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. INTRODUCTIONGlycerol, a C 3 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;Mü llner and Daum, 2004), redox balance (Ansell et al., 1997;Valadi et al., 2004), osmoadaptation (reviewed by Hohmann, 2002, oxidative stress protection (Påhlman et al., 2001), and response to heat shock (Siderius et al., 2000). 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;Wojda et al., 2003).In cells ranging from mammals (Lang et al., 1998) to archea (Kempf and Bremer, 1998), 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 andWang et al., 2001). Their production, consumption and retention are consequently tightly regulated and dynamic processes (reviewed by Hohmann, 2002). 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). Glycerol has also...

Many yeast species can utilize glycerol, both as a sole carbon source and as an osmolyte. In Saccharomyces cerevisiae, physiological studies have previously shown the presence of an active uptake system driven by electrogenic proton symport. We have used transposon mutagenesis to isolate mutants affected in the transport of glycerol into the cell. Here we present the identification of YGL084c, encoding a multimembrane‐spanning protein, as being essential for proton symport of glycerol into S. cerevisiae. The gene is named GUP1 (glycerol uptake) and, for growth on glycerol, is important as a carbon and energy source. In addition, in strains deficient in glycerol production it also provides osmotic protection by the addition of glycerol. Another open reading frame (ORF), YPL189w, presenting a high degree of homology to YGL084c, similarly appears to be involved in active glycerol uptake in salt‐containing glucose‐based media in strains deficient in glycerol production. Analogously, this gene is named GUP2. To our knowledge, this is the first report on a gene product involved in active transport of glycerol in yeasts. Mutations with the same phenotypes occurred in two other ORFs of previously unknown function, YDL074c and YPL180w.

A comparison of 42 yeast species with respect to growth in the presence of high NaCl concentration and characteristics of glycerol uptake is presented. The yeast species were classified into four classes on the basis of their ability to grow in the presence of 1, 2, 3 or 4 M NaCl. Considering that two different types of active-transport systems for glycerol uptake have been described, Na M /glycerol and H M /glycerol symports, glycerol transport was investigated by testing for proton uptake upon glycerol addition in cells incubated in the absence and in the presence of NaCl. Only strains belonging to the two higher classes of salt tolerance showed constitutive active glycerol uptake, and could accumulate glycerol internally against a concentration gradient. Five of these strains exhibited a H M /glycerol symport. All the other strains showed evidence of the activity of a salt-dependent glycerol uptake similar to that described in the literature for Debraryomyces hansenii. The strains within the two lower classes of salt tolerance showed, to varying degrees, glycerol active uptake only when glycerol was used as the carbon and energy source, suggesting that this uptake system is involved in glycerol catabolism. The results within this work suggest that active glycerol uptake provides a basis for high halotolerance, helping to maintain a favourable intracellular concentration of glycerol. The relation between the constitutive expression of such carriers and a higher level of salt-stress resistance suggests that this may be an evolutionary advantage for growth under such conditions.

Evidence is presented here that in Saccharomyces cerevisiae IGC 3507, grown either on glycerol, ethanol or acetate, glycerol is transported by a high affinity uptake system of the electrogenic proton symport type, with Km of 1.7 +/- 0.7 mM, Vmax 441 +/- 19 micromolh(-1) g(-1) dry weight and a stoichiometry of 1:1 proton per molecule of glycerol, at 30 degrees C and pH 5.0. No competitors were found among other polyols and sugars. Glycerol maximum accumulation ratios followed p.m.f. with extracellular pH. CCCP prevented glycerol accumulation, and inhibited uptake. NaCl did not interfere with H+/glycerol kinetics and energetics. This transport system was shown to be under glucose repression and inactivation. Glucose-grown cells presented, instead, a lower affinity permease for glycerol, probably a facilitated diffusion. Growth on glucose in the presence of NaCl did not induce the high affinity carrier. The stringent control of cell physiological condition over induction suggests for glycerol proton symport rather a physiological role connected with growth under gluconeogenic conditions.

Eadie-Hofstee plots of glycerol uptake in wild-type Saccharomyces cerevisiae W303-1A grown on glucose showed the presence of both saturable transport and simple diffusion, whereas an fps1⌬ mutant displayed only simple diffusion. Transformation of the fps1⌬ mutant with the glpF gene, which encodes glycerol transport in Escherichia coli, restored biphasic transport kinetics. Yeast extract-peptone-dextrose-grown wild-type cells had a higher passive diffusion constant than the fps1⌬ mutant, and ethanol enhanced the rate of proton diffusion to a greater extent in the wild type than in the fps1⌬ mutant. In addition, the lipid fraction of the fps1⌬ mutant contained a lower percentage of phospholipids and a higher percentage of glycolipids than that of the wild type. Fps1p, therefore, may be involved in the regulation of lipid metabolism in S. cerevisiae, affecting membrane permeability in addition to fulfilling its specific role in glycerol transport. Simultaneous uptake of glycerol and protons occurred in both glycerol-and ethanol-grown wild-type and fps1⌬ cells and resulted in the accumulation of glycerol at an inside-to-outside ratio of 12:1 to 15:1. Carbonyl cyanide m-chlorophenylhydrazone prevented glycerol accumulation in both strains and abolished transport in the fps1⌬ mutant grown on ethanol. Likewise, 2,4-dinitrophenol inhibited transport in glycerol-grown wild-type cells. These results indicate the presence of an Fps1p-dependent facilitated diffusion system in glucose-grown cells and an Fps1p-independent proton symport system in derepressed cells.Glycerol crosses all biological membranes by passive diffusion due to its lipophilic nature. In addition, specific transport proteins are frequently produced by microorganisms, resulting in more rapid transport of glycerol across the membrane. Active glycerol transport systems requiring the expenditure of metabolic energy have been identified in Zygosaccharomyces rouxii, Debaryomyces hansenii and Pichia sorbitophila (21,23,36), whereas glycerol crosses the Escherichia coli cytoplasmic membrane via a proteinaceous pore mechanism which is encoded by glpF (15).It has been assumed that glycerol is taken up by Saccharomyces cerevisiae by passive diffusion only. Recently FPS1, which encodes a protein belonging to the MIP family, has been shown to affect the movement of glycerol across the membrane of S. cerevisiae (24). The FPS1 gene was isolated as a multicopy suppressor of the growth defect on fermentable sugars of a yeast fdp1 (FDP1 is also known as CIF1 and GGS1) mutant (33). Fps1p seems to play an important role in glycerol efflux, since mutants lacking FPS1 fail to rapidly release excess glycerol when hyperosmotic stress is relieved and during glycerol overproduction (reference 24 and unpublished results).The MIP family is a group of channel proteins present in organisms ranging from bacteria to humans (28). Most of these proteins are around 250 to 280 amino acids long and consist of six membrane-spanning segments. Fps1p differs from most members of the MIP family b...

Glycerol has been shown to cross the plasma membrane of Saccharomyces cerevisiae through (1) a H(+)/symport detected in cells grown on non-fermentable carbon sources, (2) the constitutively expressed Fps1p channel and (3) by passive diffusion. The Fps1p channel has been named a facilitator for mediating glycerol low affinity transport of the facilitated diffusion type. We present experimental evidence that this kinetic is an artefact created by glycerol kinase activity. Instead, the channel is shown to mediate the major part of glycerol's passive diffusion. This is not incompatible with Fps1p's major role in vivo, which has been previously shown to be the control of glycerol export under osmotic stress or in reaction to turgor changes. We also verified that FPS1 overexpression caused an increase in H(+)/symport V(max). Furthermore, yfl054c and fps1 mutants were equally affected by exogenously added ethanol, being the correspondent passive diffusion stimulated. For the first time, to our knowledge, a phenotype attributed to the functioning of YFL054c gene is presented. Glycerol passive diffusion is thus apparently channel-mediated. This is discussed according to glycerol's chemical properties, which contradict the widely spread concept of glycerol's liposoluble nature. The discussion considers the multiple roles that the intracellular levels of glycerol and its pathway regulation might play as a central key to metabolism control.

Pichia sorbitophila is a halotolerant yeast capable of surviving to extracellular NaCl concentrations up to 4 M in mineral medium when glucose or glycerol are the only carbon and energy sources. Evidence is presented here that glycerol, the main compatible solute this yeast accumulates so as to maintain osmotic balance, is actively co-transported with protons. This transport system was shown to be constitutive, not needing induction by either glycerol or salt, and was not repressible by glucose. In glucose- or glycerol-grown cells, a simple diffusion was detectable, and iterative calculations were performed to calculate kinetic parameters, in the presence and in the absence of NaCl. At 25 degrees C, pH 5.0, in glucose-grown cells these were: Km = 0.81 +/- 0.11 mM and Vmax = 634.2 +/- 164.8 mumol h-1 per g (glycerol); Km = 1.28 +/- 0.60 mM and Vmax = 558.6 +/- 100.6 mumol h-1 per g (protons). Correspondent stoichiometry was approximately 1, either for these conditions or in the presence of 1 M-NaCl. An increase in accumulation capacity was evident when different concentrations of NaCl were present. This capacity was shown to be dependent on delta pH and membrane potential, consistently with an electrogenic character. We suggest that the main role of this system is in osmoregulation, by keeping glycerol accumulated inside the cells, compensating for leakage, due to its liposoluble character.

The toxic effect of NaCl and KCI on growth of the marine yeast Debaryomyces hansenii on glucose or glycerol was studied. Above a threshold value, both salts reduced the specific growth rate, specific glucose and glycerol respiration rates and specific glucose fermentation rate, as well as biomass yields. The exponential inhibition constant, k, and minimum toxic concentration, cmin, were similar for all physiological parameters assayed. The effect of either salt on the specific activity of several glycolytic enzymes showed a similar inhibition pattern, although at much lower salt concentrations compared with the physiological parameters. In agreement with published results on glycerol phosphate dehydrogenase stimulation by salt, we present evidence that a general glycolytic flux deviation could occur naturally during salt stress, due to the intrinsic sensitivity of the glycolytic enzymes to intracellular ion concentrations.