Uptake of sucrose by Saccharomyces cerevisiae

Sucrose transport has been shown to occur in several Suc^? and Suc⁺Saccharomyces cerevisiae strains as an energy-dependent process. Assay conditions have been established to avoid both extra- and intracellular hydrolysis of the disaccharide thus allowing the identification of sucrose as such inside the cell immediately after the uptake; acid pH values (4.0–5.0) were optimal for transport although significant uptake was also detected at neutral pH. Transport of sucrose was not dependent on ATP and seemed to be driven by protonmotive force supplied by the electrochemical gradient of protons across the plasma membrane. The actual symport of protons along with sucrose was directly detected by continuous pH measurement of the reaction mixtures and the initial rate of proton movement in the symport process was determined. KC1 inhibited transport of sucrose suggesting that exit of K⁺ ions might well be involved in maintaining the electroneutrality of the process. On the other hand, NaCl stimulated transport by 50% in our experimental conditions. The specificity of sucrose transport was also tested using different disaccharides.     

Uptake of trehalose by Saccharomyces cerevisiae.

Trehalose, a storage sugar of baker's yeast, is known not to be metabolized when added to a cell suspension in water or a growth medium and to support growth only after a lag of about 10 h. However, it was transported into cells by at least two transport systems, the uptake being active, with a pH optimum at 5.5. There was no stoicheiometry with the shift of protons into cells observed at high trehalose concentrations. Trehalose remained intact in cells and was not appreciably lost to a trehalose-free medium. The uptake systems were present directly after growth on glucose, then decayed with a half-life of about 25 min but could be reactivated by aerobic incubation with trehalose, maltose, alpha-methyl-D-glucoside, glucose or ethanol. The uptake systems thus induced were different as revealed by competition experiments. At least one of the systems for trehalose uptake showed cooperative kinetics. Comparative anaysis with other disaccharides indicated the existence in Saccharomyces cerevisiae, after induction with trehalose, of at least four systems for the uptake of alpha-methyl-D-glucoside, four systems for maltose, together with the two for trehalose, variously shared by the sugars, the total of alpha-glucoside-transporting systems being five.     

Mechanism of Benzoic Acid Uptake by Saccharomyces cerevisiae

A fast uptake of the preservative benzoic acid was observed in Saccharomyces cerevisiae, reaching saturation in about two min and then remaining constant at this level. The strong dependence of benzoic acid uptake on pH was due to the relative distribution of molecular and ionic forms in solution and not to the pH itself. The molecular form was the only one taken up by the cells. The specificity of the uptake mechanism was evidenced by the pattern of irreversible heat inactivation of the uptake system resembling protein denaturation by heat. Furthermore, the effect of temperature on the uptake was similar to that observed in enzymic reactions, whereas the kinetic data of uptake conformed to the Michaelis-Menten curve of saturation with a Km of 1.54 X 10(-2) M and Vmax of 3 X 10(-3) M/10s. The evidence presented in this paper indicates that compounds of protein nature are involved in the uptake of this preservative.     

Uptake and utilization of lyso-phosphatidylethanolamine by Saccharomyces cerevisiae

Phosphatidylethanolamine (PtdEtn) is synthesized by multiple pathways located in different subcellular compartments in yeast. Strains defective in the synthesis of PtdEtn via phosphatidylserine (PtdSer) synthase/decarboxylase are auxotrophic for ethanolamine, which must be transported into the cell and converted to phospholipid by the cytidinediphosphate-ethanolamine-dependent Kennedy pathway. We now demonstrate that yeast strains with psd1Delta psd2Delta mutations, devoid of PtdSer decarboxylases, import and acylate exogenous 1-acyl-2-hydroxyl-sn-glycero-3-phosphoethanolamine (lyso-PtdEtn). Lyso-PtdEtn supports growth and replaces the mitochondrial pool of PtdEtn much more efficiently than and independently of PtdEtn derived from the Kennedy pathway. Deletion of both the PtdSer decarboxylase and Kennedy pathways yields a strain that is a stringent lyso-PtdEtn auxotroph. Evidence for the specific uptake of lyso-PtdEtn by yeast comes from analysis of strains harboring deletions of the aminophospholipid translocating P-type ATPases (APLTs). Elimination of the APLTs, Dnf1p and Dnf2p, or their noncatalytic beta-subunit, Lem3p, blocked the import of radiolabeled lyso-PtdEtn and resulted in growth inhibition of lyso-PtdEtn auxotrophs. In cell extracts, lyso-PtdEtn is rapidly converted to PtdEtn by an acyl-CoA-dependent acyltransferase. These results now provide 1) an assay for APLT function based on an auxotrophic phenotype, 2) direct demonstration of APLT action on a physiologically relevant substrate, and 3) a genetic screen aimed at finding additional components that mediate the internalization, trafficking, and acylation of exogenous lyso-phospholipids.     

Proton Gradient-Driven Nickel Uptake by Vacuolar Membrane Vesicles of Saccharomyces cerevisiae

A vacuolar H⁺-ATPase-negative mutant of Saccharomyces cerevisiae was highly sensitive to nickel ion. Accumulation of nickel ion in the cells of this mutant of less than 60% of the value for the parent strain arrested growth, suggesting a role for this ATPase in sequestering nickel ion into vacuoles. An artificially imposed pH gradient (interior acid) induced transient nickel ion uptake by vacuolar membrane vesicles, which was inhibited by collapse of the pH difference but not of the membrane potential. Nickel ion transport into vacuoles in a pH gradient-dependent manner is thus important for its detoxification in yeast.     

Mutation of Saccharomyces cerevisiae Preventing Uptake of S-Adenosylmethionine

A mutant has been isolated whose aberration severely restricts the ability of cells of Saccharomyces cerevisiae to take up S-adenosylmethionine. The mutation apparently also affects adenosylhomocysteine uptake, but not that of the S-adenosylmethionine moieties adenine, homocysteine, homoserine, or methionine, nor the sulfonium compound, S-methylmethionine. It is a single, chromosomal mutation whose expression is not dependent on the presence of ammonium ions.     

Uptake of nystatin by protoplasts of sensitive and resistant cells of Saccharomyces cerevisiae

The uptake of nystatin by protoplasts derived from sensitive and resistant cells of Saccharomyces cerevisiae has been studied as a function of nystatin concentration, temperature and pH. The presence or absence of glucose in the uptake experiments was also studied. Activation energies (Ea) for nystatin uptake revealed profound differences between protoplasts derived from sensitive and resistant cells. Those for the latter closely resembled their whole cell counterparts. The values of Ea for the uptake of nystatin under all the conditions studied indicate the importance of the cell wall in the uptake process.     

Sulfate Uptake in Saccharomyces cerevisiae: Biochemical and Genetic Study

Sulfate uptake is the first step of the sulfate assimilation pathway, which has been shown in our laboratory to be part of the methionine biosynthetic pathway. Kinetic study of sulfate uptake has shown a biphasic curve in a Lineweaver-Burk plot. The analysis of this plot indicates that two enzymes participate in sulfate uptake. One (permease I) has a high affinity for the substrate (K(m) = 0.005 mM); the other (permease II) shows a much lower affinity for sulfate (K(m) = 0.35 mM). Regulation of the synthesis of both permeases is under the control of exogenous methionine or S-adenosylmethionine. It was shown, moreover, that synthesis of sulfate permeases is coordinated with the synthesis of the other methionine biosynthetic enzymes thus far studied in our laboratory. An additional specific regulation of sulfate permeases by inhibition of their activity by endogenous sulfate and adenosyl phosphosulfate (an intermediate metabolite in sulfate assimilation) has been shown. A mutant unable to concentrate sulfate has been selected. This strain carried mutations in two independent genes. These two mutations, separated in two different strains, lead to modified kinetics of sulfate uptake. The study of these strains leads us to postulate that there is an interaction in situ between the products of these two genes.     

Uptake and phosphorylation of 2-deoxy-D-glucose by wild-type and single-kinase strains of Saccharomyces cerevisiae

The role of phosphorylation in sugar transport in baker's yeast was studied using 2-deoxy-D-glucose. In wild-type baker's yeast, 2-deoxy-D-glucose is accumulated as a mixture of the free sugar and several derivatives. Pool labeling experiments, designed to determine the temporal order of appearance of labeled 2-deoxy-D-glucose in the intracellular pools, have confirmed previous reports that 2-deoxy-D-glucose first appears in the sugar phosphate pool. Such results are consistent with a transport associated phosphorylation mechanism. Since wild-type yeasts contain three enzymes which could participate in such a process, hexokinase isozymes PI and PII and glucokinase, pool labeling experiments were carried out with single-kinase mutant strains containing only one of these enzymes. Results similar to those for wild-type strains were obtained for all three single-kinase strains, suggesting that if transport associated phosphorylation does occur in baker's yeast, it is not a function of the specific kinase present in the cell. While the results of the pool labeling experiments are consistent with a transport associated phosphorylation mechanism for 2-deoxy-D-glucose, caution is urged in interpreting the results of experiments with whole cells where problems of compartmentation and multiple pools are difficult to assess.     

Enhanced Iron Uptake of Saccharomyces cerevisiae by Heterologous Expression of a Tadpole Ferritin Gene

We genetically engineered Saccharomyces cerevisiae to express ferritin, a ubiquitous iron storage protein, with the major heavy-chain subunit of tadpole ferritin. A 450-kDa ferritin complex can store up to 4,500 iron atoms in its central cavity. We cloned the tadpole ferritin heavy-chain gene (TFH) into the yeast shuttle vector YEp352 under the control of a hybrid alcohol dehydrogenase II and glyceraldehyde-3-phosphate dehydrogenase promoter. We confirmed transformation and expression by Northern blot analysis of the recombinant yeast, by Western blot analysis using an antibody against Escherichia coli-expressed TFH, and with Prussian blue staining that indicated that the yeast-expressed tadpole ferritin was assembled into a complex that could bind iron. The recombinant yeast was more iron tolerant in that 95% of transformed cells, but none of the recipient strain cells, could form colonies on plates containing 30 mM ferric citrate. The cell-associated concentration of iron was 500 μg per gram (dry cell weight) of the recombinant yeast but was 210 μg per gram (dry cell weight) in the wild type. These findings indicate that the iron-carrying capacity of yeast is improved by heterologous expression of tadpole ferritin and suggests that this approach may help relieve dietary iron deficiencies in domesticated animals by the use of the engineered yeast as a feed and food supplement.     

Uptake of Selenite by Saccharomyces cerevisiae Involves the High and Low Affinity Orthophosphate Transporters

Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake. When cells are grown at low P_i concentrations, the high affinity phosphate transporter Pho84p is the major contributor to selenite uptake. When phosphate is abundant, selenite is internalized through the low affinity P_i transporters (Pho87p, Pho90p, and Pho91p). Accordingly, inactivation of the high affinity phosphate transporter Pho84p results in increased resistance to selenite and reduced uptake in low P_i medium, whereas deletion of SPL2, a negative regulator of low affinity phosphate uptake, results in exacerbated sensitivity to selenite. Measurements of the kinetic parameters for selenite and phosphate uptake demonstrate that there is a competition between phosphate and selenite ions for both P_i transport systems. In addition, our results indicate that Pho84p is very selective for phosphate as compared with selenite, whereas the low affinity transporters discriminate less efficiently between the two ions. The properties of phosphate and selenite transport enable us to propose an explanation to the paradoxical increase of selenite toxicity when phosphate concentration in the growth medium is raised above 1 mm.     

Uptake of GABA and putrescine by UGA4 on the vacuolar membrane in Saccharomyces cerevisiae

The product of the UGA4 gene in Saccharomyces cerevisiae, which catalyzes the transport of 4-aminobutyric acid (GABA), also catalyzed the transport of putrescine. The Km values for GABA and putrescine were 0.11 and 0.69 mM, respectively. The UGA4 protein was located on the vacuolar membrane as determined by the effects of bafilomycin A1 and by indirect immunofluorescence microscopy. Uptake of both GABA and putrescine was inhibited by spermidine and spermine, although these polyamines are not substrates of UGA4. The UGA4 mRNA was induced by exposure to GABA, but not putrescine over 12h. The growth of an ornithine decarboxylase-deficient strain was enhanced by putrescine, and both putrescine and spermidine contents increased, when the cells were expressing UGA4. The results suggest that a substantial conversion of putrescine to spermidine occurs in the cytoplasm even though UGA4 transporter exists on vacuolar membranes.     

Xylose fermentation by Saccharomyces cerevisiae

We have performed a comparative study of xylose utilization in Saccharomyces cerevisiae transformants expressing two key enzymes in xylose metabolism, xylose reductase (XR) and xylitol dehydrogenase (XDH), and in a prototypic xylose-utilizing yeast, Pichia stipitis. In the absence of respiration (see text), baker's yeast cells convert half of the xylose to xylitol and ethanol, whereas P. stipilis cells display rather a homofermentative conversion of xylose to ethanol. Xylitol production by baker's yeast is interpreted as a result of the dual cofactor dependence of the XR and the generation of NADPH by the pentose phosphate pathway. Further limitations of xylose utilization in S. cerevisiae cells are very likely caused by an insufficient capacity of the non-oxidative pentose phosphate pathway, as indicated by accumulation of sedoheptulose-7-phosphate and the absence of fructose-1,6-bisphosphate and pyruvate accumulation. By contrast, uptake at high substrate concentrations probably does not limit xylose conversion in S. cerevisiae XYL1/XYL2 transformants. Correspondence to: M. Ciriacy     

D-xylose utilization by Saccharomyces cerevisiae

Although it is generally accepted that Saccharomyces cerevisiae is unable to assimilate D-xylose, four strains were found to utilize xylose aerobically at different efficiencies in the presence of a mixture of substrates. The degree of D-xylose utilization by S. cerevisiae ATCC 26602 depended upon the presence of other substrates or yeast extract. The greatest amount of xylose (up to 69% over 7 d) was utilized when sugar substrates such as D-ribose were co-metabolized. Much lower degrees of utilization occurred with co-metabolism of organic acids, polyols or ethanol. A mixture of D-glucose, D-ribose, D-raffinose, glycerol and D-xylose resulted in greater xylose utilization than the presence of a single substrate and xylose. The absence of growth on a co-substrate alone did not prevent the utilization of xylose in its presence. Xylose was co-metabolized with ribose under anaerobic conditions but at a much slower rate than under aerobic conditions. When [14C]xylose was utilized in the presence of ribose under anaerobic conditions, the radioactive label was detected mainly in xylitol and not in the small amounts of ethanol produced. Under aerobic conditions the radioactive label was distributed between xylitol (91.3 +/- 0.8%), CO2 (2.6 +/- 2.3%) and biomass (1.7 +/- 0.6%). No other metabolic products were detected. Whereas most xylose was dissimilated rather than assimilated by S. cerevisiae, the organism apparently possesses a pathway which completely oxidizes xylose in the presence of another substrate.     

Cadmium biosorption by Saccharomyces cerevisiae

Cadmium uptake by nonliving and resting cells of Saccharomyces cerevisiae obtained from aerobic or anaerobic cultures from pure cadmium-bearing solutions was examined. The highest cadmium uptake exceeding 70 mg Cd/g was observed with aerobic baker's yeast biomass from the exponential growth phase. Nearly linear sorption isotherms featured by higher sorbing resting cells together with metal deposits localized exclusively in vacuoles indicate the possibility of a different metal-sequestering mechanism when compared to dry nonliving yeasts which did not usually accumulate more than 20 mg Cd/g. The uptake of cadmium was relatively fast, 75% of the sorption completed in less than 5 min. (c) 1993 Wiley & Sons, Inc.     

Maltotriose fermentation by Saccharomyces cerevisiae

Maltotriose, the second most abundant sugar of brewer's wort, is not fermented but is respired by several industrial yeast strains. We have isolated a strain capable of growing on a medium containing maltotriose and the respiratory inhibitor, antimycin A. This strain produced equivalent amounts of ethanol from 20 g l⁻¹ glucose, maltose, or maltotriose. We performed a detailed analysis of the rates of active transport and intracellular hydrolysis of maltotriose by this strain, and by a strain that does not ferment this sugar. The kinetics of sugar hydrolysis by both strains was similar, and our results also indicated that yeast cells do not synthesize a maltotriose-specific α-glucosidase. However, when considering active sugar transport, a different pattern was observed. The maltotriose-fermenting strain showed the same rate of active maltose or maltotriose transport, while the strain that could not ferment maltotriose showed a lower rate of maltotriose transport when compared with the rates of active maltose transport. Thus, our results revealed that transport across the plasma membrane, and not intracellular hydrolysis, is the rate-limiting step for the fermentation of maltotriose by these Saccharomyces cerevisiae cells. Journal of Industrial Microbiology & Biotechnology (2001) 27, 34–38. Received 13 January 2001/ Accepted in revised form 29 May 2001     

Maltotriose metabolism by Saccharomyces cerevisiae

Saccharomyces cerevisiae grew slower but reached higher cellular densities when grown on 20 g maltotriose l^–1 than on the same concentration of glucose or maltose. Antimycin A (3 mg l^–1) prevented growth on maltotriose, but not on glucose or maltose, indicating that it is not fermented but is degraded aerobically. This was confirmed by the absence of ethanol and glycerol production. Active uptake of maltotriose across the plasma membrane is the limiting step for metabolism, and the low rate of maltotriose transport observed in maltotriose-grown cells is probably one of the main reasons for the absence of maltotriose fermentation by S. cerevisiae cells.     

Uptake of putrescine and spermidine by Gap1p on the plasma membrane in Saccharomyces cerevisiae

It has been reported that Gap1p on the plasma membrane of Saccharomyces cerevisiae can catalyze the uptake of many kinds of amino acids. In the present study, we found that Gap1p also catalyzed the uptake of putrescine and spermidine, but not spermine. The Km and Vmax values for putrescine and spermidine were 390 and 21 microM, and 4.6 and 0.59 nmol/min/mg protein, respectively. The uptake of putrescine was strongly inhibited by basic amino acids, lysine, arginine, and histidine, whose Ki values were 25-35 microM. Thus, it is deduced that spermidine and basic amino acids have almost the same affinity for Gap1p. When the concentrations of amino acids in the medium were reduced to one-third and 0.5 mM putrescine or 0.1 mM spermidine was added to the medium, accumulation of putrescine or spermidine by Gap1p was observed. Furthermore, when yeast was transformed with the GAP1 gene and cultured in the presence of 60 mM putrescine, cell growth was inhibited through overaccumulation of putrescine. GAP1 mRNA was found to be induced by polyamines. This is the first report of the identification, at a molecular level, of a polyamine uptake protein on the plasma membrane in eukaryotes.