FEBS Letters 581 (2007) 19231927

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 of the Saccharomyces cerevisiae glycerol/H+ symporter encoded by STL1, which correlates well with Stl1p levels. This happens in both fermentable and respiratory metabolic growth conditions, though the induction in the latter is much higher. The relief of glucose repression by high temperature at the level of protein expression and activity (Stl1p) is reported for the rst time. We reason that the glycerol internal levels ne-tuning, under heat-stress as in other physiological condition, can be achieved with the contribution of the tight regulation of the symporter. 2007 Federation of European Biochemical Societies. Published by 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 glycerol transport. Glycerol 3P dehydrogenase, from glycerol production pathway, is encoded by two isogenes GPD1 and GPD2 [1]. The gpd1Dgpd2D mutant is unable to produce enough 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 reported so far [2], overcoming glucose repression [3]. Also, the glycerol kinase (Gutlp) from glycerol consumption pathway interferes in glycerol transport creating measurement artefacts [2,4]. When this gene is deleted the transport Vmax is highly reduced [2,4]. In addition, in the pkc1D mutant (defective in the MAPKinase from PKC pathway), the transport Vmax is also diminished, possibly, due to the inability this mutant presents to derepress GUT1 under induction conditions [5]. Saccharomyces cerevisiae Stl1p, has been assigned as encoding the glycerol H+/symporter [3]. STL1 is induced in a Cat8p-dependent manner during diauxic shift. Cat8p is a transcription 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], symptomatic 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 proton 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 (citric, temperature, and oxidative) as well as carbon source regulation [17,18], just to mention the ones with more determinant inuence 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 observation of increased glycerol intracellular levels at high temperatures, and that temperature-induced osmoresistance of HOG pathway mutants could be related with this increase [20]. Accordingly, mutants impaired in glycerol production by deleting either GPD1/2 above mentioned, or GPP isogenes, encoding glycerol-3-P-phosphatases, exhibit a thermosensitivity that was suppressed by growth on glycerol-based media [19]. The present work reveals the contribution of the active glycerol transporter, Stl1p, for the maintenance of S. cerevisiae glycerol intracellular levels at high temperatures. Additionally, it reports for the rst time, a phenomenon of glucose repression alleviation caused by high temperature, both at the level of protein expression and activity.

2. Materials and methods

2.1. Strains, media and growth conditions S. cerevisiae strains used in this work were W303-1A [14], FVVY28 [3], gpd1Dgpd2D [1]; FVVY39 [3] and gpp1Dgpp2D [18]. Batch cultures 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 energy sources. Incubation and drop tests were performed as before [3,21]. 2.2. Glycerol transport studies The assays were performed as described before [7,22]. The S. cerevisiae intracellular volume used to calculate the intracellular glycerol concentrations was determined previously [22].

Corresponding author. Fax: +351 253678980. E-mail addresses: celiaf@bio.uminho.pt, 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

1924 2.3. Western blotting The 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 ChemiDoc XRS (Bio-Rad, Laboratories, Inc.) with Quantity-One 4.5.0 Software (Bio-Rad, Laboratories, Inc.).

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

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 transcription factors include Hap1p, Nrg1p and Sko1p and Smp1p, besides the already stated Hog1p/Hot1p and Cat8p. These are associated with redox regulation, glucose sensing/lamentous 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 temperature stress response, prompting us to study STL1/Stl1p at high temperatures. 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 more pronounced 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 mutant cells were provided with external glycerol (Fig. 1-YPG). Such improvement was more evident for gpp1Dgpp2D than for 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, accumulation 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 gpd1Dgpd2D and gpp1Dgpp2D 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 diffusion rates were measured and maintained constant in all conditions and strains (not shown). 3.3. Glucose repression over Stl1p is overcome at high temperatures To verify if the previously reported regulation of the symporter, 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), accumulation 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 spotted onto YPD, YPE and YPG. Cells were cultured for 5 days at 30 C and 37 C.

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

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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) wt cells 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.

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 identical results to wt (Fig. 3B, inset). Yet, the maximum in/out ratios at 37 C were considerably lower, respectively, 2 and 11 (Fig. 3B). As before, Fps1-mediated diusion 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 glucose-based media (Figs. 2 and 3). 3.4. Stl1p expression is enhanced at high temperatures The same batches of cells used to measure glycerol accumulation were analyzed by Western Blot for Stl1p as described before [3]. Signicant 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 control 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 ne-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 putative 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

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

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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) wt cells 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 and 37 C. The same batches of cells were used to detect the levels of Stl1p expression by Western blot using STL1-ZZ construction (A) and measure the radiolabeled glycerol maximum accumulation ratios (B).

showed that gpd1Dgpd2D and gpp1Dgpp2D mutant strains recovered the ts phenotype when external glycerol was provided, and that gpp1Dgpp2D mutant strain accumulates intracellular 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 eective for the gpp1Dgpp2D mutant.

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

1927 Pkcl activity aects glycerol metabolism in Saccharomyces cerevisiae. FEMS Yeast Res. 8, 767776. Rep, M., Krantz, M., Thevelein, J.M. and Hohmann, S. (2000) The transcriptional response of Saccharomyces cerevisiae to osmotic shock. Hotlp and Msn2p/Msn4p are required for the induction of subsets of high osmolarity glycerol pathway-dependent genes. J. Biol. Chem. 275, 82908300. Lages, F. and Lucas, C. (1997) Contribution to the physiological characterization of glycerol active uptake in Saccharomyces cerevisiae. Biochim. Biophys. Acta 1322, 818. Ferea, T.L., Botstein, D., Brown, P.O. and Rosenzweig, R.F. (1999) Systematic changes in gene expression patterns following adaptive evolution in yeast. Proc. Natl. Acad. Sci. USA 96, 9721 9726. Gasch, A.P., Spellman, P.T., Kao, C.M., Carmel-Harel, O., Eisen, M.B., Storz, G., Botstein, D. and Brown, P.O. (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol. Biol. Cell 11, 42414257. Yoshimoto, H., Saltsman, K., Gasch, A.P., Li, H.X., Ogawa, N., Botstein, D., Brown, P.O. and Cyert, M.S. (2002) Genome-wide analysis of gene expression regulated by the calcineurin/Crzlp signalling pathway in Saccharomyces cerevisiae. J. Biol. Chem. 277, 3107931088. Larsson, C., Pahlman, I.-L., Ansell, R., Rigoulet, M., Adler, L. and Gustafsson, L. (1998) The importance of the glycerol 3phosphate shuttle during aerobic growth of Saccahromyces cerevisiae. Yeast 14, 347357. van Dijken, J. and Scheers, W. (1986) Redox balance in the metabolism of sugars in yeasts. FEMS Microbiol. Rev. 32, 199 224. Sanders, D. and Slayman, C. (1982) Control of intracellular pH. Predominant role of oxidative metabolism, not proton transport, in the eukaryotic microorganism Neurospora. J. Gen. Physiol. 80, 377402. Van Aelst, L., Hohmann, S., Zimmermann, F., Jans, A. and Thevelein, J. (1991) A yeast homologue of the bovine lens bre MIP gene family complements the growth defect of a Saccharomyces cerevisiae mutant on fermentable sugars but not its defect in glucose-induced RAS-mediated AMPc signalling. EMBO J. 10, 20952104. Luyten, K., Albertyn, J., Skibbe, W., Prior, B., Thevelein, J. and Hohmann, S. (1995) Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and eux and it is inactive under osmotic stress. EMBO J. 14, 13601371. Tao, W., Deschenes, R. and Fassler, J. (1999) Intracellular glycerol levels modulate the activity of Slnlp, a Saccharomyces cerevisiae two component regulator. J. Biol. Chem. 274, 360 367. Hohmann, S. (2002) Osmotic stress signalling and osmoadaptation in yeasts. Microbiol. Mol. Biol. Rev. 66, 300372. Pahlman, A.K., Granath, K., Ansell, R., Hohmann, S. and Adler, L. (2001) The yeast glycerol 3-phosphatases Gpp1p and Gpp2p are required for glycerol biosynthesis and dierentially involved in the cellular responses to osmotic, anaerobic, and oxidative stress. J. Biol. Chem. 276, 35553563. Siderius, M., van Wuytswinkel, O., Reijenga, K.A., Kelders, M. and Mager, W.H. (2000) The control of intracellular glycerol in Saccharomyces cerevisiae inuences osmotic stress response and resistance to increased temperature. Mol. Microbiol. 36, 1381 1390. Wojda, I., Alonso-Monge, R., Bebelman, J.P., Mager, W.H. and Siderius, M. (2003) Response to high osmotic conditions and elevated temperature in Saccharomyces cerevisiae is controlled by intracellular glycerol and involves coordinate activity of MAP kinase pathways. Microbiology 149, 11931204. Ferreira, C., Silva, S., von Voorst, F., Aguiar, C., KiellandBrandt, M.C., Lucas, C. and Brandt, A. (2006) Absence of Guplp in Sacharomyces cerevisiae results in a defective cell wall composition, assembly, stability and morphology. FEMS Yeast Res. 6, 10271038. Lages, F., Silva-Graca, M. and Lucas, C. (1999) Active glycerol uptake is a mechanism underlying halotolerance in yeasts: a study of 42 species. Microbiology 145, 25772585.

Such recovery, being due to intracellular glycerol accumulation, 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 rescued when grown on YPG, and that the activity of the transporter is improved at high temperatures as inferred from the higher accumulation ratios. The increase observed on transporter activity, higher for wt, correlates directly with Stl1p protein levels. Based on these results, we propose that Stl1p contributes to the ne-tuning of glycerol internal levels, applying to heatstress as to other physiological conditions, like diauxic transition. In this phase in particular, the cells go through a switch from fermentative to respiratory metabolism, which must correspond to a considerable signalling eort. This could explain the fact of STL1 being highly and transiently expressed during diauxic-shift, regardless of the needs for stress response [3]. Consistently, the glycerol produced during the previous exponential growth phase is not consumed [7]. In this way, glycerol could be implicated in crucial signalling steps towards the entry 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 rst time that glucose repression is reported to be alleviated by high temperatures, both at the level of protein expression and activity, which imply proper trac and localization besides synthesis. A similar eect 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 phenomenon 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 critical reading of the manuscript. C. Ferreira is supported by a Pos-Doc Grant from the FCT, Ref. SFRH/BPD/20908/2004/W7D7.

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