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Mol Biol Cell. Apr 2005; 16(4): 2068–2076.
PMCID: PMC1073684

A Member of the Sugar Transporter Family, Stl1p Is the Glycerol/H+ Symporter in Saccharomyces cerevisiae

Benjamin Glick, Monitoring Editor

Abstract

Glycerol and other polyols are used as osmoprotectants by many organisms. Several yeasts and other fungi can take up glycerol by proton symport. To identify genes involved in active glycerol uptake in Saccharomyces cerevisiae we screened a deletion mutant collection comprising 321 genes encoding proteins with 6 or more predicted transmembrane domains for impaired growth on glycerol medium. Deletion of STL1, which encodes a member of the sugar transporter family, eliminates active glycerol transport. Stl1p is present in the plasma membrane in S. cerevisiae during conditions where glycerol symport is functional. Both the Stl1 protein and the active glycerol transport are subject to glucose-induced inactivation, following identical patterns. Furthermore, the Stl1 protein and the glycerol symporter activity are strongly but transiently induced when cells are subjected to osmotic shock. STL1 was heterologously expressed in Schizosaccharomyces pombe, a yeast that does not contain its own active glycerol transport system. In S. pombe, STL1 conferred the ability to take up glycerol against a concentration gradient in a proton motive force-dependent manner. We conclude that the glycerol proton symporter in S. cerevisiae is encoded by STL1.

INTRODUCTION

Glycerol, a C3 polyalcohol, is an important intermediate in energy metabolism in both prokaryotes and eukaryotes. It has long been used for therapeutic and industrial processes. Aspects of glycerol metabolism are also important in biotechnology, e.g., for bio-alcohol production yields or wine smoothness. Essential roles of glycerol in basic biochemical aspects have been extensively studied in several yeasts and fungi. These include biosynthesis of glycerophospholipid and triacylglycerol from glycerol 3-phosphate and dihydroxyacetone phosphate (Kohlwein et al., 1996 blue right-pointing triangle; Müllner and Daum, 2004 blue right-pointing triangle), redox balance (Ansell et al., 1997 blue right-pointing triangle; Valadi et al., 2004 blue right-pointing triangle), osmoadaptation (reviewed by Hohmann, 2002 blue right-pointing triangle), oxidative stress protection (Påhlman et al., 2001 blue right-pointing triangle), and response to heat shock (Siderius et al., 2000 blue right-pointing triangle). Responses to elevated temperatures and high osmolarity involve several signaling pathways including the protein kinase C pathway and the HOG pathway, which regulates intracellular levels of glycerol (Hohmann, 2002 blue right-pointing triangle; Wojda et al., 2003 blue right-pointing triangle).

In cells ranging from mammals (Lang et al., 1998 blue right-pointing triangle) to archea (Kempf and Bremer, 1998 blue right-pointing triangle), osmolytes play an important role in the response to osmotic stress caused by low water availability in environments as diverse as poorly irrigated soils or high-sugar musts. In eukaryotic microorganisms like algae or yeasts, polyols, primarily glycerol, act as osmolytes (reviewed by Brown, 1977 blue right-pointing triangle and Wang et al., 2001 blue right-pointing triangle). Their production, consumption and retention are consequently tightly regulated and dynamic processes (reviewed by Hohmann, 2002 blue right-pointing triangle). Magnaporthe grisea (rice blast) a phytopathogenic fungus with a strong impact on world economy, accumulates glycerol, which allows the penetration of the appressorium into the plant host cell (Thines et al., 2000 blue right-pointing triangle). Glycerol has also been shown to accumulate in the human pathogenic yeast Candida albicans in response to several types of osmotic stress in a HOG pathway–dependent manner (San José et al., 1996 blue right-pointing triangle). This pathway, besides controlling osmotic sensitivity in C. albicans as in other yeasts, also plays a role in dimorphic differentiation and virulence (Alonso-Monge et al., 1999 blue right-pointing triangle). Moreover, Ochiai et al. (2002 blue right-pointing triangle) showed that glycerol accumulates in C. albicans as a consequence of treatment with azole fungicides, suggesting a role for stress-signaling pathways in the control of hypha formation. In human physiology, plasma glycerol levels are correlated with obesity (reviewed by Large et al., 2004 blue right-pointing triangle). Glycerol has also been found to stimulate glucose-responsive neurons (Yang et al., 1999 blue right-pointing triangle). Furthermore, fasting glycerolemia is a predictor of impaired glucose tolerance in certain families (Gaudet et al., 2000 blue right-pointing triangle; see also review by Brisson et al., 2001 blue right-pointing triangle).

Glycerol can permeate the cell membrane of many organisms by specific members of the aquaporin family, the aquaglyceroporin channel proteins (Engel and Stahlberg, 2002 blue right-pointing triangle). In the yeast Saccharomyces cerevisiae, the gene FPS1 encodes a channel-type protein belonging to the major intrinsic protein (MIP) family (Luyten et al., 1995 blue right-pointing triangle). Fps1p allows regulated glycerol release under osmotic down-shift (Tamás et al., 1999 blue right-pointing triangle, 2000 blue right-pointing triangle). Furthermore, the channel has been shown to close upon osmotic up-shock, thus contributing to fast glycerol retention (Tamás et al., 1999 blue right-pointing triangle). Fps1p has recently been shown to mediate the first-order kinetics of glycerol uptake (Oliveira et al., 2003 blue right-pointing triangle), formerly considered a passive diffusion process (Lages et al., 1999 blue right-pointing triangle).

In addition, an active glycerol transport system of the proton symport type is present in S. cerevisiae when grown on nonfermentable carbon sources like glycerol or ethanol (Lages and Lucas, 1997 blue right-pointing triangle). This transport system operates independently of Fps1p-mediated diffusion (Sutherland et al., 1997 blue right-pointing triangle).

GUP1 and its homologue GUP2, encoding proteins with multiple membrane spans, have been suggested to be involved in active glycerol transport in S. cerevisiae (Holst et al., 2000 blue right-pointing triangle). GUP1 is essential for efficient growth on glycerol as sole carbon and energy source. GUP1 was also found to be important for glycerol-dependent survival upon salt stress in a mutant strain defective in glycerol synthesis. The strain was deleted for both isogenes of glycerol 3P-dehydrogenase, GPD1 and GPD2, and was therefore unable to produce sufficient amounts of glycerol to counteract osmotic stress (Albertyn et al., 1994 blue right-pointing triangle; Ansell et al., 1997 blue right-pointing triangle; Oliveira and Lucas, 2004 blue right-pointing triangle). Such mutants can be rescued by inclusion of small amounts of glycerol in the medium. Additional deletion of GUP1 makes these strains unable to grow on high-osmotic media with glycerol, suggesting a role of GUP1 in glycerol uptake (Holst et al., 2000 blue right-pointing triangle). GUP2 is a paralogue of GUP1. Its disruption did not yield similar phenotypes (Holst et al., 2000 blue right-pointing triangle). The deletion of GUP1 yielded a decrease in glycerol transport Vmax. Additional deletion of GUP2 further decreased the Vmax but was not enough to abolish transport. A remaining saturable component of glycerol uptake kinetics in these strains was attributed to an artifact created by the glycerol kinase (Gut1p), the first enzyme of glycerol catabolism, because the triple mutant gup1 gup2 gut1 did not show active uptake of glycerol when grown in ethanol-based medium (Holst et al., 2000 blue right-pointing triangle). However, it was later found that this mutant has the ability to take up glycerol actively during the diauxic shift if grown on glucose-based media containing high amounts of salt (Neves et al., 2004a blue right-pointing triangle). Therefore, the roles of GUP1 and GUP2 in active glycerol uptake remain unclear. In addition, both GUP1 and GUP2 are expressed constitutively, an expression pattern that is distinct from glycerol transport activity (Oliveira and Lucas, 2004 blue right-pointing triangle). Furthermore, Gup1p and Gup2p are, by sequence homology, members of a superfamily of membrane-bound O-acyl transferases (Hofmann, 2000 blue right-pointing triangle; Neves et al., 2004b blue right-pointing triangle). gup1 deletion mutants exhibit a number of additional phenotypes apparently unrelated to glycerol uptake, like defects in bipolar bud site selection (Ni and Snyder, 2001 blue right-pointing triangle), defects in sorting of carboxypeptidase Y, proteinase A, and alkaline phosphatase (Bonangelino et al., 2002 blue right-pointing triangle) and changes in telomere length (Askree et al., 2004 blue right-pointing triangle). Taking these different data into account, it is unlikely that GUP1 and GUP2 encode glycerol transporters, although they are involved in glycerol uptake under some circumstances. We presently have no explanation for this involvement.

A deeper understanding of the ways glycerol is produced, transported, and retained in model organisms like yeasts will contribute to an improved understanding of glycerol-related aspects ranging from human health to biotechnology. With the view that the active glycerol transporter in S. cerevisiae still remained to be identified, we designed a screen to find it in the EUROSCARF yeast deletion collection. We identified a mutant lacking a membrane protein in the sugar permease family (Nelissen et al., 1997 blue right-pointing triangle) that was unable to transport glycerol. The present work presents consistent evidence that the gene STL1, sugar transporter like (Zhao et al., 1994 blue right-pointing triangle), encodes the S. cerevisiae glycerol/H+ symporter.

MATERIALS AND METHODS

Strains

S. cerevisiae strains used in this study comprise two different genetic backgrounds: BY4742 (Brachmann et al., 1998 blue right-pointing triangle) and W303-1A (Thomas and Rothstein, 1989 blue right-pointing triangle). The collection of single deletion mutants in BY4742 was obtained from EUROSCARF. The strains with W303-1A genetic background are listed in Table 1. All stl1 deletion mutants were made by transformation with a PCR-generated fragment containing the KanMX cassette (Wach et al., 1994 blue right-pointing triangle) and a 200-base pair flanking region amplified from the corresponding BY4742 mutant obtained from EUROSCARF. Deletions were verified by PCR. The construct used to complement the stl1 mutation was made by amplification of the gene and flanking regions from genomic DNA using primers 5′ CCG GCT CGA GTT GCA GAT TAC GAA AGA ATC 3′ and 5′ GGC CTC TAG AGA ATA TCA YGG CAA GAC CGC 3′. The resulting PCR product was cloned between the XhoI and XbaI of pRS316. The construct was verified by DNA sequencing. Schizosaccharomyces pombe EG573 (mat1-M mat2/3-del::leuura4-D18 ade6) was used for heterologous expression of STL1. Escherichia coli strains XL1-Blue and DH5α were used for plasmid selection and propagation.

Table 1.
S. cerevisiae strains used in the present work

Yeast Media and Growth Conditions

Batch cultures of yeast were grown aerobically in complex medium (YP: 1% [wt/vol] yeast extract; 2% [wt/vol] peptone) supplemented with 2% (wt/vol) glucose (YPD), ethanol (YPE), or glycerol (YPG) as carbon and energy sources. Incubation was performed at 30°C, 180 rpm, orbital shaking with air/liquid ratio 2.5/1. The inability to utilize glycerol as a carbon and energy source was recognized on glycerol-based defined media supplemented with 0.05% peptone (Rønnow and Kielland-Brandt, 1993 blue right-pointing triangle). Synthetic complete (SC) and SC drop-out media were made according to Burke and collaborators (2000 blue right-pointing triangle). Drop tests were performed from cellular suspensions containing ~1 × 106 cells/ml. Tenfold serial dilutions were made, and 5 μl of each suspension was applied. S. pombe EG573 was cultivated on SC with 2% (wt/vol) glucose in identical physiological conditions as S. cerevisiae.

Transformation and DNA Manipulation

S. cerevisiae cells were transformed with the lithium acetate method (Ausubel et al., 1996 blue right-pointing triangle). Transformants were selected on selective synthetic medium (SC without uracil) or YPD containing 300 μg/ml G418. E. coli strains were transformed with a CaCl2/heat shock-based protocol (Ausubel et al., 1996 blue right-pointing triangle). Transformants were selected on LB agar supplemented with 100 μg/ml ampicillin. S. pombe EG573 was transformed using the method of Bähler et al. (1998 blue right-pointing triangle). Transformants were selected on SC without uracil, supplemented with 2% (wt/vol) agar and 15 μM thiamine.

DNA isolation and manipulation was performed by standard procedures (Sambrook et al., 1989 blue right-pointing triangle). DNA fragments obtained by PCR were purified using the QIAquick PCR product Purification Kit (Qiagen, Hilden, Germany). The concentration and purity of DNA was checked spectrophotometrically (Sambrook et al., 1989 blue right-pointing triangle).

Glycerol Transport Studies

The methods of Lages and Lucas (1995 blue right-pointing triangle, 1997 blue right-pointing triangle) and Lages et al. (1999 blue right-pointing triangle) were used to measure initial rates of radiolabeled glycerol uptake and the ratios of intracellular-to-extracellular glycerol concentrations (in/out accumulation ratios). To measure initial uptake rates of [14C]glycerol (Amersham, Freiburg, Germany), cells were washed twice and resuspended in ice-cold water to a final concentration of ~30 mg/ml (dry weight). The yeast suspension (10 μl) was mixed with 100 mM Tris/citrate buffer, pH 5.0 (10 μl), in a conical centrifuge tube and incubated for 2 min at 30°C in a water bath. The reaction was started by addition of 5 μl of a 20 mM aqueous solution of [14C]glycerol with a specific activity of 2000 dpm/nmol, corresponding to a final concentration of 4 mM. Uptake at this concentration is close to Vmax (Lages and Lucas, 1997 blue right-pointing triangle; Sutherland et al., 1997 blue right-pointing triangle). After 10 s, the reaction was stopped by dilution with 5 ml of ice-cold water. Cells were immediately collected on Whatman GF/C filters (Whatman, Maidstone, England), washed twice with 5 ml water, and immersed in vials containing 5 ml of scintillation fluid (Optiphase Hisafe2, Wallac Manufactured Products, Turku, Finland). The radio-labeled glycerol taken up by cells was measured in a Packard Tri-Carb 2200 CA liquid scintillation counter (Canberra Packard International, Zurich, Switzerland). The assays were made in triplicate and referenced to a blank made by inverting the sequence of addition of glycerol and water.

To determine the transport-driven ratios of [14C]glycerol inside versus outside the cells (in/out accumulation ratios), the cell suspension (80 μl) and 110 μl 100 mM Tris/citrate (pH 5.0) were transferred to conical glass centrifuge tubes and incubated at 30°C with magnetic stirring. After 2 min of incubation the experiment was started by addition of 10 μl (200 mM) of [14C]glycerol (specific activity 300 dpm/nmol). At appropriate time intervals, 10-μl aliquots were taken and filtered through Whatman GF/C filters. The filters were then washed twice with 5 ml ice-cold water and transferred to vials containing 5 ml scintillation fluid.

The protonophores/ionophores carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) were used to prevent accumulation of glycerol through glycerol/proton symport activity, as well as to induce efflux of intracellular radiolabeled glycerol, in assay concentration of 50 μM. The S. pombe and S. cerevisiae intracellular volumes used to calculate the intracellular glycerol concentrations were determined previously (Lages et al., 1999 blue right-pointing triangle).

Subcellular Localization Using Green Fluorescent Protein as Reporter

For expression of the STL1-GFP fusion protein we made use of pUG35 (U. Güldener and J. Hegemann, unpublished results), a centromere-based vector containing the MET25 promoter, followed by a polylinker and a sequence encoding yeast-enhanced green fluorescent protein (GFP). STL1 was amplified by PCR using primers 5′ GGC CTC TAG AAT GAA GGA TTT AAA ATT ATCG3′ and 5′GGC CGA ATT CAC CCT CAA AAT TTG CTT TAT C 3′. The resulting PCR product was cloned between the XbaI and EcoRI sites of pUG35, in frame with GFP, yielding pUG35-STL1. The construct was verified by DNA sequencing. Strains were transformed with pUG35-STL1. Cells were grown overnight in SC medium without uracil, at 30°C, with glycerol as carbon source. Cells were then diluted in the same medium to an OD600 ≈0.25 and allowed to reach OD600 = 0.6. Subsequently the cells were shifted to medium without uracil and methionine to induce the MET25 promoter. After 1–2 h of induction, cells were visualized by fluorescence microscopy. The remaining culture was then shifted to medium containing 2% glucose and methionine to switch off the promoter. Cells were visualized after 1 h of growth in glucose. A Leica TCS SP2 confocal scanning microscope (Heidelberg, Germany) was used to obtain the images. GFP was excitated at 488 nm and emitted light between 500 and 570 nm.

Western Blotting

A PCR-generated fragment encoding a duplicated IgG binding domain (Z) of protein A, and containing a KanMX cassette, was amplified from plasmid pFZ according to Whyte and Munro (2001 blue right-pointing triangle) using primers 5′ CAA ACA TCA AAA ATG AAG ATA CAG TGA ACG ATA AAG CAA ATT TTG AGG GTG GAG CAG GGG CGG GTG 3′ and 5′ AAT GCT TTC TTA AGT AAA TTA CAA AAT ATG ATT TGT GAG TTG TGT GTG AAG GTC GAC GGT ATC GAT AAG 3′. This was used to transform yeast to construct the epitope-tagged strains. Correct integration was verified by PCR. For Western blots, cells (0.2 mg dry weight) were collected by centrifugation, and protein extracts were prepared according to Burke et al. (2000 blue right-pointing triangle), separated by SDS-PAGE (10%), and transferred to nitrocellulose filters (Invitrogen, Carlsbad, CA) according to Kyhse-Andersen (1994 blue right-pointing triangle). The filters were incubated with a commercial mixture of horseradish peroxidase and antiperoxidase rabbit antibody (PAP, cat. no. Z0113, Dako-Cytomation A/S, Copenhagen, Denmark), and reacting polypeptides were visualized using ECL Plus Western Blotting Detection system (Amersham Biosciences) and a Storm 860 Scanner (Molecular Dynamics, Sunnyvale, CA). GFP-containing proteins were detected using rabbit antibodies raised against rx-YFP (Østergaard et al., 2001 blue right-pointing triangle) by Pineda Antikörper (Berlin, Germany; a kind gift of Drs. Jakob Winther and Henrik Østergaard).

Expression of STL1 in S. pombe

A chromosomal copy of STL1 was obtained by PCR using the primers 5′GGC CTC GAG ATG AAG GAT TTA AAA TTA TCG 3′ and 5′GGC CCC GGG TCA ACC CTC AAA ATT TGC 3′, including restriction sequences. It was inserted into E. coli/S. pombe shuttle multicopy expression vectors pREP82X and pREP4X between the XhoI and XmaI sites. These plasmids contain a thiamine-repressible promoter, nmt1, and ura4+, and Ampr as selective markers. They differ in promoter strength, weak and strong, respectively, because of increased expression-modulating mutations (Maundrell, 1993 blue right-pointing triangle). The integrity of the constructs was verified by sequencing. S. pombe EG573 was transformed with pREP4X-STL1 or with pREP82X-STL1. Transformants were selected and cultivated in liquid SC medium without uracil and supplemented with 2 nM thiamine for the lower expression plasmid pREP82X and 15 μM thiamine for the higher promoter strength plasmid pREP4X. Cells were harvested by centrifugation at OD600 = 1.2 (exponential growth phase), resuspended in the same medium without added thiamine (Sieiro et al., 2003 blue right-pointing triangle), and grown for 6 h for cultures from medium with 2 nM thiamine or for 30 h for cultures from medium with 15 μM thiamine. Cells were recollected by centrifugation and resuspended in water to a cell density of 30 mg/ml (dry weight) and kept on ice before glycerol transport assays.

RESULTS

Identification of Mutants Impaired in Glycerol Utilization

We began our screen with the assumptions that a mutant lacking the glycerol transporter would grow less efficiently on glycerol than the parent and that the transporter would be a multispanning membrane protein. On the basis of these assumptions, we screened a subset of the EUROSCARF single-deletion mutant collection, comprising 321 open reading frames with six or more predicted membrane spans, for glycerol utilization. Ten deletion mutants with a growth defect on a defined medium containing glycerol as the sole carbon source were found. Five did not grow (PER1, PCP1, DRS2, PMT5, and RHK1), whereas the remaining five grew poorly (PMR1, GEF1, ERG4, GUP1, and STL1). The strains deleted for PER1 (protein processing in the ER), PCP1 (processing of cytochrome c peroxidase), PMR1 (Ca2+/Mn2+ P-type ATPase, Golgi), and GEF1 (chloride channel localized to Golgi involved in intracellular iron metabolism) are listed at the SGD database (http://www.yeastgenome.org/) as having growth defects on respiratory substrates. Deletions of DRS2 (integral membrane Ca2+-ATPase) and PMT5 (protein O-mannosyl transferase of the ER) are listed as having a growth defect on lactate as a carbon source. Thus deletion of six of the genes identified in the present screen are known to be generally compromised for growth on nonfermentable carbon sources and thus probably not directly involved in glycerol uptake. In addition, RHK1 (involved in synthesis of the dolichol-linked oligosaccharide donor for N-linked glycosylation of proteins) and ERG4 (C24-sterol reductase) are both predicted to have 9 transmembrane domains and to be located in the ER. Interestingly, ergosterol is required for targeting of the tryptophan permease to the membrane (Umebayashi and Nakano, 2003 blue right-pointing triangle); this may also apply to the glycerol transporter. STL1 (Zhao et al., 1994 blue right-pointing triangle) and GUP1 (Holst et al., 2000 blue right-pointing triangle) are the only genes from this group encoding proteins with 12 putative transmembrane domains, and STL1 is the only one with an unknown function. Stl1p belongs to the sugar transporter family in S. cerevisiae (Nelissen et al., 1997 blue right-pointing triangle).

Most studies on active glycerol uptake have been performed in the W303-1A genetic background. Therefore STL1 was disrupted with the KanMX cassette (Wach et al., 1994 blue right-pointing triangle) in this strain. Deletion of STL1 in both BY4742 and W303-1A genetic backgrounds resulted in cells that grew poorly on glycerol (Figure 1). A copy of the STL1 gene comprising 2 kbp of the promoter region and 200 base pairs of the terminator region was introduced into the stl1-deleted strain using a centromere-based plasmid. The resulting strain grew like the wild-type strain, demonstrating functional complementation of the stl1 growth defect (Figure 1).

Figure 1.
Growth phenotypes on glycerol-based medium (2% wt/vol). Tenfold serial dilutions from left to right. Plates were incubated at 30°C for 5 d.

Stl1p Is Essential for Active Glycerol Uptake

The glycerol utilization phenotype prompted us to analyze glycerol transport in the stl1 mutant. Wild-type cells and stl1 cells were grown in glucose-based complex medium containing 1 M NaCl and 15 mM glycerol. Cells were harvested at the diauxic shift, and glycerol transport was measured. These conditions were chosen because they are known to allow detection of glycerol accumulation even in a strain lacking GUP1, GUP2, and GUT1 (Neves et al., 2004a blue right-pointing triangle). Deletion of STL1 eliminated active transport-driven glycerol accumulation (Figure 2A). The same occurred in the mutant strains stl1 fps1 and stl1 gut1 (data not in the figure). Interestingly, the gpd1 gpd2 stl1 strain, unlike the gpd1 gpd2 strain, did not grow on salt even in the presence of small amounts of glycerol. Additionally, cells deleted for all three genes previously known to be directly or indirectly involved in glycerol transport in S. cerevisiae, GUP1, GUP2, and FPS1 (Holst et al., 2000 blue right-pointing triangle; Tamás et al., 1999 blue right-pointing triangle), accumulated glycerol against a chemical gradient (Figure 2B). This accumulation was sensitive to the depletion of proton motive force by CCCP. Moreover, glycerol symport activity has previously been observed on nonfermentable carbon sources like ethanol (Lages and Lucas, 1997 blue right-pointing triangle). To avoid interference of metabolism with measurements of transport kinetics (Holst et al., 2000 blue right-pointing triangle; Oliveira et al., 2003 blue right-pointing triangle), experiments were also performed in a gut1 background. The deletion of STL1 in this background eliminated the active transport-driven glycerol accumulation (Figure 2C). This was also observed in the mutant strains stl1 gpd1 gpd2 and stl1 fps1 grown on ethanol (data not in the figure). Thus, under all growth conditions where S. cerevisiae is known to take up glycerol by active transport, deletion of STL1 abolished this capacity.

Figure 2.
(A) Accumulation ratios of [14C]glycerol in W303-1A (•) and W303-1A stl1 cells ([filled triangle]) at the diauxic shift after growth in glucose-based complex medium (YPD) containing 1 M NaCl and 15 mM glycerol. Efflux of radiolabel after the addition ...

Stl1p and Active Glycerol Transport Are Induced in Response to Osmotic Shock

Transcription of STL1 in glucose-grown cells is induced upon a shift to high salt conditions. The expression is transient and dependent on Hog1p and Hot1p (Rep et al., 2000 blue right-pointing triangle). Glycerol/H+ symport activity is absent in wild-type cells during exponential growth in glucose-based complex medium irrespective of the presence of NaCl (Holst et al., 2000 blue right-pointing triangle). It becomes measurable during the diauxic shift when glucose is exhausted (Lages and Lucas 1997 blue right-pointing triangle; Neves et al., 2004a blue right-pointing triangle).

To analyze the expression of STL1 in more detail, we integrated DNA encoding a duplicated IgG-binding domain (Z) of the Staphylococcus aureus protein A to the chromosomal copy of STL1. The strain expressing the fusion protein (Stl1p-ZZ) grew on glycerol medium like the wild-type, indicating that the fusion protein is functional. Subsequently, we analyzed the Stl1p level and measured uptake of glycerol under osmotic shock conditions. Cells were grown to exponential phase on glucose and shifted to medium containing 0.7 M NaCl. Samples were taken at time intervals and the Vmax of active transport using labeled glycerol at a single concentration was estimated. The same cells were used to determine the maximum in/out concentration ratio, reflecting active transport-driven accumulation (Figure 3A). Osmotic shock led to a sharp increase in glycerol accumulation capability. After 1.5 h of incubation at 30°C, significant amounts of Stl1p were produced in cells experiencing osmotic shock (Figure 3B). Stl1p was not produced in cells that were not osmotically shocked. Importantly, the levels of Stl1p and the glycerol uptake activity directly correlated. The high levels of Stl1p appearing after 1.5 h shift to NaCl gradually disappeared, and the protein was no longer detectable after 4 h (Figure 3B, GPD1 GPD2). These results are in agreement with the transient appearance of the STL1 transcript after salt shock, reported by Rep et al. (2000 blue right-pointing triangle). They found that the transcript appeared 0.5 h after the shock, but was undetectable after 1.5 h. Synthesis and accumulation of glycerol are major responses of yeast to sudden increases in the osmolarity (Hohmann, 2002 blue right-pointing triangle). The glycerol synthesis is accomplished by induction of the genes GPD1 and GPP2, encoding a glycerol 3-phosphate dehydrogenase and a glycerol 3-phosphate–specific phosphatase, respectively (Ansell et al., 1997 blue right-pointing triangle). The closure of the Fps1p channel ensures that glycerol does not exit the cell during osmotic shock (Tamás et al., 1999 blue right-pointing triangle). Interestingly, in cells that are deleted for the two isogenes GPD1 and GPD2, Stl1p appeared during the osmotic shock, but the down-regulation/degradation was prevented (Figure 3B, gpd1 gpd2), presumably because the osmotic imbalance persisted in the absence of glycerol synthesis. The gpd1 gpd2 strain exhibits a significant lag phase and somewhat slower growth after salt shock compared with the parent, so we also analyzed a strain that is deleted only for GPD1 and that is not affected in growth rate after salt shock. Under these conditions, the presence of Stl1p was also stabilized (Figure 3B, gpd1 GPD2).

Figure 3.
(A) Transient induction of active glycerol transport by salt shock in W303-1A (GPD1 GPD2) cells grown in YPD. NaCl to 0.7 M was added at t = 0 during the exponential growth phase. [14C]Glycerol initial uptake rates (at 4 mM; gray bars) and maximum accumulation ...

If cells were grown continuously in glucose-based medium with a high concentration of salt, Stl1p was detectable only when the cells entered the diauxic shift (Figure 4A, arrow, and B), consistent with active glycerol transport (Holst et al., 2000 blue right-pointing triangle; Neves et al., 2004a blue right-pointing triangle). However, gpd1 and gpd1 gpd2 cells, which produce only residual amounts of glycerol (Oliveira and Lucas, 2004 blue right-pointing triangle), made significant amounts of Stl1p during exponential growth in 0.7 M NaCl (Figure 4C). This is consistent with the fact that gpd1 gpd2 cells display glycerol uptake during exponential growth in glucose-based medium with NaCl (Holst et al., 2000 blue right-pointing triangle). This is not surprising, considering the growth defect of the gpd1 gpd2 stl1 strain on media containing 1 M NaCl and 15 mM glycerol. Thus, the absence of glycerol synthesis apparently prevented the down-regulation and degradation of Stl1p in this mutant.

Figure 4.
Induction of Stl1p in W303-1A cells containing STL1-ZZ during growth in YPD in the continuous presence of 0.7 M NaCl. Samples were taken at the points included in the growth curve shown in A and analyzed by Western blot (B). The arrow indicates the time ...

Stl1p Is Localized to the Plasma Membrane and Is Subject to Glucose Inactivation

The subcellular localization of Stl1p was analyzed using the GFP fused to the carboxy-terminus of Stl1p, with the gene under the control of the methionine-repressible MET25 promoter. The fusion protein was functional in uptake of [14C]glycerol. The majority of the fluorescence was detected at the plasma membrane in cells growing exponentially on glycerol-based medium (Figure 5A). This is consistent with a function of Stl1p as a plasma membrane-localized glycerol transporter. The symporter activity (Vmax) decreases progressively after transfer from ethanol- to glucose-containing medium. The activity is reduced to 10% within 30 min after transfer (Lages and Lucas, 1997 blue right-pointing triangle). In full accordance, when cells were shifted from glycerol to glucose, and the promoter driving the expression of the fusion protein was turned off by the addition of methionine, the fluorescence signal disappeared from the plasma membrane within 30 min. Most fluorescence was detected in the vacuolar lumen 1 h after the shift to glucose (Figure 5A). Western blot analysis using anti-GFP antibodies showed a 90-kDa band, presumably reflecting the full-length fusion protein. In addition, a 38-kDa degradation product was detected. On shift to glucose-containing medium the full-length protein disappeared, and the degradation product accumulated and appeared to be stable for at least 2.5 h (Figure 5B). These observations indicate that the protein is internalized into the vacuole, and that the GFP moiety is detached from Stl1p, releasing it from the membrane. Inactivation of Stl1p-ZZ by growth in glucose was also followed by Western blot analysis. When glucose was added to cells growing exponentially in ethanol-based complex medium, Stl1p progressively disappeared (Figure 5C, WT) and was no longer detected 1.5 h after the addition of glucose. This is consistent with the behavior of the GFP fusion protein. If, however, the fate of Stl1p was followed in an end3 mutant, which is defective in endocytosis (Raths et al., 1993 blue right-pointing triangle), Stl1p was not degraded within 4 h, and a larger molecular species appeared (Figure 5C, end3). This larger form of Stl1p may represent one or more ubiquitinated species of Stl1p. We conclude that Stl1p is inactivated by growth in glucose, internalized to the vacuole by endocytosis in an END3-dependent manner, where it is subsequently degraded.

Figure 5.
(A) Localization in vivo of fluorescent Stl1-GFP in W303-1A cells containing a centromere-based vector with the STL1-GFP fusion. Cells were grown in glycerol-based synthetic medium (left panels) and shifted to glucose and visualized after 1 h (right panels). ...

The S. cerevisiae STL1 Gene Provides Active Uptake of Glycerol When Expressed in S. pombe

Fission yeast, S. pombe, does not take up glycerol by active transport (Lages et al., 1999 blue right-pointing triangle). This yeast does not have any genes with high sequence homology to STL1. Therefore, we chose to introduce the S. cerevisiae STL1 gene into S. pombe to test if this gene indeed codes for the glycerol/H+ symporter. S. pombe transformants expressing STL1 from two promoters of different strength were obtained. The time needed for optimal expression of STL1 was initially determined by glycerol uptake experiments using a single concentration of radiolabeled glycerol in the transport Vmax range. For each construct, a short time interval was found in which glycerol accumulation could be measured after the promoter was turned on. This was 6 and 30 h for the weaker and the stronger promoter, respectively.

We determined glycerol accumulation ratios in a single transformant for each construct (Figure 6A) and compared it with untransformed cells as well as with control transformants harboring plasmids without STL1 insert (Figure 6B). Accumulation exceeded equilibrium and was higher for the transformants expressing the gene with the stronger promoter. The accumulation was sensitive to the dissipation of proton motive force through the action of the protonophores/ionophores CCCP and FCCP (Figure 6A). Furthermore, these drugs elicited glycerol efflux (Figure 6C). We conclude that the expression of the S. cerevisiae STL1 gene in S. pombe introduces uptake of glycerol in this yeast that is dependent on the proton motive force.

Figure 6.
(A) Accumulation ratios of [14C]glycerol in Schizosaccharomyces pombe transformants harboring the plasmids pREP82X-STL1 ([filled square]) (n = 5) and pREP4X-STL1 (○) (n = 8) and the transformant harboring pREP82X-STL1 in the presence of CCCP ([filled triangle]) ...

DISCUSSION

STL1 was identified in a screen for genes encoding membrane proteins involved in glycerol utilization. It has not previously been assigned a function. We propose that Stl1p is the active glycerol/H+ symporter in S. cerevisiae based on the following arguments. First, stl1 mutants are not able to efficiently utilize glycerol as a sole carbon and energy source. Second, active uptake of glycerol is absent in stl1 mutants. Third, the expression data from the literature as well as protein data presented here show that STL1 expression and Stl1 protein correlate directly with glycerol uptake activity. Fourth, the localization of Stl1p in the plasma membrane and its glucose inactivation are fully consistent with the function as a transporter. Fifth, heterologous expression of STL1 in S. pombe enables this yeast to take up glycerol via an active mechanism. Notably, the glycerol utilization phenotype of the stl1 mutant proved to be a direct consequence of its involvement in the glycerol transport, as the deletion of STL1 abolished glycerol accumulation.

Yeast utilizes glucose by fermentation and produces ethanol and small amounts of glycerol. The production of glycerol under fermentative conditions provides the cell with an electron sink that enables it to maintain redox balance (Ansell et al., 1997 blue right-pointing triangle). The produced glycerol exits the cell via the Fps1p channel. When all glucose is consumed, the yeast culture enters the diauxic shift, during which major changes in gene expression alter the fermentative to oxidative metabolism, allowing the yeast to utilize the produced ethanol and glycerol before entry into the stationary phase. Cat8p is a transcription factor that regulates a subset of gluconeogenic genes when cells are grown on nonfermentable carbon sources such as ethanol (Hedges et al., 1995 blue right-pointing triangle). STL1 is induced, in concert with 36 other genes, in a Cat8p-dependent manner during the diauxic shift (Haurie et al., 2001 blue right-pointing triangle). Most of these Cat8p-induced genes are related to ethanol utilization but in addition to STL1, the gene encoding the lactate permease, JEN1 is also induced (Makuc et al., 2001 blue right-pointing triangle). Correspondingly, glycerol symport has previously been shown to be repressed by glucose, induced by growth on nonfermentable carbon sources and transiently detectable during diauxic shift upon growth on glucose (Lages and Lucas, 1997 blue right-pointing triangle). STL1 is also highly and transiently induced by osmotic shock during exponential growth on glucose-based media (Posas et al., 2000 blue right-pointing triangle; Rep et al., 2000 blue right-pointing triangle; Yale and Bohnert 2001 blue right-pointing triangle; this work). The rapid appearance of Stl1p under these conditions suggests a role for the glycerol symporter during the immediate response to osmotic shock. This might be important in nature, considering the extreme, diverse and rapid changes in environmental conditions yeasts may experience. Because yeast cells leak a substantial amount of the produced glycerol into the medium (Shen et al., 1999 blue right-pointing triangle), this induction of Stl1p is not surprising. Degradation of Stl1p involves END3-mediated endocytosis. Several plasma membrane proteins with transport functions are subject to down-regulation via ubiquitination and subsequent endocytosis (Katzmann et al., 2002 blue right-pointing triangle; Hicke and Dunn, 2003 blue right-pointing triangle). Among other examples in yeast are the glucose inactivation of the lactate permease Jen1p (Andrade and Casal, 2001 blue right-pointing triangle; Paiva et al., 2002 blue right-pointing triangle) and the galactose transporter Gal2p (Horak and Wolf, 2001 blue right-pointing triangle). The occurrence of higher-molecular-weight Stl1-ZZ species, in particular in the end3 mutant (Figure 5), is consistent with the idea that Stl1p degradation is also signaled by ubiquitination.

The STL1 gene product is one of the 34 members of the sugar permease family in S. cerevisiae (Nelissen et al., 1997 blue right-pointing triangle), which is a part of the major facilitator superfamily (MFS). However, Stl1p is distinct from the hexose transporter subfamily, the maltose transporter subfamily and the inositol permease subfamily of the sugar permeases in S. cerevisiae. Hence the sugar permease family in yeast includes members that transport hexoses and inositol by facilitated diffusion and members that transport maltose and now glycerol by proton symport. Stl1p is predicted to contain 12 membrane-spanning helices. The elucidation of the structure of two members of the MFS, the glycerol 3-phosphate transporter and the lactose/H+ symporter, both from E. coli, revealed that they have very similar structures despite a low degree of sequence similarity (Abramson et al., 2003 blue right-pointing triangle, Huang et al., 2003 blue right-pointing triangle). Differences in residues that are involved in substrate binding and translocation are expected to generate the different transporter functions within the MFS family. It is thus not unexpected that a member of the sugar permease family in S. cerevisiae transports glycerol, given the structural resemblance between sugars and glycerol.

Polyols accumulate in plants and fungi in response to salt, low temperature, and drought. Polyols increase the osmotic potential of the cytoplasm and protect cellular structures and metabolism. In a number of plant species, mannitol and sorbitol are, in addition to sucrose, primary products of photosynthesis (Cheng et al., 2004 blue right-pointing triangle). As these plants experience salt or water stress, they synthesize polyols that are transported from the leaves, where they are synthesized, to nonphotosynthetic organs. Apium graveolans (celery) synthesizes and transports mannitol from leaves to other organs during water or salt stress. Mannitol is transported by a proton symport mechanism across the plasma membrane of the phloem strands. The transporter is a member of the sugar permease family of the MFS (Noiraud et al., 2001 blue right-pointing triangle). In addition, a sorbitol proton symporter has been identified in Prunus cerasus (sour cherry; Gao et al., 2003 blue right-pointing triangle) and in Malus domestica (apple; Ramsperger-Gleixner et al., 2004 blue right-pointing triangle; Watari et al., 2004 blue right-pointing triangle). Recently a polyol proton symporter in Arabidopsis thaliana has also been identified (Reinders et al., 2004 blue right-pointing triangle). These plant polyol proton symporters all belong to the sugar permease family of the MFS.

Debaryomyces hansenii (Lucas et al., 1990 blue right-pointing triangle) and Candida albicans (G. Kayingo, personal communication) also exhibit active glycerol transport. Stl1p homologues in D. hansenii (Accession no. CAG87598.1) and C. albicans (Accession no. CA0472) share 63 and 59% identity with Stl1p, respectively. Other yeasts for which genomic sequences are partially available via the Génolévures Project (Souciet et al., 2000 blue right-pointing triangle) that display active glycerol transport (Lages et al., 1999 blue right-pointing triangle) also contain sequences encoding putative proteins with a significant degree of identity to Stl1p, e.g., P. angusta, Zygosaccharomyces rouxii, and Kluyveromyces marxianus. K. lactis contains a gene (Accession no. XP_456249) encoding a protein with 71% identity to Stl1p. However, the type of glycerol uptake mechanism in this yeast is presently not known. Furthermore, several phytopathogenic fungi contain genes encoding putative proteins with a considerable degree of similarity, in particular Eremothecium gossypii (Accession no. NP_984235.1), Aspergillus nidulans (Accession no. XP_413305.1), and Magnaporthe grisea (Accession no. EAA53889.1), which are 70, 53, and 44% identical to Stl1p, respectively. Eremothecium and Magnaporthe depend on glycerol for appressorial turgor, allowing invasiveness (de Jong et al., 1997 blue right-pointing triangle). Aspergillus is able to take up glycerol, although it is not known whether this operates via an active mechanism (Visser et al., 1988 blue right-pointing triangle). These interspecies homologies are high considering that the closest relatives in S. cerevisiae, members of the hexose transporter family, only share 27% or less sequence identity with Stl1p. This suggests that active glycerol uptake is a mechanism that operates in many different yeasts and fungi.

Acknowledgments

We thank Dr. J. H. Hegemann from Heinrich-Heine-Universität, Institut für Mikrobiologie, Düsseldorf; Dr. H. Riezman from University of Geneva, Biochemistry Institute; and Dr. R. Egel, from the University of Copenhagen, for kindly providing the pUG35 GFP expression vector, S. cerevisiae RH266–1D, and S. pombe EG573, respectively. We thank J. Greisvold for skilful technical assistance, Dr. J. Winther for help with the bio-informatical analysis, and Dr. C. Tachibana for critical reading of the manuscript. C.F. is a PhD student from FCT (Fundação para a Ciência e Tecnologia), Ministério da Ciência e do Ensino Superior, Portugal, Ref. SFRH/BD/4714/2001. L.N. is PRODEP (Portugal) PhD student no. 53/258.002/00.

Notes

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E04-10-0884) on February 9, 2005.

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