con-ditions [5].

Saccharomyces cerevisiae Stl1p, has been assigned as encod-

ing the glycerol H+/symporter [3]. STL1 is induced in a

Cat8p-dependent manner during diauxic shift. Cat8p is a tran-

scription factor regulating gluconeogenic genes, activating

transcription when cells are grown on non-fermentable carbon

sources. STL1 is also induced by osmotic shock by Hot1p/

Hog1p [6]. Accordingly, the glycerol symporter activity had

been described to be under glucose repression and inducible

by growth on non-fermentable carbon sources [7]. Latest data

suggest a more complex regulation for this gene [810], symp-

tomatic of an involvement in: (1) temperature and oxidative

stress response [8], (2) carbon source regulation upon entry

into stationary phase [8], and (3) calcium regulation through

calcineurin [10]. This is in accordance with glycerol complex

roles in diverse cell functions and metabolism as: cytosolic/

mitochondrial redox balance [11,12], intracellular pH and pro-

ton sink regulation [12,13], intracellular Pi availability [14,15],

lipid synthesis, modulation of HOG sensor Sln1p signalling

[16], response to several stress conditions besides osmotic (cit-

ric, temperature, and oxidative) as well as carbon source regu-

lation [17,18], just to mention the ones with more determinant

influence on cell maintenance.

A new and important task of glycerol in maintaining the

signalling competent state of the cells has been suggested in

the literature [19]. This was proposed based on the observa-

tion of increased glycerol intracellular levels at high tempera-

tures, and that temperature-induced osmoresistance of HOG

pathway mutants could be related with this increase [20].

Accordingly, mutants impaired in glycerol production bydeleting either GPD1/2 above mentioned, or GPP isogenes,

encoding glycerol-3-P-phosphatases, exhibit a thermosensitiv-

ity that was suppressed by growth on glycerol-based media

[19].

The present work reveals the contribution of the active glyc-

erol transporter, Stl1p, for the maintenance of S. cerevisiae

glycerol intracellular levels at high temperatures. Additionally,

it reports for the first time, a phenomenon of glucose repres-

sion alleviation caused by high temperature, both at the level

of protein expression and activity.

2. Materials and methods

2.1. Strains, media and growth conditionsS. cerevisiae strains used in this work were W303-1A [14], FVVY28

[3], gpd1Dgpd2D [1]; FVVY39 [3] and gpp1Dgpp2D [18]. Batch cul-tures of yeast were grown aerobically on complex medium (YP: 1%(w/v) yeast extract; 2% (w/v) peptone) supplemented with 2% (w/v)glucose (YPD), ethanol (YPE), or glycerol (YPG) as carbon and en-ergy sources. Incubation and drop tests were performed as before[3,21].

2.2. Glycerol transport studiesThe assays were performed as described before [7,22]. The S. cerevi-

siae intracellular volume used to calculate the intracellular glycerolconcentrations was determined previously [22].

*Corresponding author. Fax: +351 253678980.E-mail addresses: [email protected], celiamjf@gmail. com (C.

Ferreira).

0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

doi:10.1016/j.febslet.2007.03.086

FEBS Letters 581 (2007) 19231927


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2.3. Western blottingThe assay was performed as before [3]. The reacting polypeptides

were visualized using ECL Plus Western Blotting Detection System(Amersham Biosciences) and an Image Analysis System ChemiDocXRS (Bio-Rad, Laboratories, Inc.) with Quantity-One 4.5.0 Software(Bio-Rad, Laboratories, Inc.).

3. Results

STL1 has a long promoter region, around 2 kb, according to

our group previous studies in which the minimal functional

size was determined [3]. We performed an in silico study of this

promoter, that revealed a high number of putative response

elements (http://www.yeastract.com). The corresponding tran-

scription factors include Hap1p, Nrg1p and Sko1p and

Smp1p, besides the already stated Hog1p/Hot1p and Cat8p.

These are associated with redox regulation, glucose sensing/fil-

amentous growth/alkaline pH response, oxidative, osmotic

stress response and carbon source/diauxic shift transition.

Additionally, one response element for Hsf1p can also be

found suggesting a relation of STL1 expression with tempera-

ture stress response, prompting us to study STL1/Stl1p at hightemperatures.

3.1. Thermosensitivity phenotype of stl1D mutant

Several glycerol-related mutants were previously shown to

be sensitive to high temperatures [1921]. These include hog1D,

gpd1Dgpd2D, gpp1Dgpp2D and gup1D. Moreover, intracellular

accumulation of glycerol has been observed during growth at

high temperatures on some of these mutants, as well as in

the wt [19,20]. This led us to check the thermosensitivity (ts)

at 37 C of the mutant defective on the symporter Stl1p, when

grown on YPD, YPE or YPG. In addition, gpd1Dgpd2D and

gpp1Dgpp2D mutants were also investigated.

We found that the stl1D mutation caused moderated ts on

YPD at 37 C (Fig. 1). This ts phenotype, at 37 C, was morepronounced on the non-fermentable carbon sources (Fig. 1).

This result was predicted, since stl1D mutant already exhibits

a growth defect on YPE and YPG when grown at 30 C

(Fig. 1, [3]). We also found just partial ts for gpd1Dgpd2D

and gpp1Dgpp2D mutants at 37 C on YPD (Fig. 1). Again,

the phenotype was more distinct on YPE than on YPD

(Fig. 1). However, in these mutants, but not in stl1D mutant,

the ts observed on ethanol was partially rescued when the mu-

tant cells were provided with external glycerol (Fig. 1-YPG).

Such improvement was more evident for gpp1Dgpp2D thanfor gpd1Dgpd2D mutant cells.

3.2. Stl1p is active at high temperatures

To analyze glycerol transport at high temperatures, wt cells

were grown at 37 C on YPE. Ethanol was chosen since at

30 C, both the STL1 gene and the transport activity presented

a high induction [2,3]. We found that, on YPE at 37 C, accu-

mulation ratio exceeded equilibrium and was sensitive to

CCCP, reaching 50 times in/out (Fig. 2A), whereas at control

temperature, 30 C, the maximum in/out ratio was just 12

(Fig. 2A inset). The same results were obtained in cells cultured

on YPG (not shown).

Identical assays were done with the gpd1D

gpd2D

andgpp1Dgpp2D mutants. These behaved like wt, except for the

maximum ratios attained which were respectively, 5.5 and

27 times in/out at 37 C (Fig. 2B), and 4 and 10 times in/

out at 30 C (Fig. 2B inset). Furthermore, Fps1-mediated dif-

fusion rates were measured and maintained constant in all con-

ditions and strains (not shown).

3.3. Glucose repression over Stl1p is overcome at high

temperatures

To verify if the previously reported regulation of the sym-

porter, namely the glucose repression [2,3,7], was maintained

at 37 C, wt cells were grown on YPD and again, the glycerol

transport-derived accumulation was measured. We found that,

in opposition to what happens at 30 C (Fig. 3A inset), accu-mulation exceeded equilibrium, presenting a maximum in/out

Fig. 1. Temperature-sensitivity of glycerol-related mutants. Ten fold serial dilutions of cells collected during exponential growth-phase were spottedonto YPD, YPE and YPG. Cells were cultured for 5 days at 30 C and 37 C.

1924 C. Ferreira, C. Lucas / FEBS Letters 581 (2007) 19231927
http://www.yeastract.com/http://www.yeastract.com/


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ratio of 20, once more sensitive to CCCP (Fig. 3A). This

occured in spite of the presence of glucose, i.e., regardless of

the predicted glucose repression. Additionally, gpd1Dgpd2D

and gpp1Dgpp2D mutants were assayed on YPD, yielding iden-

tical results to wt (Fig. 3B, inset). Yet, the maximum in/out ra-

tios at 37 C were considerably lower, respectively, 2 and

11 (Fig. 3B). As before, Fps1-mediated diffusion rates were

measured and subsisted constant for the three strains (not

shown).

Regardless of the strain, the maximum in/out ratios attained

at 37 C on YPE and YPG, i.e. non-fermentable carbon

sources, were 2.5 times higher than the ones observed on glu-

cose-based media (Figs. 2 and 3).

3.4. Stl1p expression is enhanced at high temperatures

The same batches of cells used to measure glycerol accumu-

lation were analyzed by Western Blot for Stl1p as described be-

fore [3]. Significant amounts of Stl1p were produced in wt cells

cultivated on YPD at 37 C (Fig. 4A, 37 C). The quantities of

Stl1p produced in cells grown, also at 37 C, on YPE (Fig. 4A,

37 C) or on YPG (not shown) were even higher. At the con-

trol temperature, 30 C, glucose repression was active, there-

fore no protein was found in YPD, whereas in YPE some

was produced (Fig. 4A, 30 C, [3]). In the gpd1Dgpd2D mutant

at 37 C, a decrease in protein levels was observed either on

YPD, YPE (Fig. 4A, 37 C) or on YPG (not shown). At

30 C, the mutant behaved as the wt, i.e., no protein could

be detected on YPD, whereas some was detected on YPE

(Fig. 4A, 30 C). The levels of Stl1p and the glycerol uptake

activity are directly correlated (Fig. 4A/B).

4. Discussion

Considering the complex roles of glycerol and the recurrent

suggestion in the literature that glycerol levels fine-tuning may

play a crucial part in several aspects of yeast cell life [1118], it

urges to understand the regulation of the active transporter in

detail. Accordingly, STL1 appears to be regulated by an

unusually long promoter harbouring a high number of puta-

tive response elements.

STL1 defective strain, as other glycerol-related mutants [19

21], displays a growth limitation at high temperature, both on

fermentable or respiratory conditions. Siderius and co-authors

B

Accumulationratio

(in/out)

Time (min)

0

10

20

30

40

50

60

0 10 20 30 40 50 60 70

A

0 10 20 30 40 50 60 70

0

10

20

30

40

50

60

Time (min)

Accumulationratio

(in/out)

0 10 20 30 40 50 60 700

10

20

30

40

50

60

Time (min)

Accumulationratio

(in/out)

Time (min)

0 10 20 30 40 50 600

10

20

30

40

50

60

Accumulationratio

(in/o

ut)

70

Fig. 2. Activity of the symporter at 37 C and ethanol. Accumulation ratios of [14C] glycerol on exponential growing cells on YPE at 37 C: (A) wtcells and (B) gpp1Dgpp2D (n) and gpd1Dgpd2D () mutants cells. Cells were assayed in the absence (close symbols) or in the presence (open symbols)of CCCP. In the insets are represented the control experiments at 30 C.

C. Ferreira, C. Lucas / FEBS Letters 581 (2007) 19231927 1925


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showed that gpd1Dgpd2D and gpp1Dgpp2D mutant strains

recovered the ts phenotype when external glycerol was pro-

vided, and that gpp1Dgpp2D mutant strain accumulates intra-

cellular glycerol at high temperatures [19]. This is consistent

with our results, since we observed a rescue of gpd1Dgpd2D

and gpp1Dgpp2D mutant strains ts phenotype on YPE when

cells were grown on YPG. The rescue was somehow more

effective for the gpp1Dgpp2D mutant.

A

0

10

20

30

0 10 20 30 40 50 60 70

Time (min)

Accumulationratio

(in/ou

t)

B

0 10 20 30 40 50 60 70

Time (min)

Accumulationratio

(in/out)

0

10

20

30

Time (min)

0 10 20 30 40 50 60 700

10

20

30

Accumulationratio

(in/out)

Accumulationratio

(in/out)

0 10 20 30 40 50 60 70

0

10

20

30

Time (min)

Fig. 3. Activity of the symporter at 37 C and glucose. Accumulation ratios of [14C] glycerol on exponential growing cells on YPD at 37 C: (A) wtcells and (B) gpp1Dgpp2D (n) and gpd1Dgpd2D () mutants cells. Cells were assayed in the absence (close symbols) or in the presence (open symbols)of CCCP. In the inserts are represented the control experiments at 30 C.

Fig. 4. Induction of active glycerol transport. Cells of wt and gpd1Dgpd2D mutant were grown to exponential phase on YPD or YPE at 30 C and37 C. The same batches of cells were used to detect the levels of Stl1p expression by Western blot usingSTL1-ZZ construction (A) and measure theradiolabeled glycerol maximum accumulation ratios (B).

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Such recovery, being due to intracellular glycerol accumula-

tion, could be to a certain extent obtained by the contribution

of the symporter activity. We propose this based on the fact

that the ts phenotype of the STL1 defective strain is not res-

cued when grown on YPG, and that the activity of the trans-

porter is improved at high temperatures as inferred from the

higher accumulation ratios. The increase observed on trans-

porter activity, higher for wt, correlates directly with Stl1p

protein levels.Based on these results, we propose that Stl1p contributes to

the fine-tuning of glycerol internal levels, applying to heat-

stress as to other physiological conditions, like diauxic transi-

tion. In this phase in particular, the cells go through a switch

from fermentative to respiratory metabolism, which must cor-

respond to a considerable signalling effort. This could explain

the fact ofSTL1 being highly and transiently expressed during

diauxic-shift, regardless of the needs for stress response [3].

Consistently, the glycerol produced during the previous expo-

nential growth phase is not consumed [7]. In this way, glycerol

could be implicated in crucial signalling steps towards the en-

try into stationary phase. All taken, through a complex and

tight regulation of STL1 expression, Stl1p would play a sine

qua non role for signalling.

Moreover, in this work, we show that at high temperatures

the Stl1p is active on YPD exponential cells. Genome-wide

analyses have shown that STL1 RNA is produced under these

conditions [9]. However, to our knowledge, this is the first time

that glucose repression is reported to be alleviated by high tem-

peratures, both at the level of protein expression and activity,

which imply proper traffic and localization besides synthesis. A

similar effect on glucose repression was before observed on

gpd1Dgpd2D mutant grown in high salt media [3]. For the time

being, the molecular nature of this glucose non-repression phe-

nomenon remains obscure, though one can speculate about the

possible change in nuclear localization of factors like Cat8.

Acknowledgements: We thank L. Neves and H. Johnson for criticalreading of the manuscript. C. Ferreira is supported by a Pos-DocGrant from the FCT, Ref. SFRH/BPD/20908/2004/W7D7.

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