AGT1 , Encoding an α-Glucoside Transporter Involved in Uptake and Intracellular Accumulation of Trehalose in Saccharomyces cerevisiae (original) (raw)
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Two Distinct Pathways for Trehalose Assimilation in the Yeast Saccharomyces cerevisiae
Applied and Environmental Microbiology, 2004
The yeast Saccharomyces cerevisiae can synthesize trehalose and also use this disaccharide as a carbon source for growth. However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear. By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose. We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route. First, the AGT1/MAL11 gene is controlled by the MAL system, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains. Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease. Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0. The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast.
Kinetics and Energetics of Trehalose Transport in Saccharomyces cerevisiae
European Journal of Biochemistry, 1996
Cells of Saccharomyces cerevisiae are able to transport trehalose against a concentration gradient, without efflux or counterflow of the labeled substrate. Uptake was inhibited by uncouplers, acetic acid, and organic mercury compounds. The addition of trehalose resulted in alkalinization of the medium. The ratio of H' depletion to trehalose uptake by yeast cells was approximately 1 : 1, which indicates the existence of a trehalose-H' symporter in these cells. The optimum pH for this active H+-trehalose symport was 5.0, and both the K,,, and the V,,, were negatively affected by increasing or decreasing the extracellular pH from its optimum value. Kinetic studies showed the existence of at least two different trehalose transport activities in yeast cells: a high-affinity Hi -trehalose symporter ( K , = 4 mM), and a low-affinity transport activity (K, > 100 mM) that could be a facilitated diffusion process. The high-affinity H+trehalose symporter was repressed by glucose, whereas the low-affinity uptake was constitutively expressed in S. cerevisiae.
FEBS Letters, 1994
When yeast strains were cultivated on maltose, the synthesis of trehalose already started in the exponential phase of growth, well before exhaustion of the sugar from the medium. This active pattern of trehalose accumulation was also observed in a maltose constitutive mutant strain growing on &cose. However, this accumulation was completely prevented by deletion of the TPSI gene coding for the catalytic subunit of the UDPgluccse-linked trehalose-6-phosphate synthase, indicating that no alternative pathway for trehalose synthesis exists in yeast. The active pattern of trehalose accumulation seems to be consistent with the finding that trehalose-6-phosphate synthase is more active in strains growing on maltose than on glucose.
Applied and Environmental Microbiology, 2007
In the yeast Saccharomyces cerevisiae , the synthesis of endogenous trehalose is catalyzed by a trehalose synthase complex, TPS, and its hydrolysis relies on a cytosolic/neutral trehalase encoded by NTH1 . In this work, we showed that NTH2 , a paralog of NTH1 , encodes a functional trehalase that is implicated in trehalose mobilization. Yeast is also endowed with an acid trehalase encoded by ATH1 and an H + /trehalose transporter encoded by AGT1 , which can together sustain assimilation of exogenous trehalose. We showed that a tps1 mutant defective in the TPS catalytic subunit cultivated on trehalose, or on a dual source of carbon made of galactose and trehalose, accumulated high levels of intracellular trehalose by its Agt1p-mediated transport. The accumulated disaccharide was mobilized as soon as cells entered the stationary phase by a process requiring a coupling between its export and immediate extracellular hydrolysis by Ath1p. Compared to what is seen for classical growth cond...
Applied and Environmental Microbiology, 2008
In the yeast Saccharomyces cerevisiae, the synthesis of endogenous trehalose is catalyzed by a trehalose synthase complex, TPS, and its hydrolysis relies on a cytosolic/neutral trehalase encoded by NTH1. In this work, we showed that NTH2, a paralog of NTH1, encodes a functional trehalase that is implicated in trehalose mobilization. Yeast is also endowed with an acid trehalase encoded by ATH1 and an H ؉ /trehalose transporter encoded by AGT1, which can together sustain assimilation of exogenous trehalose. We showed that a tps1 mutant defective in the TPS catalytic subunit cultivated on trehalose, or on a dual source of carbon made of galactose and trehalose, accumulated high levels of intracellular trehalose by its Agt1p-mediated transport. The accumulated disaccharide was mobilized as soon as cells entered the stationary phase by a process requiring a coupling between its export and immediate extracellular hydrolysis by Ath1p. Compared to what is seen for classical growth conditions on glucose, this mobilization was rather unique, since it took place prior to that of glycogen, which was postponed until the late stationary phase. However, when the Ath1p-dependent mobilization of trehalose identified in this study was impaired, glycogen was mobilized earlier and faster, indicating a fine-tuning control in carbon storage management during periods of carbon and energy restriction.
Biochimica et Biophysica Acta (BBA) - General Subjects, 1997
In Saccharomyces cereÕisiae, trehalose-6-phosphate synthase converts uridine-5 X-diphosphoglucose and glucose 6-phosphate to trehalose 6-phosphate which is dephosphorylated by trehalose 6-phosphatase to trehalose. These two steps take place within a complex consisting of three proteins: trehalose-6-phosphate synthase encoded by the GGS1r TPS1 () s FDP1,s BYP1,s CIF1 gene, trehalose 6-phosphatase encoded by the TPS2 gene and by a third protein encoded by both the TSL1 and TPS3 genes. Using three different methods for trehalose determination, we observed trehalose accumulation in ggs1 r tps1D, tps2D and tsl1D mutants, and in the double mutants ggs1 r tps1D r tps2D and also in ggs1 r tps1D deleted mutants suppressed for growth on glucose. All these mutants harbor MAL genes. Trehalose synthesis X Ž in these mutants is probably performed by the adenosine-5-diphosphoglucose-dependent trehalose synthase, ADPG-dependent nt trehalose synthase which was detected in all strains tested. It is noteworthy that trehalose accumulation in these mutants was detected only in cells grown on weakly repressive carbon sources such as maltose and galactose or during the transition phase from fermentable to non-fermentable growth on glucose. a-Glucosidase activity was always present in high Ž. amounts. We also describe an adenosine-diphosphoglucosepyrophosphorylase ADPG-pyrophosphorylase activity in Saccharomyces cereÕisiae which increased concomitantly with the accumulation of trehalose during the transition phase Ž c. from fermentable to non-fermentable growth in MAL-constitutive MAL2-8 strains. The same was observed when Ž. MAL-induced MAL1 strains were compared during growth on glucose and maltose. These results led us to conclude that maltose-induced trehalose accumulation is independent of the UDPG-dependent trehalose-6-phosphate synthaserphosphatase complex; that the ADPG-dependent trehalose synthase is responsible for maltose-induced trehalose accumulation probably by forming a complex with a specific trehalose-6-phosphatase activity and that ADPG synthesis is activated during trehalose accumulation under these conditions.
Expression of Saccharomyces cerevisiae α-glucoside transporters under different growth conditions
Brazilian Journal of Chemical Engineering, 2014
Important biotechnological processes depend on the efficient fermentation by Saccharomyces cerevisiae yeasts of starch hydrolysates rich in maltose and maltotriose. The rate-limiting step for fermentation of these α-glucosides is the transport across the plasma membrane of the cells. In order to contribute to a better understanding of maltose and maltotriose metabolism by S. cerevisiae, we analyzed the expression of the main α-glucoside transporter genes in two different yeast strains grown on media with glucose, maltose or maltotriose as carbon source. Although both yeast strains have higher α-glucoside transport activity during growth on maltotriose, our results show similar expression levels of the analyzed genes on either maltose or maltrotriose media. Thus, our results indicate that, although the transport capacity of maltotriose grown cells is higher than that of maltose grown cells, maltotriose cannot be considered a better inducer of α-glucoside transporter genes.
Expression of high-affinity trehalose–H+ symport in Saccharomyces cerevisiae
Biochimica et Biophysica Acta (BBA) - General Subjects, 1998
The expression of the high-affinity trehalose-H q symport was investigated in various Saccharomyces cereÕisiae strains and culture conditions. Previous kinetic studies of trehalose transport in yeast have revealed the existence of at least two different uptake mechanisms: a high-affinity trehalose-H q symport activity repressed by glucose, and a constitutive low-affinity transport activity, a putative facilitated diffusion process. Exogenously added trehalose was not an inducer of the high-affinity transport activity, and a correlation between trehalose and maltose uptake by yeast cells was found. Our results indicate that the maltose-H q symporters encoded by MAL11, MAL21, and MAL41 are not responsible for the trehalose transport activity. The analysis of both trehalose and maltose transport activities in wild-type and in laboratory strains with defined MAL genes showed that the trehalose-H q symporter was under control of MAL regulatory genes. Our results also suggest that the recently characterized AGT1 gene of S. cereÕisiae may encode the high-affinity trehalose-H q symporter. During diauxic growth on glucose the transport activity was low during the first exponential phase of growth, increased as glucose was exhausted from the medium, and decreased again as the cells reached the late stationary phase. This pattern was coincident with that of the intracellular levels of trehalose. The strong correlation between these two parameters may be of physiological significance during adaptation of yeast cells to stress conditions. q 1998 Elsevier Science B.V.